Beruflich Dokumente
Kultur Dokumente
MONOGRAPHS ON SYSTEMATIC
BACTERIOLOGY
A Compilation of Culture
Media
FOR THE CULTIVATION OF
MICROORGANISMS
BY
MAX LEVINE, Ph.D.
Professor of Sanitaryand Technical Bacteriology, Bacteriologist,
Iowa Engineering Experiment Station
AND
H. W. SCHOENLEIN, M.S.
Formerly Fellow in the Department of Bacteriology,
Iowa State College, Ames, Iowa
BALTIMORE
THE WILLIAMS & WILKINS COMPANY
1930
Copyright 1930
THE WILLIAMS & WILKINS COMPANY
-i 35b i) 1
Viii CONTENTS
materials of ani-
c. Agar media containing commercial digests and
mal origin other than extracts or infusions.
Section 5 (Med. 1628-1660) ....... 469
media containing commercial digests and extracts or in-
d Agar
1661-2028) 481
fusions of animal origin. Section 6 (Med.
digests.
e. Agar media containing non-commercial
Section 7 (Med. 2029-2078) 650 .
animal origin
e Aear media containing unknown constituents of
Section 9 (Med. 2142-2192) 690
but no digests.
MEDIA.
VII KEY TO THE SUB-GROUPS OF REVERSIBLY SOLID GELATIN
2199-2371) 711
Group III (Med.
A Sub-group IIIA. Key to gelatin media containing no other organic
(Med. 2199-2204) 711
materials.
composition other
3 Gelatin media containing materials of unknown
Section 3 (Med. 2324-2371) 752
than digests.
MEDIA; INITIALLY
IX KEY TO SUB-GROUPS OF IRREVERSIBLE SOLID
LIQUID; SOLIDIFYING AGENT ORGANIC.
Group V (Med. 2383-2466) 768
of plant
A Sub-group VA. Irreversibly solid media, solidified by materials
(Med. 2383-2397) 768
origin.
deriva-
B Sub-group. VB. Irreversibly solid media, solidified by blood or its
(Med. 2398-2430) 772
thres.
AGENT INOR-
X KEY TO IRREVERSIBLE SOLID MEDIA; SOLIDIFYING2467-2485)
Group VI (Med. 803
GANIC.
X CONTENTS
which are really old, the authors will feel well repaid for their time and efforts.
To Mr. H. G. Dunham, of the Digestive Ferments Company, we are greatly
indebted for helpful criticisms, and suggestions.
To Mrs. C. H. Werkman, Miss Ruth Confare and Miss Lois Kratoska, the
authors extend cordial thanks for assistance in typewriting and correcting the
manuscript.
To Dr. R. E. Buchanan the authors wish to particularly express their indebt-
edness and gratitude for the fatherly advice, encouragement, and assistance, so
graciously rendered at all times.
Max Levine,
h. w. schoenlein.
Iowa State College,
Ames, Iowa,
July 1, 1929
INTRODUCTION
At the request of the Society of American Bacteriologists, the Hterature was
surveyed for formulae of culture media reported useful for the growth of bacteria
and other microorganisms. The standard periodicals such as the various Cen-
tralblatts fiir Bakteriology, ZeitschriftHygiene, Annals of the Pasteur
ftir
conjunction with a specific name, the latter including the chief nitrogen and
carbon source, where possible, and the name of the author who first listed the
medium. Thus "273 Ayers' Glucose Ammonium Phosphate Solution," indi-
cates that Ayers first described the medium, the nitrogen source was ammonia,
the carbon source glucose, and that the medium is No. 273 in this collection.
In addition to the carbon and nitrogen source the nature of the solidifying
agent is also employed in naming culture media, as, for example, "1553 Greig
able author's name appears in the medium name, but the other authors are given
under the heading "references" at the end of the description of the medium.
Where several media differ only with respect to the relative quantities of
constituents employed, or in the method of preparation, the earliest described
medium takes precedence, and all others are described as variants.
One of the vexing questions which arose was that of the disposition of media
in which it was specified that a number of substances might be substituted for
each other. For example, if the availability of various carbon sources is being
studied, shall each new combination, as the employment of glucose, sucrose,
glycerol, etc., be considered a distinct medium? To do so would have markedly
extended the number of media to be considered.
The term "basal" was introduced to take care of this situation. This was ap-
plied to the formula exclusive of the substituted constituents which then became
added nutrients. The term "basal" occurring in the name of a medium there-
fore designates that the formula is not in iteslf a complete medium, but that it
serves as a base to which other constituents are added.
A serious difficulty in naming media arose in connection with the use of beef
extracts and meat infusions. This was particularly troublesome when authors
did not specify whether extract or infusions were employed. Where the use of
extracts was specified in the articles reviewed, these terms appear in the names
of the respective media, as e.g., "779 Dunham's Infusion Broth" or "1695
Heinemann's Meat Extract Agar." Where the author did not specify the
nature of the material employed (i.e., whether extract or infusion) the term
"bouillon" is employed, thus "936 Kendall, Day and Walker's Mannitol
Bouillon."
COMPOSITION OF CULTURAL MEDIA AND SUBSTRATES USED
IN BACTERIOLOGY
Any classification of media and substrates used in bacteriology must of
necessity be artificial and arbitrary in form. In the classification here adopted
an effort made to bring closely related media together, although it is
has been
some cases even the primary divisions tend to separate media
realized that in
having much the same essential composition and used for much the same
purposes.
The 7 primary divisions which have been adopted are as follows:
, ., . ^ , , , !
j.
metallic impurities which will inter-
is based primarily upon whether the nitro-
j-^j.^ ^^^-^ j^^ ^^^
gen supplied is inorganic or organic.
Distilled water' for the bacteriological
Subgroups of liquid media laboratory.
Ai. Water only Subgroup I-A Surface and ground water for media.
A2. Water with other constituents
Bi. All constituents of medium inorganic 1, Molisch's Basal Nutrient Solution
Subgroup I-B (Med. 2 to 113)
B2. One or more constituents organic Constituents:
Subgroup I-C (Med. 114 to 1394) 1. Sea water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Levulose 20 g.
Dextrin 20 g. 6. Locke's Solution
RafRnose 20 g. Constituents
Lactose 20 g. 1. Water 1000.0 cc.
References: Hoffmann and Hammer (1910 2. NaCl (0.9 to 1.0%)... 9.0 to 10.0 g.
p. 128). 3. KCl
(0.01%) 0.1 g.
4. CaCl2 (0.02%) 0.2 g.
5. von Wahl's Basal Salt Solution Preparation: (1) Dissolve 2, 3 and 4 in 1.
Reference: Charrin and Dissard (1893, p. through a Berkefeld filter, in some cases
182). by heat.
Use To study proteolysis by Proteus vul-
:
1. Water (River, Tap) 1000.0 cc. Uses: To study the effect of filter paper
2. Dipotassium phosphate 0.5 g. (colloid) on bacterial processes, particu-
Preparation :
Added Nutrients: The author employed
(1) Dissolve 2 and 3 in 1.
the following:
(2) Add suitable carbon source. (a) 1.0 g. glucose + 0.1 g. CaCOs
(3) Distribute in large Erlenmeyer flasks (b) 1.0 g. glucose + 0.1 g. CaCOs + 10.0 g.
(f) Specified the use of tap water, added Maltose 2.0% + sodium succinate 1.0%
1.0 g. asparagin and used 1.2 g. gyp- Maltose 2.0% + sodium asparaginate
sum. 1.0%
Reference: van Delden (1903-04, p. 83). Maltose 2.0% + ammonium citrate 1.0%
18. Czapek's Basal Solution (Waksman) Maltose 2.0% + peptone 1.0%
Constituents
Maltose 2.0% + asparagin 1.0%
Raffinose 2.0%
1. Distilled water 1000.0 ce.
Melitose 2.0%
2. K2HPO4 1.0 g.
Inulin 2.0%
3. KCl 0.5 g.
Glycerol 1.0, 3.0 or 5.0%
4. MgS04 0.5 g.
Glycerol 1.0, 3.0 or 5.0% + ammonium
5. FeS04 0.01 g.
phosphate 0.5%
Preparation
Inositol 2.0%
2, 3, 4 and 5 in 1.
(1) Dissolve
Mannitol 2.0%
As desired.
Sterilization:
Uses: Used by many investigators
Ammonium formate 1.0%, 2.0% or 5.0%
as a
basic mineral or salt solution for com-
Ammonium Tartrate 1.0%
parison of availability of various nitrogen
Ammonium citrate 1.0%
Sodium, potassium or calcium butyrate
and carbon nutrients.
0.5%, 1.0% or 2.0%
Reference: Waksman (1918, p. 479).
Sodium butyrate (0.5%) 5.0 g. + ammo-
19. Buchanan's Basal Solution nium phosphate 0.5%
Constituents: Calcium butyrate (1.0%) 1.0 g. + am-
1. Water 1000.0 cc. monium phosphate 0.5%
2. KH.PO4 2.0 g. Potassium valerianate 0.5%, 1.0% or
3. MgS04 (0.01%) 0.1 g. 2.0%
Preparation Sodium succinate 0.5%, 1.0% or 2.0%
(1) Dissolve 2, 3 and one of the added Sodium succinate 1.0% + ammonium
nutrients in 1. phosphate 0.5%
(2) Cool on ice. Calcium lactate 0.5%, 1.0% or 2.0%
(3) Filter to remove insoluble precipi- Calcium lactate 1.0% + ammonium phos-
tates. phate 0.5%
(4) Tube. Sodium citrate 1.0%
Sterilization: Intermittently in flowing Sodium citrate 1.0% + ammonium phos-
steam on each of 3 successive days. phate 0.5%
Use To study growth of Bacillus radicicola
: Asparagin 1.0%
bacteroids, and to study gum production Asparagin 1.0, 2.0, 3.0 or 5.0% + am-
by Bacillus radicicola. Author reported monium phosphate 0.5%
that generally carbohydrates and gluco- Asparagin 1.0% + sodium asparaginate
sides favored, while peptones inhibited 1.0%
the growth of B. radicicola; mannitol Sodium asparaginate 1.0, 3.0 or 5.0% +
especially favored growth. ammonium phosphate 0.5%
Added nutrients: One of the following Sodium asparaginate 0.5, 1.0,3.0 or 5.0%
materials or combinations was added: Nutrose 0.1, 0.5, 1.0, 2.0 or 5.0%
Arabinose 2.0% Nutrose 1.0% + ammonium tartrate
Rhamnose (Isodulcitol) 2.0% 0.5%
Glucose 2.0% Nutrose 1.0% + ammonium phosphate
2.0% + peptone 1.0% 0.5%
Mannose 2.0% Nutrose 1.0% + asparagin 1.0%
Galactose 2.0% Peptone (Witte) 1.0%
Levulose 2.0% Peptone (Witte) 1.0% + Ammonium
Sucrose 0.1, 10.0, 20.0, 30.0, or 50.0% phosphate 0.5%
Sucrose 2.0% + peptone 1.0% Peptone (Witte) 1.0% + sodium aspar-
Sucrose 2.0% + KNO3 0.05 to 0.5% aginate 1.0%
Sucrose 2.0% + nutrose 1.0% Peptone (Witte) 1.0% + asparagin 1.0%
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Peptone (Witte) 1.0% + KNO3 0.1 or (3) Distribute in 100.0 cc. lots in Erlen-
1.0% meyer flasks.
Amygdalin 2.0% (4) Add 0.5 g. of one of the added nu-
Amygdalin 2.0% + ammonium phos- trients to each flask.
phate 0.5% Sterilization: Not specified.
Salicin 2.0% + ammonium phosphate Use: To study the availability of nitrogen
0.5% for Actinomycetes odorifer, Act. chromo-
Salicin 2.0% genes, Act. albus I and II, Act. S. a, b,
bay ae, Bad. capri, Bad. guano, Bad. Sterilization: Not specified.
muscili, Bad. hoUandicus. Uses: Used by Beijerinck and van Del-
Reference : Buchanan (1906 p. 73). Perotti den as a mineral mixture to be added in
(1908 p. 220), Klaeser (1914 p. 38a) Stapp small quantities to a considerable variety
(1920 p. 3). of liquid media.
Reference: Beijerinck and van Delden
23. Box's Inorganic Salt Solution For Fungi (1903 p. 41).
(Tanner)
SUBGROUP I-B. SECTION 2
Constituents
Inorganic liquid media of known composi-
1. Distilled Water 3000.0 cc.
tion, nitrogen as free nitrogen only, com-
2. MgS04 1.5 g.
plete solutions, used as media without
3. K2HPO4 3.0 g.
addition of other nutrients.
4. KCl 1.5 g.
5. FeS04 "Complete" inorganic non-nitrogenous
0.03 g.
media have been described for the cultiva-
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
tion of certain iron bacteria and for certain
Sterilization : Not specified.
algae. In each case it will be noted that
Uses: Cultivation of fungi.
either "well water" or a heavy inoculum of
Reference: Tanner (1919 p. 65).
soil or soil extract is used. It is probable
therefore that ammoniacal or nitrate nitro-
24. Gage's Basal Salt Solution
gen is usually present, and may in some
Constituents cases be essential for the growth of the mi-
1. Water 1000.0 cc. croorganisms.
2. KH2PO4 0.5 g. The important media of this section may
3. MgS04 0.2 g. be differentiated as follows:
4. CaCl2 0.02 g. Ai. Medium employed primarily for iron
5. Fe2(S04)3 1 drop of 10% solution. bacteria.
Preparation Ellis' Ferric Hydroxide Solution 26
(1) Dissolve 2, 3, 4 and 5 in 1. A2. Media for algae. (Cyanophyceae or
(2) Add 1.5 g. of suitable carbon source. blue-green).
Sterilization: Method not specified. Beijerinck's Basal Phosphate Solution. 27
Uses Study of nitrogen fixation and of the
:
Heinze's Basal Solution A 28
morphology of the nitrogen fixing bacteria. Heinze's Basal Solution B 29
Added nutrients: The author suggests the
addition of 1.5 g. of one of the following 26. Ellis' Ferric Hydroxide Solution
carbon sources: Constituents:
Mannitol Galacton from slip- 1. Well water.
Kita's Basal Ammonium Sulphate (3) Distribute in 40.0 cc. lots in 100.0 cc,
Solution 39 Erlenmeyer jena glass flasks.
Grimm's Basal Ammonium Sulphate Sterilization: Not specified.
Solution 40 Uses Used to study the formation of starch
:
32. Kendall, Walker and Day's Basal Am- (2) Then add 0.5% ammonium sulphate
monium Chloride Solution to (1).
Sterilization: Sterilize on each of 3 succes-
Constituents:
sive days for 20 minutes in flowing steam.
1. Redistilled water 1000.0 cc.
Use : To study gum production of Bacillus
2. NH4CI 4.0 g.
radicicola.
3. NaCl 5.0 g.
Added nutrients: The following carbon
4. Na2HP04 sources (2.0%) were used by the author:
Preparation
arabinose levulose lactose
(1) Dissolve 2, 3 and 4 in 1.
rhamnose melitose raffinose
(2) Add one of the nutrients listed below.
glucose inositol mannitol
(3) Distribute in 100.0 cc. quantities in
mannose sucrose inulin
flasks.
galactose maltose starch
Sterilization: Method not given.
Reference: Buchanan (1909 p. 388).
Use: To study lipase production by tuber-
cle bacilli. Different esters were added 35. Cathelineau's Basal Ammonium Sul-
to clear bacteria free culture broth and phate Solution
incubated for 24 hours. Amount of acid
Constituents
produced measured in terms of N/50
1. Water 100.0 cc.
NaOH, determined lipase production.
2. (NH4)2S04 1.0 g.
Added nutrients: The authors used ethyl
Preparation
alcohol, glycerol or mannitol as carbon
(1) Dissolve 2 in 1.
sources.
(2) Add one of the following to (1).
Reference: Kendall, Walker and Day (1914
(a) Glucose 5.0 g.
p. 455).
(b) Sodium succinate 5.0 g.
(c) Sodium phosphate 5.0 g.
33. Lohnis' Basal Ammonium Chloride
(d) Sodium succinate 5.0 g.
Solution
Glucose 5.0 g.
Constituents: (e) Glucose 5.0 g.
1. Water (tap) 1000.0 cc. Sodium phosphate 5.0 g.
2. NH4CI 0.5 g. (f) Sodium succinate 5.0 g.
3. K2HPO4 0.5 g. Sodium phosphate 5.0 g.
Preparation Sterilization: Method not given.
(1) Dissolve 2 and 3 in 1. Use: To study pigment production and
(2) Add 1.0% of one of the listed nutrients fluorescence by Bacillus viridis (Lesage).
Sterilization: Not specified. Pigment production was independent of
Use: Methane production by cellulose de- the amount of phosphates present.
composers. Added nutrients: The author added the
Added nutrients: The author added 1.0% following carbon sources and salts:
of one of the following materials: (a) Glucose 5.0 g.
acetate starch (b) Sodium succinate 5.0 g.
butyrate sugar (c) Glucose 5.0 g.
lactate peptone Sodium succinate 5.0 g.
gum wool (d) Glucose 5.0 g.
Reference: Lohnis (1913 p. 93). Sodium phosphate 5.0 g.
(e) Sodium succinate 5.0 g.
34. Buchanan's Basal Ammonium Sulphate Sodium phosphate 5.0 g.
Solution Reference: Cathelineau (1896 p. 235).
Constituents:
1. Water 1000.0 cc.
36. Pere's Basal Ammonium Sulphate
Solution
2. (NH4)2S04 (0.5%) 5.0 g.
Preparation Constituents
(1) Add 2.0% of one of the carbon sources 1. Water 100.0 cc.
to water and dissolve. 2. Ammonium phosphate 1.0 g.
14 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
3. (NH4)2S04 3.0 g.
3. KH2PO4 5.0 g.
4. FeCla solution drops
4. MgS04 2.0 g.
5. Carbon source. 5. (NH4)2S04 5.0 g.
Preparation
Preparation
Dissolve 2, 3 and 4 in 1. (1) Dissolve 2, 3 and 5 in 1.
(1)
(2) Do not adjust the reaction. (2) Add several drops of a ferric chloride
solution to (1).
(3) Add any desired carbon source.
Sterilization: Not specified.
(3) Add 50.0 g. of any desired carbohy-
drate to (2).
Use : To study
a suitable carbon source for
acetic acid organisms, B. pasteurianum,
Sterilization: Not specified.
38. Omeliansky's Basal Ammonium Sul- 40. Grimm's Basal Ammonium Sulphate
phate Solution Solution
Constituents: Constituents
1. Distilled water 1000.0 cc. 1. Distilled water 1000.0 cc.
2. Potassium phosphate 0.1 g. 2. K2HPO4 0.5 g.
3. (NH4)2S04 0.1 g. 3. MgS04 0.5 g.
4. MgS04 0.05 g. 4. (NH4)2S04 5.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 15
Brom cresol purple, 2.0 cc. saturated Preparation: (1) Dissolve 2, 3 and 4 in 1.
(0.5 to
1.0%) 5.0 to 10.0 g. (2) Add 9.0 to 10.0% of one of the organic
3. KH2PO4 (0.5%) 5.0 g. acids to (1).
4. MgS04 (0.5%) 5.0 g. (3) Adjustment of reaction not specified.
5. K3PO4 (0.05%) 0.5 g. (4) Distribute into fermentation flasks
Preparation (sealed with H2SO4).
(1) Dissolve 2, 3, and 5 in 1.
4 (5) After inoculation plug the flask with
(2) Add one of the added nutrients. a cork and seal with paraffin.
Sterilization: Not specified. Sterilization: Sterilize in a steamer
Use: Cultivation of schizomycetes. (method not given).
Added nutrients: The author added 5.0% Use: To study the utilization of acids by
glycerol or 5.0% sucrose. yeast. The author found 0.1 g. tartaric,
Variants: Tanner gave the following as 0.15 g. malic and 0.26 g. citric acid was
Fermi's Solution: used in 65 days by the yeast. Acid de-
1. Distilled water 1000.0 cc. terminations were made by titration with
2. MgS04-7H20 0.2 g. normal NaOH, using litmus as an indi-
3. K2HPO4 1.0 g. cator.
4. (XH4).HP04 10.0 g. Added nutrients and variants
5. Glycerol 45.0 g. (a) The author added 9.0 to 10.0% of one
Reference: Fermi (1892 p. 26), Tanner of the following organic acids:
(1919 p. 68). tartaric malic citric
(b) Henneberg specified the use of 0.3%
50. Palladin's Basal Ammonium Phosphate KH,P04 and used 0.1% MgS04 in-
Solution stead of 0.05%. He added 5.0% glu-
Constituents cose and used it for the cultivation of
1. Water 1000.0 cc. lactic acid bacteria.
2. Ammonium phosphate 4.7 g. Reference: Schukow (1896 p. 607), Henne-
3. Potassium phosphate 3.0 g. berg (1903 p. 7).
4. MgS04 1.0 g.
5. CaClj 1.0 g. 52. Koser's Basal Ammonium Phosphate
6. FeCl2 Trace Solution
Preparation Constituents
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 1. Distilled water 1000.0 cc.
(2) Add sufficient carbohydrate to make 2. NaCl 5.0 g.
solution one fourth molar. 3. MgS04-7H20 0.2 g.
CULTURE MEDIA FOR CULTIVATIOX OF MICRO ORGAXISMS 19
53. Miinter's Basal Ammonium Nitrate 54. von Bronsart's Basal Ammonium Nitrate
Solution Solution
Constituents: Constituents
1. Water 1000.0 cc. 1. Water 1000.00 cc.
2. MgS04 0.5 g. 2. iMgS04 (0.25%) 2.5 g.
3. NaCl 0.5 g. 3. KH2PO4 (0.25%) 2.5 g.
4. CaCl. 0.2 g. 4. NH4NO3 (1.0%) 10.0 g.
5. K2HPO4 1.0 g. Preparation
6. NH4NO3 (1) Dissolve 2, 3 and 4 in 1.
7. CaCOa (2) Add one of the test materials to (1).
Preparation Sterilization: Not given.
(1) Dissolve 2, 3, 4 and 5 in 1. Use: To study utilization of various car-
(2) Neutralize by the addition of CaCOs. bon sources by Xylaria, Xylaria arhua-
(3) To each 100.0 cc. of (2) add 0.025 g. cula, Xylaria polymorpha, Xylaria hy-
of nitrogen in the form of XH4NO3. poxylon. Growth best using starch as a
(4) Add 1.0 g. of one of the test materials carbon source with Xylaria arbuscula,
to (3). and levulose produces best growth with
20 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents
1. Distilled water 1000.0 cc.
61. Winogradsky Ammonium Sulphate
Solution (Harvey)
2. NH4CI 0.5 g.
3. Potassium phosphate 0.1 g. Constituents
4. MgS04 0.02 g. 1. Distilled water 1000.0 cc.
5. CaCl. 0.01 g. 2. (NH4)2S04 1.0 g.
6. CaCOs 5.0 g. 3. K2SO4 1.0 g.
Preparation 4. MgS04 7.5 g.
(1) Dissolve 2, 3, 4 and 5 in 1. Preparation: (1) Dissolve 2, 3 and 4 in 1.
(2) Add 6 and distribute into flasks. Sterilization : Not specified.
Sterilization: Not specified. Use: Study of nitrifying organisms.
Use: For the study of nitrite formation. Reference: Harvey (1921 p. 106).
Reference: Johnson (1912 p. 219).
58. Beijerinck and Minkman Ammonium 62. Buhlert and Fickendey Ammonium
Chloride Solution Sulphate Solution
Constituents: Constituents:
1. Water 100.0 cc. 1. Water 1000.0 cc.
2. K2HPO4 0.02 g. 2. (NH4)2S04 4.0 g.
3. NH4CI 0.02 g. 3. Potassium phosphate 2.0 g.
4. NaHCOs 0.1 g. 4. Basic magnesium carbonate
Preparation (4MgC03-Mg(OH)2-5H20).. 40.0 g.
(1) Dissolve 2, 3 and 4 in 1. Preparation: (1) Dissolve 2, 3 and 4 in 1.
(c) Abbott gave the following solution: (3) Add several tenths grams of MgCOj
1. Water 1000.0 cc. to each lot.
2. (NH4)2S04 1.0 g. (4) Sterilize intermittently.
3. Potassium phosphate... 1.0 g. (5) Add 1.0 or 2.0 cc. of a sterile 2.0%
4. MgCOs (Basic) (NH4)2S04 solution to each lot under
(1) Dissolve 2 and 3 in 1. aseptic conditions.
(2) Flask in 100.0 cc. quantities. (c) Harvey modified Burri and Stutzer's
(3) Add 0.5 to 1.0 g. of basic MgCOs sus- solution in that he used 1.0 g.
pended in a little sterile water to K2HPO4 instead of KH2PO4 and used
each flask. 2.0 g. NaCl instead of 0.5 g.
References: Buhlert and Fickendey (1906 References: Winogradsky (1891 p. 577),
p. 404), Barthel (1910 p. 112), Percival Burri and Stutzer (1896 p. 106), Harvey
(1920 p. 208), Gage (1910 p. 14), Abbott (1921-22 p. 105) Heinemann (1905 p. 129).
(1921 p. 603). Modified Winogradsky's
64. Giltner's
Ammonium Sulphate Solution
63. Winogradsky's Ammonium Sulphate Constituents:
Solution 1. Distilled water 1000.0 cc.
2. (NH4)2S04 0.4 g.
Constituents:
3. MgS04 0.05 g.
1. Distilled water 1000.0 cc.
4. K2HPO4 0.1 g.
2. Potassium phos-
5. NaoCOs 0.6 g.
phate 1 g.
CaClo trace
6.
3. MgS04 0.5 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
4. CaCla trace
in 1.
5. MgCOs amount not specified
Sterilization: Not specified.
6. (NH4)2S04 2.0 to 2.5 g.
Use: Study of nitrate formation.
Preparation
Reference: Giltner (1921 p. 369).
(1) Dissolve 2, 3 and 4 in 1.
(2) Distribute in flasks.
65. Winogradsky and Omeliansky's
(3) After sterilization add MgCOs Ammonium Sulphate Solution
(amount not specified) and from 2 to Constituents:
2.5 parts per thousand of (NH4)2S04. 1. Distilled water 1000.0 cc.
Sterilization: Method not specified. 2. (NH4)2S04 2.0 g.
Use: To show nitrification by organisms 3. Potassium phosphate 1-0 g.
found in the soil. Inoculate with soil. 4. MgS04 0.5 g.
Variants 5. NaCl 2.0 g.
(a) Burri and Stutzer 6. FeS04 0.4 g.
1. Water 1000.0 cc. 7. MgCOs in excess
2. NaCi 0.5 g. Preparation
3. KH2PO4 '
1.0 g. (1) Dissolve 3, 4, 5 and 6 in 1.
ing organisms when initially inoculated (3) Distribute (1) in 5.0 cc. lots.
with soil. Giltner and Ldhnis used (4) Add 0.5 cc. of (2) to each 5.0 cc. lot
Harvey's solution (1.0 g. (NH4)2S04) and of (3).
suggested the use of either CaCOa or Sterilization: Not specified.
MgCOs. He reported that both nitrite Use: To isolate nitrite formers from the
and nitrate bacteria are stimulated if soil and to study nitrification. Tromms-
CaCOs is used instead of MgCOs. Tan- darf's reagents were used to test for the
ner used KH2PO4 in Harvey's (a) solution presence of nitrites.
instead of K2HPO4. Reference: Gowda (1924 p. 252).
OMELTANSKY
INVESTIGATOR 1899
Distilled water
(NH4)2S04
NaCl
Potassium phosphate
MgS04
FeS04
MgCO,
K2HPO4
KH2PO4
KCl
24 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
clave.
5. NaCl 2.0 g.
6. FeS04 0.4 g.
Use : Inoculate with soil to study nitrifying
Preparation
bacteria.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Reference : Christensen (1913 p. 418).
(2) During incubation at 20 to 25C.
69. Gage's Ammonium Sulphate Solution allow some ordinary gas to enter the
incubator.
Constituents
1. Water 1000.0 cc.
Sterilization: Not specified.
2. (NH4)2S04 4.0 g.
Use: To study the assimilation of Ca2 by
B. oUgocarbophilus.
3. KH2PO4 2.0 g.
4. MgS04 1.0 g.
Reference: Lohnis (1913 p. 106).
5. FeS04 0.8 g.
6. NaCI 4.0 g. 72. Stutzer's Ammonium Magnesium
Preparation Phosphate Solution
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents:
(2) Distribute in Erlenraeyer flasks.
Sterilization: Method not specified.
1. Water 2000.0 cc.
bacillus thiooxidans. The organisms can (2) Adjustment of reaction not specified.
grow at a pH = 1.0 with a maximum at (3) Inoculate with a little ditch or canal
pH = 3.0 to 4.0. water or ditch mud. Incubate at
Variants: The author added a trace of 28 to 30 C.
FeS04 and the following combinations of Sterilization: None required.
potassium salts of phosphoric acid to the Use: Enrichment and isolation of organ-
above solution containing no potassium isms utilizing carbonic acid, Thiobacillus
phosphate, thioparus. Two or three days after
(a) KH2PO4 3.0 g. pH = 4.2 inoculation the surface is covered with a
'KH2PO4 1.5 g.\ ^TT _ 5 4
layer of free sulphur filled with bacteria.
Variants
(c) K2HPO4 3.0 g. pH = 6.0 (a) When Beijerinck used an inoculum
. ,. /K2HPO4 3.0 g.l ^ _..
P^ - ^-^
from sea water, 3.0% NaCl was added
^^)
icaCOa 10.0 g./ to the solution.
At the various pH values the author (b) Waksman used the above solution
found starting with acid medium some with either 0.02 g. Na2HP04 or K2-
Th. thiooxidans give final pH = 1.2. Soil HPO4 and added one of the following
bacteria produce no acid at pH = 4.2 combinations:
but change pH = 8 to pH = 7.6. (1) KH2PO4 3.0 g. pH = 5.4
Reference: Waksman (1922 pp. 606, 609 (2) NaHCOa 1.0 g. pH = 8.8
to 613). fNaHCOalLOg. ^3 =
^'^'
\CaCO3 P^ ^-^
jlO.Og.
9 4
75. Waksman's Sulphide Solution
He found that Thiobacillus thio-
Constituents oxidans produced a pH = 1.4 in
1. Water 1000.0 cc. variant (b) (1).
2. K2S 5.0 g. (c) Trautwein substituted Na2HP04 for
3. NH4CI 0.1 g. K2HPO4 in the original solution and
4. MgClj 0.1 g. used the solution to grow Thionic acid
5. Na2HP04 or K2HPO4 0.2 g. bacteria (Omeliansky).
6. NaHCOs 1.0 g. Reference: Beijerinck (1903 p. 595), Waks-
7. CaC03 10.0 g. man (1922 p. 609), Trautwein (1921 p.
or CaClj 0.25 g. 518).
Preparation
77. Kaserer's Hydrogen Ammonium Chlor-
(1) Dissolve 2, 3, 4 and 5 in part of 1.
ide Solution
(2) Dissolve 6 and 7 in rest of 1.
Constituents
Winogradsky 's Basal Sodium Nitrite
Distilled water
Solution 90
1. 1000.0 cc.
D2. Magnesium sulphate not added.
2. (NH4)2HP04 0.2 g.
Gartner's Sodium Nitrite Solution ... 91
3. NaaCOa 1.0 g.
4. Beijerinck and van Del-
Wimmer's Sodium Nitrite Solution.. 92 .
of (1).
Bijerinck and van Delden's Potassium
Sterilization: Not specified.
Nitrite Solution A 95
Use: Bacillus oligocarbophilus grows as a
snow white scum on the surface of the
86. Hewlett's Nitrite Solution (Johnson)
medium.
Reference: Beijerinck and van Delden Constituents
(1903 p. 41). 1. Distilled water 1000.0 cc.
2. KNO2 0.3 g.
85. Beijerinck and van Delden's Ammo- Potassium phosphate
3. 0.1 g.
nium Phosphate Solution
4. MgS04 0.05 g.
Constituents 5. CaCOs 5.0 g.
1. Distilled water 100.0 cc. Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
2. K2HPO4 0.02 g. Sterilization: Not specified.
3. (NH4)2HP04 0.02 g. Use: Study of nitrate production. Bacil-
4. NazCOa 0.1 g. lus megatherium produced no nitrates
Preparation in this solution.
(1) Dissolve 2, 3 and 4 in 1. Reference: Johnson (1912 p. 219).
(2) Reaction to be slightly alkaline.
Sterilization: Not specified. 87. Cunningham's Nitrite Solution
Use: Bacillus oligocarbophilus grows as a
thin white scum on the medium. Constituents
1. Distilled water 1000.0 cc.
Reference: Beijerinck and van Delden
(1903 p. 41).
2. NaNOo 1.0 g.
3. K2HPO4 0.5 g.
(3) Add a small quantity of sterile addition of 0.1% Na2C03 gave the best
CaCOs to each tube of sterile (2). results. The addition of FeS04 had no
Sterilization: Sterilize (2) and the CaCOa effect on nitrate production.
Use: To study oxidation of nitrites and the following materials to determine the
nitrates. effect of organic material on nitrification.
Reference: Cunningham (1924 p. 151). glycerolO.l, 0.2, 0.4, 0.8, 1.0, 1.4 or 2.0%
glucose 0.05, 0.1, 0.2, 0.4, 0.8, 1.0 or 2.0%
88. Omelianski's Nitrite Solution urea 0.1, 1.0 or 1.6%
Constituents: asparagin 0.1, 0.6, 0.8, 1.0 or 2.0%
1. Distilled water 1000.0 cc. hay infusion 4.0, 8.0, 16.0 or 32.0%
2. NaNOs (Merck) 1.0 g. leaf infusion 4.0, 8.0, 16.0 or 32.0%
3. NaaCOs (ustum) 1.0 g. dirt infusion 4.0, 8.0, 16.0 or 32.0%
4. Potassium phosphate 0.5 g. meat infusion 2.0, 4.0, 8.0 or 10.0%
Reference: Omeliansky (1899 p. 548), Boul- (c) Gibbs (Gowda) used 0.5 g. K2HPO4
langer and Massol (1903 p. 494). and a trace of Fe2(S04)3 instead of
0.4 g. FeS04.
89. Winogradsky and Omeliansky's Sodium Reference: Winogradsky and Omeliansky
Nitrite Solution Wimmer (1904
(1899 p. 333), p. 140),
Constituents Smith (1905 p. 199), Harvey (1921-22 p.
1. Distilled water 1000.0 cc. 106), Gowda (1924 p. 258), Percival (1920
2. NaNOa 1-0 g- p. 149).
(4) Add 0.0, 0.1, 0.2, 0.4, 0.5 or 0.6 g. of 6. NaCl 0.5 g
peptone. Constituents:
(c) 0.1, 0.2, 0.4 or 0.8 g. urea. 1. Distilled water.. 1000.0 cc.
(d) 0.1, 0.2 or 0.4 g. asparagin. 2. Natrium nitro-
(e) 5.0 cc. of bouillon. sum puriss
The presence of organic materials gen- (NaNOo) 1.0, 2.0 or 50.0 g.
erally tends to slow up the oxidation. Preparation: (1) Dissolve 2 in 1.
WINOGRADSKY
AND
OMELIANSKY
Distilled water 1000.0 cc. 1000.0 cc. 1000.0 cc. 1000.0 cc.
NaN02 1.0 g. 2.0 g. 25.0 cc. 2.0% l.Og.
solution
Potassium phosphate 0.5 g. 1.0 g.
MgS04 0.3 g. 0.3 g. 0.5 g. 0.3 g.
NaCl 0.5 g. 0.5 g. l.Og. 0.5 g.
NaoC03 1.0 g. 0.3 g.
K2HPO4 1.0 g. l.Og.
K2CO3 0.5 g.
KCl 0.1
7. KNO2 0.05 g.
8. Soda 0.05 g. 96. Beijerinck's Basal Nitrate Solution
Preparation : Dissolve 2, 3, 4, 5, 6, 7 and 8
Constituents:
in 1.
1. Water 1000.0 cc.
Sterilization: Not specified.
2. KNO3 1.0 g.
Use: Cultivation of bacteria found in soil
3. K2HPO4 0.2 g.
on roots and rhizomes.
Preparation
Reference: Gottheil (1901 p. 432).
(1) Dissolve 2 and 3 in 1.
(2) Add one of the materials listed under
95. Beijerinck and van Delden's Potassium
added nutrients.
Nitrite Solution A
Sterilization: Not specified.
Constituents: Use: To study denitrification by Azoto-
1. Water (tap) 100.0 cc. bacter chroococcum.
2. KCl 0.01 g. Added nutrients and variants
3. K2HPO4 0.02 g. (a) The author added 20.0 to 100.0 g.
4. Kx\02 0.01 g. mannitol.
Preparation (b) Beijerinck and van Delden used 0.2 g.
(1) Dissolve 2, 3 and 4 in 1. KNO3 in the basic solution without
(2) The reaction is slightly alkaline. additional nutrients for the cultiva-
Sterilization: Not given. tion of Bacillus oligocarbophilus
Use: Bacillus oligocarbophilus grows in a (c) Beijerinck and van Delden used 0.2 g
dry thin snow white scum on the surface KNO3 and 0.2 g. Na2HP04 in the
of the medium. basic solution and added 0.2 g. KCl for
Reference: Beijerinck and van Delden the cultivation of Bacillus oligocarbo-
(1903 p. 41). philus.
(d) Beijerinck and van Delden used 0.1 g.
SUBGROUP I-B. SECTION 7
KNO3 and 0.1 g. K2HPO4 in the basic
Inorganic basal solutions of known com- solution and added 0.1 g. Na2C08 for
positions; nitrogen supplied as nitrates; the cultivation of Bacillus oligo-
incomplete solutions requiring the addition carbophilus.
of other nutrients. (e) Beijerinck and Minkman used 1.0%
A Saltsof monovalent cations only added.
I.
KNO3 and 0.05% K.2HPO4 in the
Beijerinck's Basal Nitrate Solution. 96 basic solution and added 2.0% man-
Stoklasa and Vitek's Basal Nitrate nitol, glycerol, sodium acetate or
Solution 97 sodium propionate to study deni-
A2. Salts of mono and divalent cations trification.
dans ferments arabinose, levulose, dex- Reference: Stoklasa and Vitek (1905 p.
trose, galactose, maltose, dextrin, ethyl 104) (1901 p. 262).
and propyl alcohol, erythritol. B. aceto-
sum ferments arabinose, dextrose, galac- 102. Harvey's Basal Nitrate Solution
tose, ethyland propyl alcohol. B. aceti, Constituents:
B. Kiitzingianum, B. Pasteurianum and 1. Distilled water 1000.0 cc.
B. acetigenum ferment dextrose and 2. NaNOa 1.0 g.
ethyl and methyl alcohol. 3. NaCl 0.02 g.
Added nutrients: The author added the 4. MgS04 0.02 g.
following carbon sources: 5. K2HPO4 0.5 g.
arabinose inulin 6. FeCla trace
levulose methyl alcohol Preparation
glucose ethyl alcohol (1) Dissolve 2, 3, 4, 5 and 6 in 1.
galactose propyl alcohol (2) Add one of the added nutrients.
sarobose amyl alcohol Sterilization: Not specified.
sucrose glycerol Use: Study of nitrate reduction.
maltose erythritol Added nutrients: The author added 10.0 g.
lactose mannitol glucose or 10.0 g. glycerol.
dextrin dulcitol Variants : The author used the following
starch melampyrit solution:
glycogen quercitol 1. Distilled water 3000.0 cc.
Reference: Henneberg (1898 p. 19). 2. KNO3 7.0 g.
3. K0HPO4 1.5 g.
dry thin snow white scum on the surface 3. NaN03 (0.01%) = 0.01 g.
specify the carbohydrates used. (f) 0.12 cc. of benzol or xylol in 2000.0
Reference: Kita (1913 p. 434). cc. flask.
Reference: Lantzsch (1922 p. 310).
105. Stoklasa's Basal Nitrate Solution
Constituents
SUBGROUP I-B. SECTION 8
Constituents Constituents
1. Distilled water 100.0 cc. 1. Distilled water 100.0 cc.
112. Nabokoch and Lebedeff's Hydrogen supplied. The sections recognized may be
Nitrate Solution differentiated as follows:
6. FbsCU some
C2. Nitrogen present as ammonium salts.
7. Hydrogen
Section 2 (Med. 191-323)
Preparation
Cj. Nitrogen present as nitritesj.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Section 3 (Med. 324-327)
(2) The reaction is slightly alkaline
C4. Nitrogen present as nitrates.
(about 0.050 g. H2SO4 to 100.0 cc. of
Section 4 (Med. 328-365)
solution).
B2. Nitrogen organic.
(3) Distribute in 100 or 150.0 cc. lots in
Ci. Nitrogen present as amino acids.
vacuum flasks with side tubes.
Section 5 (Med. 368-479)
(4) Inoculate.
C2. Organic nitrogen other than amino
(5) Remove all the air by means of an
acids present Section 6 (Med. 480-515)
.
pump.
. .
oil
A2. Chemical composition of one or more
(6) Replace the air by a mi.xture of oxygen
constituents not definitely known.
and hydrogen ("Knallgase") contain-
Bi.* Containing digests.
ing carbonic acid.
Ci.* Containing commercial peptones or
Sterilization: Method not given.
digests.
Use: To study the oxidation of hydrogen
Di. Additional if any,
constituents,
in-
by microorganisms.
organic Section 7 (Med. 516-555)
Reference: Nabokich and LebedefT (1907
D2. Containing additional organic constit-
p. 352), Lohnis (1913 p. 106).
uents.
113. Bokomy's Calcium Nitrate Solution El. Chemical composition of all additional
materials known.
Constituents
Fi. No additional nitrogen compounds
1. Water 100.0 cc.
added.
2. Ca(N03)2 0.1 g.
Gi. Basal solutions. Employed with the
3. MgS04 (crystalline) 0.02 g.
addition of various nutrients.
4. KH2PO4 0.02 g.
Section 8 (Med. 556-588)
5. Iron chloride trace
B2. Complete nutrient solutions.
6. KCl 0.0 or 0.05 g.
Section 9 (Med. 589-647)
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
F2. At least one additional nitrogenous
in 1.
constituent.
Sterilization : Not specified.
Section 10 (xMed. 648-683)
Use: To study the metals required for the
E2. Composition of one or more additional
growth of Spirogyra majuscula. It was
materials not definitely known.
found that potassium was necessary.
Fi. Additional organic material exclusively
Reference: Bokorny (1912 p. 125).
of plant origin
Section 11 (Med. 684-712)
SUBGROUP I-C F2. Containing additional materials of
animal origin; plant derivatives may also
Liquid Media with One or More be present.
Constituents Organic G:. Not containing extracts or infusion.
Section 12 (Med. 713-747)
The liquid media in which one or more G2. Containing extracts or infusion.
constituents are organic may be divided Section 13 (Med. 748-977)
conveniently into sections based upon the
character of the nitrogen and carbon sources See page 37 for B2 and C2.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 37
stituents of
Henneberg's Basal Sucrose Salt Solu-
containing free or elementary nitrogen
tion 135
only, carbon organic.
Czapek's Sucrose Salt Solution
Ai. Inorganic salts not added.
(Waksman) 136
Bi. Carboh3'drates added.
von Bronsart's Basal Sucrose Salt
Pringshein's Glucose Cellulose Solu-
Solution 137
tion 114
Bokorny's Basal Sucrose Salt Solu-
Waksman and Joffe's Basal Glucose
tion 138
Solution 115
Smith's Sucrose Salt Solution 139
Buchanan's Sucrose Solution 116
B2. Alcohols added.
Munter's Basal Galactose Salt Solu-
Waksman and tion 140
Joffe's Basal Glycerol
Solution 117 Buchanan's Basal Maltose Salt Solu-
Buchanan's Glycerol Solution I 118 tion 141
*See next page for C2, E2. * See ne.xt page for F3.
38 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
F3. Carbon present as organic acids or their Winogradsky's Mannitol Salt Solu-
salts. tion (Lantzsch) 175
Percival's Succinate Solution 152 G2. Glycerol added.
E2. Calcium salts added. Koser and Rettger's Basal Glycerol
Fi. Carbon present as carbohydrates. Salt Solution 176
Gi. Glucose added. F3. Carbon present as organic acids
Hi. Glucose only organic carbon source. or their salts.
Winogradsky's Salt Glucose Solution. 153 Puriewitsch's Tartaric Acid Salt
Stutzer, Burri and Maul's Glucose Solution 177
Salt Solution 154 C2. Magnesium salts not added.
Stoklasa's Basal Glucose Salt Solu- Di. Phosphates not added.
tion 155 Omeliansky's Cellulose Solution 178
Charpentier's Salt Glucose Solution. 156 Killer's Mannitol Solution 179
Krzemieniewska's Glucose Salt Solu- Ringer's Salt Solution 180
tion (Vogel) 157 D2. Phosphates added.
Kriiger and Schneidewing's Basal Ei. Calcium salts not added.
Glucose Salt Solution (Heinze) 158 Bijerinck's Glucose Salt Solution. . . . 181
Charpentier's Basal Glucose Salt Stoklasa's Glucose Salt Solution. . . . 182
Solution 159 Gag's Maltose Salt Solution 183
Stoklasa's Basal Glucose Salt Solu- Sohngen's Colloid Mannitol Solution. 184
tion 160 E2. Calcium salts added.
Henneberg's Basal Glucose Salt Solu- Fi. Carbon present as carbohydrates.
tion 161 Bijerinck's Phosphate Glucose Solu-
H2. Glucose with other organic carbon. tion 185
Gottheil's Carbohydrate Glycerol Buhlert and Fickendey's Phosphate
Solution 162 Glucose Solution 186
Harvey's Citric Acid Glucose Solu- F2. Carbon present as alcohols or acids.
tion 163 Wojtkiewicz's Mannitol Salt Solu-
Munter's Basal Mannitol Salt Solu- tion 187
tion 164 Kranisky's Mannitol Salt Solution 188 . . .
170
Use: To study nitrogen assimilation meth-
Solution
ane fermenting bacteria.
Fred and Loomis' Mannitol Salt Solu-
Variants: The author used the following
tion 171
combinations to study nitrogen assimila-
Krzemieniewska's Mannitol Salt
tion by Clostridium and azotobacter:
Solution (Vogel) 172
(a) 10.0 g. cellulose +0.4 g. de.xtrose in
Harvey's Mannitol Salt Solution. 173 . . .
(d) 5.0 g. cellulose +0.2 g. dextrose in distilled water. Repeat the precipita-
1000.0 cc. solution tion 5 times. Finally dry the precipi-
(e) 2.5 g. cellulose +0.2 g. dextrose in tate. Saccharose may be used in higher
500.0 cc. solution concentrations.
(f) 5.0 g. cellulose +0.2 g. dextrose in Reference: Buchanan (1909 p. 371).
500.0 cc. solution
117. Waksman and Joflfe's Basal Glycerol
(g) 2.5 g. cellulose +0.2 g. dextrose in
Solution
500.0 cc. solution
(h) 5.0 g. cellulose + 0.2 g. mannite in Constituents
1000.0 cc. solution 1. Water to make 1000.0 cc.
Reference: Pringsheim (1909 p. 303), (1910
2. Glycerol 30.0 g.
p. 224).
Preparation
(1) Dissolve 2 in 1.
115. Waksman and Joffe's Basal Glucose (2) Add one of the test materials (added
Solution nutrients) to (1). When using casein
Constituents: and egg albumin, dissolve casein and
1. Water 1000.0 cc. egg albumin in N/10 NaOH. In using
2. Glucose 30.0 g. fibrin, add fibrin to each tube.
Preparation (3) Solutions vary in pH = 7.2 to 7.7,
(1) Dissolve 2 in 1. urea pH = 8.0.
(2) Add 2.0 g. of one of the added nu- (4) Tube in 10-12 cc. lots.
trients. Sterilization: Autoclave for 15 minutes at
(3) Tube in 10 to 12 cc. lots. 15pounds pressure.
Sterilization: Sterilize at 15 pounds for 15 Use: To study change in reaction in
minutes. Actinomycetes metabolism.
Use: To study reaction changes in Actino- Added nutrients: The authors added 5.0 g.
mycetes metabolism. of one of the following nitrogenous com-
Added nutrients: The authors added 2.0 g. pounds:
of one of the following nitrogen sources: fibrin leucin
(NH4)2S04 (pH of solution = 5.8) casein glycocoll
(XHOsCOa (pH of solution = 6.8) egg albumin urea
Urea (pH of solution = 7.4) peptone acetamide 5.0 g.
Reference: Waksman and Joffe (1920 p. 39). asparagin
Variants: The authors used 3.0 g. of glyc-
116. Buchanan's Sucrose Solution
erol and added 2.0 g. of NaNOa, NaNOs,
Constituents or (NH4)2S04 as inorganic nitrogen
1. Water (tap) 1000.0 cc. sources.
2. Sucrose (2.0%) 20.0 g. Reference: Waksman and JofTe (1920 pp.
Preparation 36-37).
(1) Prepare a 2.0% saccharose solution
118. Buchanan's Glycerol Solution I
in tap water.
(2) Distribute in 500.0 cc. lots in liter Constituents:
1. Water (tap) 1000.0 cc.
Sterilization: Method not given. 2. Glycerol 5.0 cc.
Use: Development of gum produced by Preparation: (1) Dissolve 2 in 1.
119. Beijerinck and van Delden's Phosphate and suggested that CaCOs might be
Glucose Solution added if desired.
(d) Percival specified the use of tap
Constituents
water.
1. Water 1000.0 cc.
(e) Beijerinck and van Delden used 0.5 g.
2. Glucose 20.0 g.
3. K2HPO4 0.5 g.
K2HPO4 instead of 0.2 g. K0HPO4.
(f Stoklasa specified Moldau river water
Preparation: (1) Dissolve 2 and 3 in 1.
and used 0.5 g. potassium phosphate
Sterilization: Not given.
Use: To study nitrogen assimilation by
instead of 0.2 g. K2HPO4.
Reference: Beijerinck (1901 pp. 568, 578),
chroococcum.
Jacobitz (1903 p. 101), Christensen (1907
Variants: Stoklasa prepared a solution of
p. 110), Percival (1920 p. 179), Beijerinck
25.0 g. of glucose and 1.5 g. K2HPO4 in
He and Van Delden (1902 p. 8), Stoklasa
1000.0 cc. of Moldau river water.
(1908 p. 489).
studied the relation between the assim-
ilation of phosphoric anhydride and 122. van Delden's Basal Lactate Solution
nitrogen by azotobacter. It was deter-
Constituents
mined that 1.0 g. of phosphoric acid
1. Water (ditch) 1000.0 cc.
anhydride and 2 to 2.3 g. elementary N
were assimilated.
2. K2HPO4 0.2 g.
3. Sodium lactate 1.0 g.
Reference: Beijerinck and van Delden
Preparation
(1902 p. 8), Stoklasa (1911 p. 461).
(1) Dissolve 2 and 3 in 1.
120. Beijerinck's Phosphate Sucrose (2) Additional nutrients may be added
Solution as indicated below.
Constituents: Sterilization : Not specified.
Use: Growth of Bacillus radicicola. (b) The author used 0.5 g. K2HPO4 and
Reference: Beijerinck (1901 p. 575). 1.0 g. of calcium lactate.
Added nutrients:
121. Beijerinck's Phosphate Mannitol (a) The author did not specify the use of
Solution ditch water, and added (NH4)2S04
Constituents andO.Og. MgS04-7H20.
1. Water 1000.0 cc. (b) The author did not specify the use of
2. Mannitol 20.0 g. ditch water and added 0.2 g. glycocoU
3. K2HPO4 0.2 g. andO.Og. MgS04-7H20.
Preparation (c) The author used 0.5 g. sodium lactate
(1) Dissolve 2 and 3 in 1. with 0.5 g. K2HPO4 and added 1.0 g.
(2) Reaction is slightly alkaline. asparagin and 0.0, 0.3, 0.6, 0.9, or
(3) Inoculate with 0.1 to 0.2 g. earth. 1.2 g. gypsum.
Sterilization: Not specified. (d) The author specified the use of tap
Use: Used for the enrichment of Azotobac- water, used 0.5 g. sodium lactate with
ter chroococcum and for nitrogen assim- 0.5 K2HPO4, added NH4CI and 1.2 g.
ilation studies. gypsum.
Variants (e) The author specified the use of tap
(a) Beijerinck specified the use of ditch water, used 0.5 g. of sodium lactate
water. with 0.5 g. K2HPO4 and added 0.5 g.
(b) Jacobitz specified the use of distilled asparagin.
water and studied nitrogen fixation (f) The author specified the use of tap
hy Bacillus ellenhachensis a (Caron). water, used 0.5 g. of sodium lactate
(c) Christensen specified distilled water with 0.5 g. K2HPO4 added 1.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 41
asparagin and 1.0 g. gypsum or 4.0 g. gen organisms was very poor in this
fi.xing
137. von Bronsart's Basal Sucrose Salt of MgS04, 0.3 g. to 20.0 g. for the
Solution basic solution. The author reported
Constituents that 2.0% MgS04 did not inhibit the
1. Water 1000.0 cc. growth of yeast, but 0.03% sufficed
was slight growth using 32.0% sucrose, KNO3, 0.2 and asparagin 2.0 g.
(b)
but no growth using 50.0% sucrose. (c) KNO3, 0.1 g. and asparagin 0.3 g.
Added nutrients: The author employed Reference: Bokorny (1911 p. 183; 1912 pp.
the following nitrogen sources: 121; 148; 1917 p. 364; 1917 p. 47; 1920 p. 24).
asparagin 1 .0%
139. Smith's Sucrose Salt Solution
peptone 0.5%
Constituents
NH4NO3 1.0%
1. Triple distilled water 1000.0 cc.
(NH4)2S04 0.5%
2. Sucrose 5.0 cc.
Reference: von Bronsart (1919 p. 61).
3. KH2PO4 2.0 cc.
4. MgS04-7H20 0.1 cc.
138. Bokorny's Basal Sucrose Salt Solution
5. NaCl 0.5 cc.
Constituents: Preparation
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4 and 5 in 1.
2. Sucrose 100.0 g. (2) All apparatus should be scrupulously
3. MgS04 1.0 g. clean and only chemicals of known
4. KH2PO4 5.0 g. purity be used.
Preparation Sterilization: Not specified.
Added nutrients: The author added 0.25 g. Use: To study fermentation of cellulose
of one of the following to each 50.0 cc. by amylobacter and organisms found in
of solution: the mud of rivers.
Constituents
1. Water 1000.0 cc.
2. KH2PO4 (0.2%) 2.0 g.
3. MgSO4(0.01%) 0.1 g.
4. Glycerol (1.0, 3.0 or 5.0%) 10.0, 30.0 or
50.0 g.
Preparation :
2. MgS04 3.5 g.
ammonium mucate 2.0 g.
FeS04, omitted the MnS04 and used and alfalfa plant nodules and found that
30.0 to 40.0 g. CaCOs per liter. little, if any, nitrogen was assimilated
(d) Bredemann specified 1.0 g. K2HPO4, after 60 days incubation at 28 to 30'^C.
0.02 g. NaCl, 0.1 g. FeS04, and 0.01 g. Reference: Stutzer, Burri and Maul (1896
MnS04 but did not specify distilled p. 669).
water.
155. Stoklasa's Basal Salt Solution
(e) Percival specified 1.0 g. K..HP04,
used 30.0 g. glucose and 0.5 g. MgS04. Constituents
(f) Lantzsch specified 1.0 g. K,HP04, 1. Water 2300.0 cc
0.01 g. NaCl, 0.01 g. FeS04 and omit- 2. Sodium phosphate 0.5 g
ted the MnS04. He did not specify 3. K.SO4 0.2 g.
distilled water. 4. MgClo 0.05 g.
(g) Lantzsch specified the use of 0.01 g. 5. Glucose 2.5 g.
NaCl, 0.2 g. FeS04 used 50.0 g. 6. CaCOs 10.0 g.
CaCOa, and omitted the MnS04. Preparation
This solution was employed as a basic (1) Dissolve 0.5 g. sodium phosphate,
solution and added one of the follow- 0.2 g. potassium sulphate and 0.05 g.
ing to each 100.0 cc. of solution: magnesium chloride in 500.0 cc. of
(a) 2.0, 5.0 or 10.0 cc. of a 2.0% NaOH water.
(soil extract). (2) Dissolve 2.5 g. glucose in 500.0 cc.
(b) 0.28 g. or 0.56 g. animal charcoal. water.
(c) 0.4 g., 1.0 g. or 3.0 g. of Bolus alba (3) Mix 200.0 cc. (1) with 100.0 cc. (2).
(pure kaolin). (4) Dissolve 6 and one of the added
(d) 0.25% or 1.0% gelatin. nutrients in (3).
This medium was used for the cultivation (5) Dilute to 20C0.0 cc.
amylobacter spores.
of Sterilization: Method not given.
References: Winogradsky (1902 p. 49), von Use: To study utilization of nitrogen by
Freudenreich (1903 p. 516), Jacobitz B icillus megatherium, {Bacillus Ellen-
(1903 p. 100), Smith (1905 p. 199), Brede- The author reported
bichii) or "Alinit."
mann (1909 p. 496), Percival (1920 p. 174), that more nitrogen was found to be in
Lantzsch (1921 p. 2), Lantzsch (1921 p. 5). solution after incubation.These organ-
isms decomposed nitrogenous material to
154. Stutzer, Burri and Maul's Glucose a form that may be absorbed by the root
Salt Solution hairs.
3. Potassium phosphate 10 g.
156. Charpentier's Glucose Salt Solution
4. MgS04 0.5 g.
5. NaCl 1.0 g. Constituents:
6. CaCU 0.1 g. 1. Water 1000.0 cc.
7. Dextrose 20.0 g. 2. K2HPO4 0.25 g.
Preparation 3. KH2PO4 0.25 g.
(1) Dissolve 3, 4, 5, 6 and 7 in 1. 4. MgS04 0.37 g.
(2) Heat long fibre asbestos to glowin?. 5. Iron phosphate trace
(3) To a 2 liter flask add 10.0 g. (2) 25.0 6. CaS04 trace
water.
cc. (1), 25.0 cc. sterile 7. NaCl 0.2 g.
(4) After sterilization add a large plati- 8. Glucose
num loop of a gelatin streak culture Preparation:
of an organism to 100.0 cc. sterile (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
water. Add 2.0 cc. of (5) to (4). (2) Distribute in flasks.
Sterilization: Method not given. (3) Add 0.075 g. glucose to each flask.
Use: To determine nitrogen fixation. Amount of medium in the flask not
Authors used the bacteria from mustard specified.
52 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(2) Add one of the added nutrients to (1).
Sterilization: Not specified.
Use: To study growth requirements of
yeasts. Author reported that yeast grew
more luxuriantly in a medium containing
peptone than asparagin. Growth best,
however, in sugar beer wort medium.
Added nutrients: The author added 5.0 g.
of asparagin or 5.0 g. of peptone.
Reference: Chrzaszcz (1904 p. 148).
Constituents
1. Distilled water 1000.0
2. K2HPO4
3. MgS04-7H20
4. NaCl
5. CaCl2
6. Sucrose (0.2%)
Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(3) When using KNO3 distribute by 171. Fred and Loomis' Mannitol Solution
filling 200.0 cc. flasks. When using
Constituents:
other nitrogen sources distribute in
1. Distilled water 1000.0 cc.
thin layers in flasks.
2. Mannitol 10.0 g.
(4) Inoculate with canal water contain- 3. MgS04 0.2 g.
ing some mold or decay (moder).
4. KH2PO4 0.2 g.
Sterilization: Not specified. 5. NaCl 0.2 g.
Use: To study the decomposition of cellu- 6. CaS04 0.1 g.
lose by denitrifying organisms. The Preparation
author reported denitrification and de- (1) Dissolve 2, 3, 5 and 6 in 1.
composition of cellulose with the pro- (2) Dissolve phosphate separately in a
duction of N2 and CO2. little water and add to (1).
Added nutrients: The author used the (3) Make (2) neutral to phenolphthalein
following nitrogen sources: with N/1 NaOH.
KNO3 2.5 g. (4) After sterilization add NaOH and
NH4CI 1.0 g. H2SO4 to obtain pH value from 2.7
KNO2 amount not given to 11.1.
MgNH4P04 amount not given Sterilization: Method not specified.
peptone amount not given Use: To show the effect of hydrogen ion
Variants The author sprinkled MgNH4P04
: concentration on growth of Rhizobium.
between two Swedish filter papers and Maximum growth at pH 7.2. None in
placed them in a flask. Then he poured pH 2.77 and 3.1. Yamagata and Itano
a solution of 0.5 g. K2HPO4 in a liter of found the optimum pH for azotobacter
water over the paper. He found that to be between 6.6 and 7.7 depending on
Bacillus ferr ugineus from the soil decom- the type of azotobacter.
posed the cellulose. Variants: Yamagata and Itano used 20.0 g.
Reference: van Iterson (1903-4 p. 690). of mannitol instead of 10.0 g.
Reference: Fred and Loomis (1917 p. 629),
170. Jones' Modified Ashby's Mannitol Yamagata and Itano (1923 p. 522).
Solution
172. Krzemieniewska's Mannitol Salt
Constituents: Solution (Vogel)
1. Distilled water 1000.0 cc. Constituents:
2. Mannitol 20.0 g.
1. Water 1000.0 cc.
3. IV2HPO4 0.2 g.
2. K2HPO4 2.5 g.
4. MgS04 0.2 g.
3. MgS04 2.5 g.
5. NaCl 0.2 g.
4. CaS04 2.5 g.
6. CaS04 0.1 g.
5. Mannitol 20.0 g.
7. CaCh 5.0 g
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 Sterilization: Not specified.
in 1.
Use: Cultivation of Azotobacter.
Sterilization: Not specified. Variants: The author used 2.5 g. CaHPOi
Use: Cultivation of Azotobacter. instead of 2.5 g. CaS04, or omitted the
Variants K2HPO4 and added 2.5 g. CaHPO* instead
(a) Bonazzi specified the use of tap of CaS04.
water, used 0.408 g. MgS04-7H20; Reference: Vogel (1911-12 p. 416).
0.127 g. CaS04; 0.2 g. CaCOs and
specified that Ca(N03)2 might be 173. Harvey's Mannitol Salt Solution
added if desired (amount not given). Constituents:
(b) Giltner used 15.0 g. of mannitol and 1. Distilled water 1000.0 cc.
added 1 drop of 10.0% Fe2Cl6 solution. 2. Mannitol 15.0 g.
Reference: Jones (1913 p. 14), Bonazzi 3. MgS04 0.2 g.
(1921 p. 357), Giltner (1921 p. 376). 4. KH2PO4 0.2 g.
56 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents
176. Koser and Rettger's Basal Glycerol
1. Water (surface) 1000.0 cc.
Salt Solution
2. Mannitol 15.0 g.
phthalein. Preparation
(1) Dissolve 2, 3, 4, 5, 6 and 7 in
1.
(3) Distribute in 100.0 cc. lots in 500.0 cc.
Erlenmeyer flasks. (2) Add one of the added nutrients to (1).
glaucum. Preparation
(1) Dissolve 2, 3, 4 and 5 in ditch water
Reference: Puriewitsch (1895 p. 342 from
an abstract by Heinze 1903 p. 27). ("Grabenwasser") containing 45 mg.
SO3 per liter.
178. Omeliansky's Cellulose Solution (2) Distribute into flasks or beaker.
Constituents: Cover the beaker with a glass plate
1. Water or the flask full to reduce the sur-
fill
extract instead of water in variant Added nutrients: The author added 0.5 g.
(a). Nitrogen assimilation was bet- NH4CI or 0.5 g. peptone.
ter in plain solution however, Reference: Butcher (1924 p. 295).
(c) Hanzawa suggested the addition of
humus or saltpeter in varying SUBGROUP I-C. SECTION 2
amounts to variant (a).
Liquid media or basal solutions with con-
References: Wojtkiewicz (1914 p. 255),
stituents of known chemical composition;
Hanzawa (1914 p. 574).
nitrogen supplied as ammonium salts,
188. Krainsky's Mannitol Salt Solution carbon organic.
Ai.* Media primarily for growth of yeast,
Constituents:
fungi, or algae; not bacteria.
1. Water 1000.0 cc.
KH2PO4 Bi. Ammonia supplied as ammonium
2. 0.5 g.
chloride.
3. NaCl 0.5 g.
Wilder's Sugar Salt Solution
4. CaCOa 0.5 g.
(Chrzaszcz) 19I
5. FeSOj trace
6. Mannite 10.0 g. to 20.0 g.
LaGarde's Sucrose Ammonium Chlo-
ride Solution 192
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
Fulmer and Nelson's Sucrose Am-
in 1.
Ci. Only one type of organic carbon added. Bs. Ammonia present as ammonium salts
Di. Organic carbon supplied as carbo- of organic acids.
hydrate. Ci. No other types of organic carbon added.
El. Monosaccharides added. Bokorny's Ammonium Tartrate Solu-
Charpentier's Glucose ammonium tion 222
Sulphate Solution 204 C2. Other types of organic carbon added.
Artari's Cane Syrup Ammonium Sul- Pasteur's Sucrose Ammonium Tar-
phate Solution (Owen) 205 trate Solution (Pringsheim) 224
Ei. Disaccharides added. Bokorny's Sucrose Ammonium Tar-
Bokorny's Sucrose Ammonium Sul- trate Solution 225
phate Solution (acid) 206 Pringsheim's Sucrose Ammonium
Mortensen's Sucrose Ammonium Sul- Tartrate Solution 226
phate Solution 207 A2. Media Primarily for growth of bacteria.
Woltje's Sucrose Ammonium Sulphate Bi. Ammonia supplied as ammonium hy-
Solution (Zikes) 208 droxide.
D2. Organic carbon supplied as alcohols. Hulton-Frankel, Barber and Pyle's
Bokorny's Ethyl Alcohol Ammonimn Acetic Acid and Ammonia Solu-
Sulphate Solution 209 tion 227
Bokorny's Glycerol Ammonium Sul- B2. Ammonia supplied as ammonium chlo-
phate Solution 210 ride.
Dj. Organic carbon supplied only as acids Ci. Only one type of organic carbon
or their salts. supplied.
Behrens' Basal Malic Acid Ammo- Di.* Organic carbon supplied as carbo-
nium Phosphate Solution 211 hydrate.
C2. More than one type of organic carbon Wherry's Levulose Ammonium Chlo-
added. ride Solution 228
Di. Containing carbohydrates and organic Schardinger's Sucrose Ammonium
acids. Chloride Solution 229
Dombrowski's Glucose Ammonium Klecki's Lactose Ammonium Phos-
Sulphate Solution 212 phate Solution 230
Bokorny's Tartrate Ammonium Sul- Krainsky's Starch Ammonium Chlo-
phate Solution 213 ride Solution 231
Raulin's Solution (Lode) 214 Vierling's Cellulose Ammonium Chlo-
D2. Containing alcohols and organic acids. ride Solution 232
Proskauer and Beck's Basal Citrate D2. Organic carbon supplied as alcohols.
Ammonium Sulphate Solution. Proskauer and Beck's Basal Glycerol
(Mendel) 215 Ammonium Chloride Solution 233
B4. Ammonia supplied as ammonium salts Higgins's Glycerol Ammonium Chlo-
of phosphoric acid. ride Solution 234
Ci. Organic carbon present as carbohy- Revis' Glycerol Ammonium Chloride
drates (other types of organic carbon may Solution 235
also be present). Proskauer and Beck's Mannitol Am-
Pasteur's Glucose Ammonium Phos- monium Chloride Solution 236
phate Solution (Simanowsky) 216 D3. Organic carbon supplied as salts of
Omeliansky's Glucose Ammonium organic acids,
Phosphate Solution 217 van Delden's Lactate Ammonium
Henneberg's Sucrose Ammonimn Chloride Solution 237
Acid Phosphate Solution 218 Sohngen's Basal Ammonium Chloride
Kossowicz's Sucrose Ammonium Salt Solution 238
Phosphate Solution (Will) 219 Wherry's Ammonium Acetate Solu-
Schukow's Glucose Ammonium Phos- tion 239
phate Solution 220 D4. Organic carbon supplied as hydro-
C^. Organic carbon supplied as fat. carbons.
Rahn's Fat-Ammonium Phosphate
Solution 221 *See D2, D3 and D4.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 61
Proskauer and Beck's Basal Glycerol 193. Fulmer and Nelson's Sucrose Ammo-
Ammonium Tartrate Solution 319 nium Chloride Solutions
Uschinsky's Glycerol Ammonium Lac-
Constituents
tate Solution 320
1. Water 1000.0 cc.
Fermi and Montesano's Glycerol Am- NH4CI
2. 1.88 g.
monium Tartrate Solution 321
3. Sucrose 100.0 g.
D2. Containing more than one additional
4. K2HPO4 1.0 g.
carbon source.
Preparation
Gottheil's Ammonium Tartrate Solu-
(1) Dissolve 2, 3 and 4 in 1.
No. VI
tion. 322
(2) Adjustment of reaction not given.
MacKensie's Ammonium Tartrate
Sterilization: Method not specified.
Solution (Smith) 323
Use: Continuous cultivation of Saccharo-
myces cerevisiae. Authors found that
191. Wilder's Sugar Salt Solution these solutions gave continuous growth.
(Chrzaszcz) Variants
(a) Authors added 1.0 g. CaCl2 to above
Constituents:
solution.
1. Water 1000.0 cc.
(b) Authors added 1.0 g. CaClo and 6.0 g.
2. Sugar 100.0 g.
dextrin to the above solution.
3. MgS04 2.5 g.
Reference: Fulmer and Nelson (1923 p.
4. KCl 2.5 g.
132).
5. NH4CI 2.5 g.
6. Na2HP04 2.5 g.
Mayer's Sucrose Ammonium Nitrate
194.
7. CaCOa 0.5 g.
Solution (Pringsheim)
Preparation
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. Constituents
(2) A white precipitate of Ca3P04 forms 1. Water 1000.0 cc.
when CaCO'; is added. This may or 2. Sugar 100.0 g.
may not be filtered out. 3. KH2PO4 5.0 g.
yeasts. The filtered medium showed less Preparation: (1) Dissolve 2, 3, 4, 5 and 6
growth of yeast than the non-filtered in 1.
(2) Distribute in 50.0 cc. lots. (c) Add an equal amount of (1) to cane
syrup.
(3) Prepare a series of flasks by adding
10.0 g. filter paper (Swedish) or 10.0 g.
(d) To 1 part cane syrup add 2 parts
(1).
ordinary filter paper, or 10.0 g. of
pine shavings or 10.0 g. of wadding (3) Tube in about 10.0 cc. lots.
6. MgCOs 0.4 g
217. Omeliansky's Glucose Ammonium
7. (NH4)2S04 0.25 g
Phosphate Solution
8. FeS04 0.07 g
9. ZnS04 0.07 g Constituents
10. Calcium silicate 0.07 g 1. Water 1000.0 cc.
11. Ammonium acetate 4.0 g 2. Glucose 100.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8, 3. Ammonium phosphate 2.0 g.
9, 10 and 11 in 1. 4. KCl 0.1 g.
Sterilization: Not specified. 5. MgS04 0.1 g.
Use: Cultivation of molds, Aspergillus. Preparation
Reference: Lode (1902 p. 125). (1) Dissolve 2, 3, 4 and 5 in 1.
third day add 1.0% of glucose and steri- 5. Sucrose (15.0%) 150.0 g.
Added nutrients: The author used 1.0% of Variants: The author suggested the addi-
the following carbohydrates as carbon tion of one of the following:
sources: Gypsum (2.0%) 20.0 g.
Constituents: Constituents
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Saccharose (5.0%) 50.0 g. 2. K2HPO4 5.0 g.
3. KCl (0.4%) 4.0 g. 3. (NH4)3P04 5.0 g.
4. (NH4)2HP04 (0.4%) 4.0 g. 4. MgS04 1.0 g.
5. MgS04 (0.4%) 4.0 g. 5. CaCl2 1.0 g.
6. CaHP04 (0.04%) 0.4 g. 6. FeCls trace
Preparation: 7. HCl trace
(1) Dissolve 2, 3, 4, 5, and 6 in 1. 8. Fat
Sterilization: Not specified. Preparation
Use: Cultivation of non-spore forming (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Saccharormjces. (2) Melt fat and pour in an Erlenmeyer
Reference: Will (1908 p. 387). flask and
incline the flask in such a
position so that when it solidifies only
220. Schukow's Glucose Ammonium a portion of the fat will be immersed
Phosphate Solution in the solution.
Constituents
(3) Neutralize (1) with 10.0% NaOH
using litmus as an indicator. Do not
1. Water 1000.0 cc.
filter.
2. Ammonium phos-
Add the flasks containing fat,
(4) (3) to
phate 5.0 g.
solidified on the wall of the flask.
3. Potassium phos-
Do not add enough (3) to immerse
phate 10 g.
all the fat.
4. MgS04 0.5 g.
Sterilization: Not specified.
5. Glucose 10.0 g.
Use: To study fat decomposition by
6. Organic acid 90.0 g. to 100.0 g.
Penicillium glaucum. Fat was decom-
Preparation
(1) Dissolve 2, 3, 4 and 5 in 1.
Reference: Rahn (1905 p. 423).
(2) Add 9 to 10.0% of an organic acid to
(1).
222. Bokomy's Ammonium Tartrate
(3) Adjustment of reaction not specified.
Solution
(4) Distribute into fermentation flasks
(sealed with H2SO4). Constituents:
(5) After sterilization and inoculation 1. Distilled water 1000.0 cc.
plug the flasks with a cork and sear 2. Ammonium tartrate . . 2.0 g.
with paraffin. 3. KH2PO4 1.0 g.
Sterilization: Sterilize in the steamer 4. MgS04 0.5 g.
(method not given). 5. Iron chloride trace
Use To study utilization of acids by yeast.
: 6. CaS04 0.0 or 1.0 g.
After 75 days 0.11 g. tartaric, 0.193 g. Preparation: Dissolve 2, 3, 4, 5 and 6 in
malic, 0.34 g. citric and 0.09 g. succinic water that has been distilled in quartz
acid were used. Acid determined by containers.
titration with N/10 NaOH using litmus Sterilization: Not specified.
as an indicator. Use: To study effect of metallic salts on
Variants: The author used one of the fol- j'east. No growth took place in the
lowing organic acids: solution lacking calcium.
tartaric acid Reference: Bokorny (1912 p. 135).
70 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
224. Pasteur's Sucrose Ammonium Solution 227. Hulton-Frankel, Barber and Pyle's
(Pringsheim) Acetic Acid and Ammonia Solution
Constituents Constituents
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. Sucrose 100.0 g. 2. H3PO4 M soln
(12.79 g) 129.5 cc.
3. Ammonium tartrate 10.0 g. 3. CH3COOH (1.13 g) M soln. . 18.8 cc.
4. KH2PO4 5.0 g. 4. NH4OH (0.62 g) M soln 17.8 cc.
5. Ca3(P04)2 1.0 g. 5. 0.01% CaClo (0.001 g) 10.0 cc.
6. Mg3(P04)2 1.0 g. 6. 0.01% FeCU (0.001 g) 10.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 7. 0.01% MgS04 (0.001 g) . . . . 10.0 cc.
in 1. 8. NaOH M soln
(4.0 g) 100.0 cc.
Sterilization: Not specified. 9. KOH (5.6 g) M soln 100.0 cc.
Use: To study bios requirements of yeast. Preparation
Fermentation took place after one day. (1) Dilute solutions 23, and 4 to 500.0 cc.
Reference: Pringsheim (1906 p. 114). with 1.
meta or pyro sodium phosphate for (2) Adjustment of reaction not given.
calcium phosphate. These solutions (3) Distribute in Pasteur fermentation
gave the following results: tubes.
Primary salt growth good at 48 hours Sterilization : Method not given.
part are strongly acid proof, some Use: Cultivation of Bacillus saccharo-
"spores." butyricus. Fermentation takes place after
Secondary salt growth good at 48 hours about 60 hours after inoculation.
part are strongly acid proof, some Reference: Klecki (1896 p. 255).
"spores."
Tertiary salt growth good. Non-acid 231. Krainsky's Starch Ammonium Chloride
proof. Solution.
Meta no growth.
Constituents
Pyrophosphate growth good. Rods
1. Water 1000.0 cc.
and non-acid proof.
are chiefly short
2. Starch (1.0%) 10.0 g.
Some are larger and longer and are
3. NH4CI (0.05%) 0.5 g.
strongly acid proof.
4. K2HPO4 (0.05%) 0.5 g.
(b) Substituted 2.0 g. K2HPO4 for cal-
non-acid
Preparation: (1) Dissolve 2, 3 and 4 in 1.
cium phosphate. Bacilli
Sterilization:Not specified.
proof.
Use: Isolation and enrichment of Actino-
(c) Substituted 2.0 g. KH2PO4 for cal-
myces. After 2 days because of bacterial
cium phosphate. Acid proof rods
increase, a weak coloration and a slight
were often club shaped.
H2S odor appears. The actinomycetes
Reference: Wherry (1913 p. 150).
swim on the surface. Inoculate
colonies
229. Schardinger's Sucrose Ammonium new medium of the
these colonies in a
Chloride Solution same composition and a heavy scum is
Constituents formed.
1. Distilled water 1000.0 cc. Variants: The author substituted 2 to 3.0%
2. Sucrose 50.0 to 80.0 g. 1.0% starch.
cellulose for
3. NH4CI 5.0 g. Reference: Krainsky (1914 p. 657).
4. KH2PO4 1.0 g.
5. MgS04 0.5 g. 232. Vierling's Cellulose Ammonium
6. CaCOs 10.0 to 15.0 g. Chloride Solution
Preparation: (1) Dissolve 2, one of 3, 4, 5
Constituents
and 6 in 1.
1. Water 1000.0 cc.
Sterilization: Not specified.
2. CaC03 (0.1%) 1.0 g.
Use: Study of slime production. Fer-
3. (NH4) CI (0.1%) 1.0 g.
mentation starts after 24 hours. The
4. Filter paper.
slime forms at the bottom of the flask.
Preparation
Variants: The author suggested the use of
(1) Dissolve 2 and 3 in 1.
(NH4)2S04 instead of NH4CI.
(2) Place strips of filter paper in flasks.
Reference: Schardinger (1902 p. 177).
(3) Pour (1) into each flask containing
230. Klecki's Lactose Ammonium Phosphate filter paper.
Solution (4) Seal the flasks with paraffin after
Constituents inoculation.
1. Water 1000.0 cc. Sterilization: Not specified.
2. Lactose 20.0 g. Use: Decomposition of cellulose by Mycch
72 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Add one of the listed carbon sources. 1. Water 1000.0 cc.
Sterilization not specified. 2. Mannitol (0.6%,) 6.0 g.
Reference: Proskauer and Beck (1894 p. (2) Cool and add sugar (Kind and amount
142). not specified).
Sterilization: Not specified.
243. Conn's Glycerol Ammonium Chloride
Use: General culture medium.
Solution.
Variants: Add 3.0% NaCl if the medium is
Preparation Constituents
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 1. Water 1000.0 cc.
(2) Adjust the reaction to about neutral 2. NH4NO3 (0.2%) 2.0 g.
(pH = 7.0) by the addition of NaOH 3. Mannite (0.2%) 2.0 g.
(about 10.0 cc. of normal solution). 4. NaCl (0.02%) 0.2 g.
Sterilization: Not specified. 5. KH2PO4 (0.02%) 0.2 g.
Use: Cultivation of Actinomycetes. 6. MgS04 (0.01%) 0.1 g.
Variants: The author omitted the CaCOs Preparation: (1) Dissolve 2, 3, 4, 5 and 6
and used citric acid instead of malic acid. in 1.
Preparation Preparation
(1) Dilute HCl so that 1.0 cc. is not quite (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
neutralized by 1.0 cc. of silicate solu- (2) Adjustment of reaction not specified.
tion made by dissolving 24.0 g. (3) Distribute into Drechsel's flasks with
KsSiOs and 8.4 g. NasSiOa in 500.0 g. a ground cover.
distilled water. (Phenolphthalein as (4) Add an excess of chalk to each flask.
indicator.) (5) After sterilization connect the flask
(2) Add to the HCl the following salts: with a nitrogen generator and after
MgS04 0.5 g. inoculation replace the air with
CaO 0.01 g. nitrogen.
Fe2(S04)3 0.01 g. an autoclave.
Sterilization: Sterilize in
MnS04 0.01 g. Use: Cultivation of Clostridium Pastori-
(NH4)2S04 1.0 g. anum.
(3) Standardize (2) against the silicate Reference: Winogradsky (1902 p. 49).
solution so that 1.0 cc. silicate equiva-
lent to 1.0 cc. (2) using methyl orange 253. Gage's Glucose Ammonium Sulphate
as indicator. Solution
(4) Standardize a solution of H2SO4 in
Constituents:
same way as HCl omitting the salts.
1. Water 1000.0 cc.
(5) Standardize H3PO4 in similar way as
2. (NH4)2S04 4.0 g.
HCl omitting salt and using phenol-
3. Glucose 1.0 g.
phthalein as indicator.
4. KH2PO4 2.0 g.
(6) Mix the acids in the following ratio:
5. MgS04 1.0 g.
HCl 153.5 cc.
6. Iron sulphate 0.8 g.
H2SO4 77.0 cc.
7. NaCl 4.0 g.
H3PO4 116.0 cc.
8. MgCOa
(7) Mix equal quantities of N/0.6205
Preparation
KOH and N/0.2578 NaOH.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
(8) 1.0 cc. of (7) should neutralize 1.0 cc.
(2) Distribute in 100.0 cc. lots in 150.0 cc.
of (6), using phenolphthalein as
flasks.
indicator.
(3) To each flask add 1.0 cc. of a heavy
(9) Draw acid and base into separate
suspension of MgCOs.
plugged burettes, allow to stand
Sterilization: Not specified.
several hours to sterilize.
Use: To study nitrification by Nitroso-
(10) Add enough sterile glucose solution
hacter and Pseudomonas radicicola.
to equal amounts of (6) and (7) to give
Reference: Gage (1910 p. 33).
concentration of glucose of 10.0 grams
per liter to a mixture of equal amounts
254. Stoklasa's Glucose Ammonium
of (6) and (7).
Sulphate Solution
Use: General synthetic culture medium.
Reference: Doryland (1916 pp. 146-148). Constituents:
1. Distilled water 1000.0 cc.
252. Winogradsky's Glucose Ammonium d-glucose
2. 25.0 g.
Sulphate Solution
3. K2SO4 1.0 g.
Constituents 4. MgCl2 0.5 g.
1. Distilled water 1000.0 cc. 5. Iron sulphate 0.1 g.
2. Dextrose 20.0 g. 6. (NH4)2S04
3. Potassium phosphate 1.0 g. 7. One of the following: 1.0 g.
4. MgS04 0.2 g. Monodiferric-phosphate Fe203(P20s)
5. NaCl ] Monodialuminum-phosphate
'""^^^
6. FeS04 , A1203P205-8H20
^'"*'
7. MnS04 J
Preparation
8. (NH4)2S04 0.01 g. (1) Dissolve 2, 3, 4, 5 and 2.0 g. nitrogen
9. Chalk in the form of (NH4)2S04 in 1.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 77
(2) Add 1.0 g. of one of 7 to (1). Reference: Lipman, Waksman and Joffe
(3) Adjustment of reaction not given. (1921 p. 475) taken from (1922 p. 108).
Sterilization: In the autoclave.
257. Bokorny's Sucrose Ammonium Sul-
Use: To study the cycle of the phosphate
phate Solution (Basic)
ion in the soil using Bac. proteus and
vulgaris, Bac. mesentericus vulgatus. Constituents:
PO4 ions were probably formed. 1. Distilled water 1000.0 cc.
Reference: Stoklasa (1911 p. 479). 2. Sucrose 100.0 g.
3. K,HP04 5.0 g.
255. Percival's Glucose Ammonium Sul-
4. MgS04 10 g.
phate Solution
5. (XH4)2S04 5.0 g.
Constituents: Dissolve 4 and 5 in
Preparation: (1) 2, 3, 1.
1. Distilled water 1000.0 cc.
Sterilization: Not specified.
2. (NH4)2S04 2.0 g.
Use: To study yeast growth.
3. K2HPO4 1-0 g.
Variants Zikes used the modification given
:
4. NaCl 2.0 g.
below to study volutin formation by wine
5. MgS04 0.5 g.
yeast, Saccharomyces Frohberg, Sac-
6. FeS04 0.4 g.
charomyces anamensis, Saccharomyces
7. MgCOs 10.0 g.
ilicis,Saccharomyces, Will (Bajonus)
8. De.xtrose 0.01, 0.03, 1.0 or (3.0%) 0.1,
Pichia membranaefaciens, Cholara myco-
0.3, 3.0, 10.0 or 30.0 g.
derma, Monilia Candida, Oidium laclis,
Preparation
Endomyces Magnusii.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
1. Distilled water 1000.0 cc.
(2) Distribute in 50.0 cc. into conical 100.0 g.
2. Sucrose
flasks.
3. K2HPO4 10 g.
(3) Add 0.5 g. of basic MgCOs to each
4. MgS04 0.3 g.
flask.
5. (NH4)2S04 2.5 g.
To each flask add 0.01, 0.03, 0.3, 1.0
CH =
(4)
(pH = 5.3718, 4.25 X lO--^).
or 3.0% glucose.
Reference: Bokorny (1911 p. 183), Zikes
Sterilization: Not specified.
(1922 p. 29).
Use: To study nitrification by bacteria
from the soil. 258. Wellman's Starch Ammonium Sulphate
Reference: Percival (1920 p. 144). Solution
(2) Adjust to pH between 6.0 and 6.2. Reference: Wellman (1912 p. 616).
7. FeCla (dilute solution) ... 20 drops (2) Fill tubes with (1).
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and (3) Add about 0.1 g. of filter paper,
7 in 1. cotton wool or straw to each tube.
Sterilization: Not specified. Sterilization: Not specified.
Use: Cultivation of Aspergillus and other Use: Cultivation of cellulose decomposers.
molds. Author inoculated with rotten dung.
Variants Variants: Cunningham tubed the solution
(a) The author used 2.5 g. glycogen in 15.0 to 20.0 cc. quantities and added a
instead of 10.0 g. small quantity of finely cut filter paper to
(b) The author used 1.3 g. (NH4)2S04 each tube. Fermentation tubes with a
and 2.5 g. of glycogen instead of glass rod inside to hold them above the
0.1 g. (NH4)2S04 and 10.0 g. of glyco- CaCOs were added. The medium was
gen. used to grow anaerobes.
(c) The author used 4.0 g. MgS04, 2.5 g. Reference: Lohnis (1913 p. 93), Cunning-
glycogen and omitted the FeCls ham (1924 p. 142).
solution.
263. Lohnis' Pectin Ammonium Sulphate
Reference: Heinze (1904 p. 182).
Solution
261. McBeth's Cellulose Ammonium Sul-
Constituents
phate Solution (Sanborn) Water (tap) 1000.0 cc.
1.
(6) Dissolve the pectin by adding dilute (2) Add one of the carbohydrates listed
ammonia and precipitate it from to (1).
solution by the addition of CaCU. Sterilization: Not specified.
(7) Thoroly wash the pectin precipitate Use: To study the constituents essential
with distilled water. for the growth of the tubercle bacillus.
(8) Add 0.1 g. of (7) to a series of flasks. Added nutrients:
(9) Dissolve 2, 3 and 4 in 1. (a) The authors added one of the follow-
(10) Add 2.0 cc. of (9) to each flask of (8). ing carbohydrates.
Sterilization: Not specified. no added carbon
Use: Enrichment of B. amylobader raffinose 1-0%
Reference: Lohnis (1913 p. 92). sucrose .0%1
maltose 1-0%
264. Beijerinck's Glucose Starch Ammo- lactose 1.0%
nium Sulphate Solution
glucose 1.0%
Constituents: manno.se 1-0%
1. Water 1000.0 cc. levulose 1.0%
2. Glucose 30.0 g. dulcitol 0.6%
3. Starch 1.0 g. isodulcitol
4. KH2PO4 0.5 g. (b) Tiffeneau and Marie added 0.6% man-
5. (NH4)2S04 0.5 g. nitol as a carbon source.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. Reference: Proskauer and Beck (1894
Sterilization: Not specified. pp. 142-146), Tiffeneau and Marie (1912
Use: Cultivation of Streptothrix chromo- p. 48).
gena. The author found that the medium
supported growth very well.
267. Doryland's Acetic Acid Ammonium
Sulphate Solution
Reference: Beijerinck (1900 p. 11).
Constituents:
265. Proskauer and Beck's Mannitol Glyc-
1. Water 500.0 cc.
erol Ammonium Sulphate Solution 2. NaOH, N/0.2578
Constituents 3. KOH, N/0.6205
1. Water 1000.0 cc. 4. HCl
2. Mannitol (0.6%) 6.0 g. 5. MgSOi 0.5 g
3. Glycerol (1.5%) 1.5 g. 6. CaO 0.01 g
4. KH2PO4 (0.5%) 5.0 g. 7. Fe2(S04)3 0.01 g
5. MgS04 (0.25%) 2.5 g. 8. MnS04 0.01 g
6. (NH4)2S04 (0.2%) 2.0 g. 9. (NH4)2S04 1.0 g
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 10. H2SO4
in 1. 11. H3PO4
Sterilization: Not specified. 12. CH3COOH
Use: To determine constituents essential Preparation
for the growth of the tubercle bacillus. (1) Dilute the HCl so that 1.0 cc. is not
Reference: Proskauer and Beck (1894 quite neutralized by 1.0 cc. of a
p. 147). silicate solution made by dissolving
24.0 g. KaSiOs and 8.4 "g. NaoSiOa
266. Proskauer and Beck's Basal Glycerol
in 500.0 g. water. Phenolphthalein
Ammonium Sulphate Solution
is used as an indicator.
Constituents: (2)Add to the HCl the following salts:
1. Water 1000.0 cc. MgS04 0.5 g
2. Glycerol (1.5%) 15.0 g. CaO 0.01 g
3. KH2PO4 (0.5%) 5.0 g. Fe2(S04)3 0.01 g
4. Magnesium citrate (0.25%). 2.5 g. MnS04 0.01 g
5. (NH4)2S04 (0.2%) 2.0 g. (NH4)2S04 1.0 g
Preparation (3) Standardize the resulting HCl solu-
(1) Dissolve 2, 3, 4 and 5 in 1. tions against the silicate, using
80 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
272. Beijerinck's Glucose Ammonium ferent esters with clear bacteria free
Phosphate Solution culture broth were incubated for 24 hours;
the amount of acid produced (measured
Constituents;
in terms of N/50 NaOH), determined
1. Water 1000.0 g.
lipase production.
2. Glucose 20.0 g.
References: Kendall, Day and Walker
3. K2HPO4 0.2 g.
(1914 p. 434), Kendall, Walker and Day
4. (XH4)2HP04 0.2 g.
(1914 p. 455).
Preparation: (1) Dissolve 2, 3 and 4 in 1.
2. Ammonium phosphate 0.08 g. (2) Pour into 250.0 cc. flasks and add 5.0 g.
3. Potassium phosphate 0.04 g. of CaCOs.
4. MgS04 0.04 g. (3) Cut about 3.0 g. of filter or blotting
5. (NH4)2S04 0.02 g. paper into small pieces and add
6. Starch, potato 30.0 g. to (2).
7. CaC03 20.0 g. (4) Inoculate with a piece of fresh horse
Preparation : dung or mud from the bottom of a
(1) Dissolve 2, 3, 4, 5 and 6 in 1. pond.
(2) Add 7 to (1). (5) Fill the flasks completely and insert
Sterilization: Not specified. a cork thru which passes a bent glass
Use: To ascertain fermentation of starch tube, arranged so as to collect any
by Bacillus hutyricus. The author found gas given off.
that gas was produced from the starch Sterilization: Not specified.
after 24 hours and that the culture Use: Cultivation of B. amylobacter, a
reduced Fehling's solution. cellulose splitting organism. Author re-
Reference: Botkin (1872 p. 431). ported the production of gas after 3 or
4 weeks.
280. Waksman and Carey's Ammonium Reference: Percival (1920 p. 231).
Phosphate Cellulose Solution
282. Loew's Formaldehyde Ammonium
Constituents
Phosphate Solution
1. Distilled water 100.0 cc
2. (NH4)2HP04 5.0 g Constituents
3. MgS04 1.0 g 1. Water 1000.0 cc.
4. FeS04 0.02 g 2. KH2PO4 2.0 g.
5. KCl 1-0 g 3. (NH4)2HP04 1.0 g.
6. Cellulose (filter paper) 4.0 g 4. MgS04 0.1 g.
Preparation : 5. CaClj 0.1 g.
(1) Dissolve 2, 3, 4 and 5 in 1. 6. Sodium salt or formaldehyd-
(2) Grind filter paper. I'| sulfurous acid ("Formalde-
(3) Add 1.0 g. ground filter paper to hyschwefligsaurem Na-
flasks containing 25.0 cc. of sterile (1). trom") 5.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 83
Constituents:
1. Water 1000.0
2. Ammonium phosphate
(0.2%)
3. Mannite (0.2%).
4. NaCl (0.02%)...
5. MgS04 (0.01%)
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 85
1. Distilled water 1000.0 cc. instead of succinic acid. The acid is used
2. K2HPO4 0.5 g. for the purpose of keeping the materials
3. MgNH4P04 1.0 g. in solution. Succinic acid does not in-
4. CaS04 0.1 g. hibit growth whereas the other mentioned
5. Methane acids do.
Preparation Reference: Janke (1916 p. 7), (1921 p. 91).
(1) Dissolve 2, 3 and 4 in 1.
(2) Flask and inoculate (garden earth, 298. Wherry's Ammonium Acetate Salt
dung water or ditch water may be Solution
used for inoculation). Constituents:
(3) Fill the flask with a known mixture 1. Redistilled water
of oxygen and methane. 2. NaCl
Sterilization: Not specified.
86 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
colon-typhoid group. No growth of cul- References: Smith (1905 p. 197), Roux and
ture studied under aerobic condition. Rochaix (1911 p. 103), Kolle and Wasser-
Modification: The author added 10.0 g. mann (1912 p. 7), Lohnis (1913 p. 44),
glucose. Tanner (1919 p. 63), Besson (1920 p. 37),
Reference: iMeyers (1920 p. 248). Dopter and Sacqu6pee (1921 p. 121),
303. Naegeli's Ammonium Tartrate Solution Giltner (192i p. 369), Klimmer (1923
p. 172).
(Smith)
Constituents: 305. Nicolle and Zia Bey's Ammonium
1. Water 1000.0 cc. Lactate Solution
2. Ammonium tartrate 10.0 g.
Constituents:
3. K2HPO4 1.0 g.
1. Distilled water 1000.0 cc.
4. MgS04 0.2 g.
2. Ammonium lactate . . . 10.0 g.
5. CaClj 0.1 g.
3. MgS04 2.5 g.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
4. Potassium phosphate . 0.0 or 5.0 g.
Sterilization: Not specified.
Preparation
Variants
(1) Dissolve 2 and 3 in 1.
(a) Harvey used 0.12 g. CaCU instead
(2) 5.0 g. of potassium phosphate may
of 0.1 g.
or may not be added.
(b) Harvey used 10.0 g. K2HPO4 instead Sterilization: Not specified.
of 1.0 g., 10.0 g. MgS04 instead of Use: To study pigment and fluorescence
0.2 g., and 0.5 g. Ca3(P04)2 instead
production by Bacillus pyocyaneiis.
of 0.1 g. CaCla.
Phosphate hastened and increased pig-
(c) Harvey used 5.0 g. KH2PO4, 0.5 g.
ment and fluorescence production.
calcium phosphate instead of CaClz
Reference: Nicolle and Zia Bey (1896
and 5.0 g. MgSOi instead of 0.2 g.
p. 671).
(d) Tanner, Devereux and Higgins used
a similar medium with 0.12 g. CaCl2 306. Braun and Cahn-Bronner's Ammonium
for the cultivation of yeast and yeast-
Lactate Solution
like fungi.
References: Smith (1905 p. 197), Harvey Constituents:
(1921-22 p. 103), Tanner, Devereu.x and 1. Water 1000.0 g.
toxin production by Bacterium enteri- (b) Gessard and Vaudremer specified the
tidis. useof K2HPO4, used 5.0 g. ammonium
References: Braun and Cahn-Bronner (1921 succinate and added 1.25 g. CaClj
pp. 4, 197), Branham (1925 p. 299). for the cultivation of tubercle bacilli.
References: Gessard (1892 p. 809), (1901
307. Lohnis' Ferric Ammonium Citrate p. 818); Gessard and Vaudremer (1922
Solution p. 1012).
(5) Sterilize in the autoclave for 15 to 20 310. Fermi's Basal Ammonium Succinate
minutes under 1.5 atmospheres pres- Solution
sure.
Constituents
References: Lohnis (1913 p. 116), Brussoff
1. Water 1000.0 cc.
(1916 p. 551).
2. Ammonium succinate
(0.5 to 1.0%) 5.0 to 10.0 g.
308. Gassard's Ammonium Succinate
3. KH2PO4 (0.5%) 5.0 g.
Solution
4. MgS04 (0.5%) 5.0 g.
8 and 9 in 1.
2. Potassium phosphate 10 g.
4. K2SO4 1.5 g.
to each 200.0 cc. lot of (3) under
325. Beijerinck and van Delden's Mannitol Reference: Beijerinck and van Delden
Nitrite Solution (1902 p. 41).
Constituents
SUBGROUP 1-C. SECTION 4
1. Water 1000.0 cc.
2. K2HPO1 0.5 g. Liquid media or basal solutions of known
3. NaNOa 0.5 g. chemical composition. Nitrogen supplied
4. Mannitol 20.0 g. as nitrates; carbon organic.
Preparation: (1) Dissolve 2, 3 and 4 in 1. Ai. Incomplete or basal solutions, em-
Sterilization: Not specified. ployed with the addition of other nu-
Use: To study denitrification by B. sub- trients.
tilis, B. mesentericus and Azotobacter Stutzer's Basal Citrate Nitrate Solu-
chroococcum. The authors reported that tion 328
B. subtilis ammonia
did not produce Mortensen's Basal Nitrate Salt Solu-
while the other organisms gave ammonia. tion 329
Reference: Beijerinck and van Delden A2. Complete media.
(1902 p. 41). Bi.* Only one type of organic carbon
supplied.
326. Beijerinck and van Delden's Acetate
Ci.* Organic carbon supplied as carbo-
Nitrite Solution
hydrates.
Constituents: Di. Monosaccharides, only, added.
1. Distilled water 1000.0 cc.
Ei. Containing inorganic salts of mono-
2. KCl 0.2 g.
valent cations only.
3. K2HPO4 0.2 g.
Stutzer's Glucose Nitrate Solution. 330 .
supplied. Constituents:
Ci. Carbon added as carbohydrates and 1. Water 1000.0 cc.
alcohols.
2. KNO3 (0.5%) 5.0 g.
Gottheil's Nitrate Solution No. VII. 358 3. Cobalt salt
C2. Carbon added as carbohydrates and
Preparation
acids.
(1) Dissolve 2 and 10.0% of one of the
Burri and Stutzer's Nitrate Solution. 359
carbon sources listed under added
Kuntze's Nitrate Solution 360
nutrients in 1.
Giltay and Aberson's Nitrate Solu-
(2) Add cobalt salts in varying amounts
tion (Stoklasa and Vitek) 361
from 4 to 1/32%.
Giltay's Nitrate
Sucrose Solution
Sterilization: Not specified.
(Fred) 362
Use: To determine the toxic properties of
Behrens' Cellulose Nitrate Solution. 363
cobalt salts for Aspergillus niger. The
C3. Carbon added as alcohols and organic
author found the toxic properties of
acids.
cobalt were different in different media.
Maassen's Malic Acid Nitrate Solu-
A 0.5% cobalt chloride solution in a
tion (Stoklasa and Vitek) 364
liquid medium was as 1.0%
toxic as
Maassen's Stearate^Nitrate Solution
medium.
cobalt chloride in a gelatin
(Vierling) 365
Added nutrients: The author added 10.0%
of one of the following to the basic
328. Stutzer's Basal Citrate Nitrate
solution:
Solution
glucose
Constituents: sucrose
1. Water 1000.0 cc. glycerol
water. 2. K2HPO4 50 g.
Giintheri, Bad. Hartlebi, Bac. pyo- add some more CaCOs. This removes
cyaneous and Bac. fluorescens. all traces of HXO3.
Variants: von Caron used the following (6) Steam in a weighted porcelain dish,
solution to study denitrification: until a solution is obtained contain-
1. Water 1000.0 cc. ing 1 part Ca(N03)2 to five parts
2. Glucose 1.0 g. water.
3. KNO3 0.1 or 3.0 g. (7) Filter and store.
4. MgS04 2.0 g. (8) Prepare solutions of dextrose and
5. K2HPO4 2.0 g. (NH4)2C03 the ratio of 1:15.
CaCla
6. 0.2 g. (9) Mix 25.0 cc. (4), 1.0 cc. of the dextrose
References : MuUer (1907 p. 472), von Caron solution, 2 drops of the (NH4)2C03
(1912 p. 96). solution and 2 drops of (7).
(10) Dilute the 250.0 cc. with distilled
335. Committee S. A. B. Glucose Nitrate
water. A liter of this solution con-
Solution
tains:
Constituents: potassium phosphate 0.1 g
1. Distilled water 1000.0 cc. MgS04 0.02 g
2. CaCIs 0.5 g. CaCl2 0.01 g
3. Nitrate (kind not given). . . 1.0 g. dextrose 0.8 g
4. Glucose 10.0 g. Ca(N03)2 0.8 g
5. K2HPO4 0.5 g. (11) Distribute in 50.0 cc. flasks that have
Preparation Dissolve 2, 3, 4 and 5 in 1.
: (1) previously been plugged and steri-
Sterilization: Method not given. lized.
Use: To study reduction of nitrates. Test Sterilization: Heat the solution and the
for nitrite with sulphanilic acid and a- steamer for 30 minutes.
flasks in the
naphthylamine and for ammonia produc- Use: Culture medium for soil, water and
tion by Thomas' method. air forms.
Variants: Committee S. A. B. specified the Reference: Heraeus (1896 p. 216).
useof l.Og. KNO3.
337. Migula's Glucose Nitrate Solution
Reference: Committee S. A. B. (1922
p. 525), (1923 p. 27). Constituents:
1. Water 1000.0 cc.
336. Heraeus' Glucose Nitrate Solution
2. KNO3 10.0 g.
Constituents 3. Magnesium phosphate 2.0 g.
1. Distilled water 1000.0 cc. 4. CaCl2 l.Og.
2. Potassium phosphate 0.1 g. 5. K2HPO4 l.Og.
3. MgS04 0.2 g. 6. Glucose 20.0 g.
4. CaCls 0.01 g. Preparation
5. Glucose 0.8 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents Preparation
1. Water 1000.0 g. (1) Dilute HCl so that 1.0 cc. is not quite
best in damp atmosphere. Also grows (3) Standardize (2) against silicate solu-
well on rice, arachis seeds, bread, roots tion so that 1.0 cc. is equivalent to
of Daucus carota, milk, etc. 1.0 cc. using methyl orange as
Reference: Went (1901 p. 546). indicator.
(4) Standardize a solution of H2SO4 in
same way as HCl omitting the salts.
340. Charpentier's Glucose Nitrate Solution
(5) Standardize H3PO4 in similar manner
Constituents as HCl omitting the salts and using
1. Water 1000.0 cc. phenolphthalein as indicator.
2. MgS04 1.0 g. (6)Mix the acids in the following ratio:
3. K2HPO4 2.0 g. HCl 153.5 cc.
4. KNO3 2.0 g. H2SO4 77.0 cc.
5. CaNOa 0.05 g. H,P04 116.0 cc.
6. FeS04 trace (7) Mix equal quantities of N/0.6205
7. Glucose 10.0 g. KOH and N/0.2578 NaOH.
Preparation: (8) 1.0 cc. of (7) should neutralize 1.0
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. cc. of (6) using phenolphthalein as
(2) Heat at 120C. indicator.
(3) Filter. (9) Draw acid and base solution in
Sterilization: Sterilize at 120C. separate burettes and allow to stand
Use: To determine nitrate reduction by several hours to sterilize.
Algae, Cystococcus humicola. The (10) Add enough sterile glucose solution
author reported that the total nitrogen to a mixture of equal parts of (6)
content is raised after 13 days incubation. and (7) to give 10 g. of glucose per
Variant: The author omitted the 2.0 g. liter solution.
KNO3 and added 1.0 g. of Ca(N03)2 in- Sterilization: Method not given.
stead of only 0.05 g. Use: A general synthetic medium.
Reference: Charpentier (1903 p. 327). Reference: Doryland (1916 pp. 146-148).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 97
342. Bokorny's Sucrose Nitrate Solution 1.0 or 2.0 g. of NH4NO3 to 100.0 cc.
of solution.
Constituents:
Reference: Stoklasa and Vitek (1905
1. Water 1000.0 cc.
p. 198).
2. KNO3 (0.4%) 4.0 g.
3. Sucrose (10.0%) 100.0 g. 344. Czapek's Sucrose Nitrate Solution
4. Phosphoric acid (PaOs)
Constituents:
(0.13%) 1.3 g.
1. Distilled water 1000.0 cc.
Preparation: (1) Dissolve 2, 3, 4 in 1.
2. NaNOs 2.0 g.
Sterilization: Not specified.
3. K2HPO4 1.0 g.
Use: Cultivation of molds.
4. KCl 0.5 g.
Variants: Bottger used 0.1% K2HPO4 in-
5. MgS04 0.5 g.
stead of phosphoric acid and used 0.2,
6. FeS04 0.01 g.
0.5 or 1.0% KNO3 to determine the
7. Sucrose 30.0 g.
toxicity of nitrates for yeast. He re-
Preparation
ported that nitrate presence had little
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
effect on loss of weight, measured in
(2) Place in 100.0 cc. portions in flasks.
grams.
Plugg.
Reference: Bokorny (1917 p. 316), Bottger
(3) Adjustment of reaction, not given.
(1921 p. 224).
Sterilization: Sterilize at 15 pounds for 15
minutes.
343. Stoklasa and Vitek's Sucrose Nitrate
Use: Cultivation of soil forms.
Solution
Variants: Tanner specified the use of 0.5 g.
Constituents: MgS04-7H20.
1. Water References: Waksman (1918 p. 479), Tan-
2. K2HPO4 1.25 g. ner (1919 p. 66).
3. K2SO4 0.2 g.
4. CaClj 0.05 g. 345. Gerlach and Vogel's Cellulose Nitrate
MgCl2 0.05 g. Solution
5.
6. NajCOa 0.1 g. Constituents
7. reP04 0.05N 1. Distilled water 1000.0 cc.
8. Saccharose 5.0 g. 2. NaNOs 5.0 g.
9. NaNOs 2.0 g. 3. Potassium phosphate 0.5 g.
Preparation 4. MgS04 0.3 g.
(c) The authors added 2.0 g. sucrose and Reference Gerlach and Vogel (1901
: p. 619).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents
Sterilization: Not specified.
Use: To study action of Azotobacter and
1. Distilled water 1000.0 cc.
Radiobacter on nitrates. Author found
2. Sodium phosphate (neu-
that nitrates were reduced.
tral) 2.5 g.
Reference: Stoklasa (1908 p. 493).
3. Glycerol 40.0 g.
4. NaCl 5.0 g.
5. NH4NO3 3.75 g. 355. Doryland's Acetic Acid Nitrate Solution
Preparation
Constituents:
(1) Dissolve 2, 3 and 4 in 1.
1. Distilled water 500.0 cc.
(2) Place 0.75 g. NH4NO3 in 175.0 cc.
Erlenmeyer flasks.
2. HCl dilute
Fill each flask containing NH4NO3 3. MgS04 0.5 g.
(3)
with
4. CaO 0.01 g.
(1).
5. re2(S04)3 0.01 g.
Sterilization: Sterilize carefully (method
not given).
6. MnS04 0.01 g.
Ci.* Organic nitrogen supplied only as Henneberg's Asparagin Salt Solution 419 .
Gg. Additional carbon supplied as organic Clark and Lubs' Solution 461
acids or their salts. Remy and Sugg's Citrate Asparagin
Hi. Acetic acid or its salts added. Solution (Proskauer and Beck).. 462 . .
Ellrodt's Acetate Asparagin Solu- G3. Alcohols and organic acids added.
tion 441a (Carbohydrates may or may not be
H2. Lactic acid or its salts added. present.)
Ii. Ammonium salts added. Uschinsky's Glycerol Asparaginate
Voges' Lactate Asparaginate Solu- Solution 463
tion 441b Sullivan's Glycerol Nitrate Aspara-
Frankel and Voges' Salt Asparagin gin Solution 464
Solution (Besson) 441c Hadley's Glycerol Asparagin Solu-
Frankel' s Lactate Asparagin Solu- tion No. V 564
tion (Tanner) 442 Lowenstein's Glycerol Asparagin
Uschinsky's Lactate Asparaginate Solution 466
Solution (Smith) 443 Lockemann's Citrate Glycerol As-
Melick's Lactate Asparagin Solution. 444 paragin Solution (Lowenstein) .... 467
van Delden's Lactate Ammonia As- Frouin's Glycerol Asparagin Solu-
paragin Solution 445 tion (Bezancon) 468
I2. Ammonium salts not added. C2. Organic nitrogen present in addition to
Beijerinck's Lactate Asparagin Solu- amino nitrogen.
tion 446 Di. Amino nitrogen present as glycocoll.
van Delden's Lactate Asparagin Solu- Hadley's Urea Glycocoll Solution... 469
tion 447 D2. Amino nitrogen present as aspartic
Schroeder and Junger's Lactate As- acid or asparagin.
paragin Solution 448 Beijerinck's Urea Asparagin Solu-
Remy's Organic Acid Asparagin tion 470
Solution 449 Beijerinck's Urea Ammonia Car-
H3. Citric acid or its salts added. bonate Solution 471
Kuntze's Citrate Asparagin Solution. 450 Sears' Uric Acid Asparagin Solution. 472
Giltay and Aberson's Citrate Aspara- Kappen's Cyanamide Asparagin Solu-
gin Solution (Tanner) 451 tion 473
Maassen's Basal Citrate Asparagin A2. More than one amino acid present.
Solution (Lohnis) 452 Bi. Incomplete or basal solutions employed
H4. Malic acid or
its salts added. with the addition of other materials.
Maassen's Malate Asparagin Solu- Davis and Ferry's Basal Cystine
tion 453 Tryptophane Solution 474
Beijerinck's Malate Asparagin Solu- B2. Complete media.
tion 454 Ci. Containing glycocoll.
F2. More than one type of additional Berthelot's Amino Acid Solution 475
organic carbon employed. Armand-Delille, et al., Glycocoll
Gi. Carbohydrates and alcohols, only, Arginine Solution (Synthetic Me-
added. dium 164) 476
Zikes' Alcohol Asparagin Solution . . 455 Armand-Delille, et al., Synthetic
Gosio's Glycerol Aspartic Acid Solu- Solution 104 477
tion 456 Armand-Delille, et al., Glycocoll As-
Gartner's Glycerol Asparagin Solu- partic Acid Solution (Synthetic
tion 457 Medium 118) 478
Borel de coulon, Boez and Quimaud's C2. Not containing glycocoll.
Glycerol Asparagin Solution 458 Zipfel's Tryptophane Asparagin Solu-
G2. Carbohydrates and organic acids, only, tion 479
added.
366. Went's Basal Glycocoll Solution
Muller, Thurgan and Osterwalder's
Malate Asparagin Solution 459 Constituents:
Maassen's Glucose Asparagin Solu- 1. Water 1000.0 cc.
tion 460 2. Glycocoll (0.66%) 6.6 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 105
Preparation
(1) Prepare 5.0% solutions of one of the
materials listed in added nutrients.
(2) Flask in 25.0 cc. quantities.
(3) Prepare a 0.66% solution of glycocoll.
(4) Add 25.0 cc. of (3) to each flask of (2).
Sterilization: Method not given.
Use: To study nutrients for Monilia sito-
phila (Mont.) Sacc. The materials
ranked as follows as suitable additional
carbon sources: maltose, glucose, lactose,
sucrose and glycerol.
Added nutrients: The author used the
following additional carbon sources:
maltose glucose
lactose glycerol
sucrose
Variants: The author used the basic solu-
tion without additional materials.
Reference: Went (1901 p. 593).
Constituents:
1. Redistilled water 1000.0 cc.
2. NaCl
3. KCl
4. CaClj
5. MgSOi
6. Glycocoll
7. (NH4)2HP04
106 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Flask in 25.0 cc. quantities. Use: To study the nutrients for Monilia
(2)
(3) Prepare a 0.66% tyrosine solution. sitophila (Mont.) Sacc. The materials
ranked as follows as suitable carbon
(4) Add 25.0 cc. of (3) to each flask of (2).
Sterilization: Method not given. sources with leucine as nitrogen source:
Use: To study nutrients for Monilia sito- glucose and maltose, sucrose, glycerol,
lactose.
phila (Mont.) Sacc. The materials
ranked as follows as suitable carbon Added nutrients: The author used the
sources with tyrosine as a nitrogen source following additional carbon sources:
maltose, glucose, sucrose, lactose, glyc- maltose glucose
erol. lactose glycerol
Added nutrients: The author used the sucrose
following additional carbon sources: Variants: The author used the basic solu-
maltose glucose tion without additional materials.
sucrose
Variants: The author used the basic solu- 373. van Delden's Basal Asparagin Salt
tion without additional materials. Solution
Reference: Went (1901 p. 593).
Constituents
371. Waksman's Basal Glycerol Leucine 1. Water, tap 1000.0 cc,
Solution 2. K2HPO4 0.5 g.
Constituents: 3. Asparagin 5.0 g.
1. Water 1000.0 cc. Preparation
2. Glycerol 30.0 g. (1) Dissolve 2 and 3 in 1.
3. K2HPO4 1.0 g. (2) Additional nutrients are added as
4. KCl 0.5 g. indicated below.
5. MgS04 0.5 g. Sterilization: Not specified.
6. reS04 0.01 g. Use: To study sulphate reduction by
7. Leucine 5.0 g. Microspira desidfuricans.
Preparation Added nutrients and variants The author :
(g) Added 10.0 g. glucose, 30.0 g. NaCl References: Kendall, Day and Walker
and 8.0 g. MgSOi-THaO to the basic (1914 p. 428), Kendall, Walker and Day
solution. (1914 p. 355).
(h) Used 2.5 g. asparagin in the basic
375. Kita's Basal Asparagin Solution
solution and added 30.0 g. NaCl,
8.0 g. MgS04-7H20 and 5.0 g. sodium Constituents:
lactate. 1. Water 1000.0 cc.
(i) Used 1.0 g. asparagin in the basic 2. MgS04 2.5 g.
solution, added 8.0 g. MgS04-7H20, 3. KH2PO4 5.0 g.
30.0 g. NaCl and 5.0 ^g. of sodium 4. FeCh solution drops
lactate or potassium succinate. 5. Asparagin 5.0 g.
(j) Used 1.0 g. of asparagin in the basic Preparation
solution and added 5.0 g. sodium (1) Dissolve 2, 3, 4 and 5 in 1.
lactate, 75.0 g. NaCl and 8.0 g. (2) Add 5.0% of any carbohydrate.
MgS04-7H20. Sterilization: Not specified.
Reference: van Delden (1903-04 p. 85, 105). Use: Cultivation of Japanese molds, Asper-
gillus Okazaki, Aspergillus candidus,
374. Uschinsky's Basal Asparagin Solution
Aspergillus albus, Aspergillus tamarii,
(Kendall, Day and Walker) Pseudorhizopus, Aspergillus glaucum.
Constituents: Added nutrients: Add 5.0% of any carbo-
1. Distilled water 1000.0 cc. hydrate.
2. Asparagin 4.0 g. Variants
3. NasHPOi 2.0 g. (a) Buromsky used the following solution
4. NaCl 5.0 g. to study effect of organic acids on
Preparation yeast, Saccharomyces ellipsoid, Sac-
(1) Dissolve 2, 3, 4 and one of the ma 4- charomyces pastorianus.
terials listed under added nutrients 1. Water 1000.0 g.
in 1. 2. MgSOi 0.5 g.
(2) Distribute in 100.0 cc. lots. 3. KH2PO4 1.0 g.
(3) Adjustment of reaction not given. 4. Asparagin 5.0 g.
Sterilization: Method not given. The solution was prepared by
Use: To study metabolism by the tubercle (1) Dissolve 2, 3 and 4 in 1.
bacilli. Authors used alizarin, neutral (2) Distribute in 100.0 cc. lots in 500.0
red and phenolphthalein to study changes cc. Erlenmeyer flasks.
in reaction; Ziehl-Neelsen stain for (3) Add one of the listed materials
staining. below as additional carbon source.
Added nutrients: The following organic (4) Sterilize on 3 successive days in a
materials were added: Koch steamer, 30 minutes the first
glucose 10.0 g. day, and 20 minutes on each the
mannitol 10.0 g. 2nd and 3rd day, (a slight turbidity
glycerol 30.0 g. develops when glycerin or mannite
The following materials were added to is added).
study lipase production: different es- The following nutrients were added:
ters were incubated for 24 hours with quinic acid 0.5%
clear bacteria free culture broth, and citric acid 1-0%
amount of acid produced, measured in tartaric acid 1-0%
terms of n/50 NaOH, determined lipase succinic acid 1-0%
production, glycerol 1.0%
ethyl alcohol and (NH4)2HP04 amounts mannitol 1 .0%
Use: To study the constituents essential (c) The authors used 0.1, 0.2, 0.3, 0.4,
0.5 or 0.6% asparagin without addi-
for the growth of tubercle bacilli.
tions.
Added nutrients The authors added one of
:
(e) 1.0 g. MgS04, 1.0 g. K2HPO4 and 380. Gottheil's Basal Asparagin Solution
10.0 g. glucose.
Constituents:
(f) 1.0 g. MgS04, 1.0 g. K2HPO4 and
1. Distilled water 1000.0 cc.
10.0 g. lactose.
2. Potassium phosphate 1-0 g.
(g) 1.0 g. MgS04, 1.0 g. K.HPO^ and
3. CaClj 0.1 g.
10.0 g. sucrose.
Reference: Hefferan (1903-4 p. 522).
4. MgS04 0.3 g.
5. NaCl 0.1 g.
6. Ifon trace
378. Zikes Basal Asparagin Solution
7. Asparagin 10.0 g.
Constituents: Preparation
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
2. Asparagin 2.5 g. (2) Add one of the added nutrients in
3. K2HPO4 1.0 g. the amount indicated.
4. MgS04 0.3 g. Sterilization: Not specified.
Preparation Use: Cultivation of organisms found in the
(1) Dissolve 2, 3 and 4 in 1. soil, on roots and on rhizomes.
. fK,HP04 l.Og.
^''^ 384. Ono's Basal Sucrose Asparagin
\Na2SO4 l.Og.
Solution
, [MgSO^ 0.2 g.
^""^ Constituents
IK2HPO4 l.Og.
1. Water 1000.0 g.
. . fMgS04 0.01 to l.Og.
^*''' 2. Sucrose 85.0 g.
\K2HPO4 l.Og.
3. Asparagin 16.0 g.
fMgS04 1.0 to 16.0 g. 4. MgS04
.J. 3.0 g.
[peptone 10.0 g.
5. KH2PO4 5.0 g.
fMgS04 0.2 g. 6. FeS04 trace
^^^ \K2HPO4 0.01 to 5.0 g. Preparation
[MgS04 0.2 g. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
(h) K2HPO4 l.Og. (2) Distribute in 30.0 cc. lots.
[peptone 5.0 g. Sterilization: Sterilize in the steamer.
Variants: The author used 5.0 g. asparagin Use: To study the effect of small amounts
and added 0.2 g. MgS04, 1.0 g. K2HPO4, of chemicals on the growth of Aspergillus
and 4.0 g. ammonium lactate. niger. The author reported that small
Reference: Sullivan (1905-06 pp. 127-140). amounts of CUSO4 tended to increase
growth while concentrations higher than
383. Miinter's Basal Glucose Asparagin
1/250 molar CUSO4 tended to hinder
Solution
growth.
Constituents: Variants and added nutrients:
1- Water 1000.0 cc. (a) The author added CUSO4 solution so
2. NaCl 0.5 g. that the medium was from 1/32,000 to
3. MgS04 0.5 g. 1/250 molar CUSO4.
4. CaCl2 Olg (b) Henneberg used 0.3% asparagin, 0.2%
5. K2HPO4 1.5 g. MgS04, 5.0% sucrose and omitted the
6. NH4NO3 l.Og. FeS04. To this solution he added
7. Asparagin 0.5 g. the following materials
8. Dextrose 9-0 g. NaCl 0.1%
Preparation CaCl2 0.1%
(1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1. K2SO4 0.1%
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 111
tion 20.0 g. mannitol and 4.0 g. aspara- (c) Added 1.0 g. KXO,.
gin were added. Reaction was dis- (d) Used 20.0 g. glycerol, 3.0 g. asparagin
tinctly acid. Filter the precipitate and added 1.0 g. XaCl.
after the first sterilization. Sterilize (e) Omitted the K2HPO4, used 10 to
for 90minutes all together, 20.0 g. glycerol, 10.0 g. asparagin,
(h) The author used 5.0 g. KH2PO4 and and added 0.2 to 1.0 g. KNO3 and
0.5 g. CaCl2 and added 5.0 g. K2HPO4 0.2 to 1.0 g. XaCl.
to the basal solution. 100.0 cc. of (f) Used 10 to 20.0 g. glycerol, 10.0 g.
this solution was diluted with 900.0 asparagin and added 0.2 to 1.0 g.
cc. of water, and 20.0 g. mannitol and HBr.
4.0 g. asparagin were added. Reac- Used 20.0 g. asparagin, 20.0 g. glycerol
(g)
tion was weakly acid. Filter the and added 0.2 to 1.0 g. KI.
precipitate after the first steriliza- (h) Used 10.0 g. asparagin, 20.0 g.
tion. Sterilize for 90 minutes all glj'cerol and made no additions, or
together, added 0.2 to 1.0 g. KCl, 0.2 to 1.0 g.
(i) The author used KH.PO4, and
5.0 g. K2SO4, or 0.2 to 1.0 g. NaoS04.
0.5 g. CaCl2, MgSOi and
omitted the Reference: Sullivan (1905-06 p. 127-140).
NaCl from the basal solution and
added 5.0 g. K2HPO4 and 3.0 g. 389. Waksman's Basal Glycerol Asparagin
MgS04. To 200.0 cc. of this solution Solution
added 800.0 cc. distilled water, 20.0 g.
Constituents:
mannitol and 4.0 g. asparagin. The
1. Water 1000.0 cc.
reaction is weakly acid. Sterilize
2. Glycerol 30.0 g
for one hour.
Reference: Peklo (1910
3. K2HPO4 1.0 g
p. 470).
4. KCl 0.5 g
388. Sullivan's Basal Glycerol Asparagin 5. MgS04 0.5 g
Solution 6. FeS04 0.01 g
Constituents:
7. Asparagin 5.0 g,
Preparation
1. Water 1000.0 cc.
2. Glycerol (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
10.0 g.
3. Asparagin 2.0 g.
(2) Add 2.0 g. of one of the added nu-
trients.
4. K2HPO4 1.0 g.
Preparation (3) Tube in 10-12 cc. lots.
Dissolve 3 and 4 in
(4) Adjustment of reaction not specified.
(1) 2, 1.
some salts to aid synthesis. For the Reference: Waksman (1920 p. 3).
medium. Constituents:
100.0 g. 1. Water 1000.0 cc.
Thiourea
2. Asparagin 2.0 g.
Taurine, amount not given.
Na2S203-5H20 3.0 g. 3. MgS04 2.0 g.
4. Citric acid 5.0 g.
Na2S03 30.0 g.
Reference: Tanner (1917 p. 586). 5. KH2PO4 2.0 g.
6. CaCl2 0.2 g.
391. Hiss' Basal Litmus Asparagin Solution Preparation
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
Constituents:
Distilled water 1000.0 cc. (2) Add NaOH until the reaction is
1.
slightly alkaline.
2. K2HPO4 2.0 g.
(3) Add 0.2% of one of the carbon sources
3. MgS04 0-4 g-
listed under added nutrients.
4. NaCl 5.0 g.
4.0 g.
Sterilization: Not specified.
5. Asparagin
Use: To study fermentation by members
6. Ammonium lactate 5.0 cc.
The authors
15.0 cc. of the colon-typhoid group.
7. Litmus (5.0% soln.)
reported no growth of Bacterium hjphi.
Preparation :
sucrose mannose
Use: To study fermentation by the dysen-
Reference: Capaldi and Proskauer (1896
tery group. Acid production turns the
p. 454).
medium from purplish blue to red.
Added nutrients: The author added 1.0% 394. Mendel's Basal Citrate Asparagin
of one of the following: Solution
glucose de.xtrin
maltose mannitol Constituents:
1. Distilled water 1000.0 cc.
sucrose
Reference: Hiss (1904 p. 32). 2. KNO3 2.0 g.
3. Asparagin 5.0 g.
392. Harvey's Basal Lactate Asparagin 4. MgS04 2.0 g.
Solution 5. Citric acid 5.0 g.
Mix and
2. MgHP04-7H20 4.93 g.
(4) (3) (1).
Sterilization: Sterilize for 20 minutes on
3. H3PO4 (1/3 molar) 20.0 cc.
4. Citric acid (3 normal) 10.0 cc.
each of two successive days. On the
third day
5. Asparagin 5.29 g.
add 1.0% of one of the added
6.Glycerol 20.0 g.
nutrients and sterilize for 15 minutes.
Preparation
Use: To study decomposition of sugars by
Bacterium coli and \Bacterium Fitzi- (1) Dissolve 2, 3, 4, 5 and 6 in 1.
anus. The author reported the following (2) Add one of the combinations given
reactions:
under added nutrients. The reaction
of the various solutions will also be
Variants: The author used 10.0 g. asparagin
instead of 5.0 g. in the basic solution. given there.
Added nutrients: The author employed Sterilization: Not specified.
Use: Cultivation of tubercle bacilli.
1.0% of one of the following as carbon
sources:
Added nutrients and modifications: The
author added the following materials:
glucose lactose
maltose sucrose
(a) K2HPO4 0.35 g. and 10.0 cc. normal
p. 37).
5. KNO3 0.25 g.
6. CaCl2 0.02 g.
399. Bokorny's Sucrose GlycocoU Solution Tyrosine 2.0 g.
7.
Constituents Constituents:
Water 1000.0 cc.
1. Distilled water 1000.0 cc. 1.
Glucose 20.0 g.
2. Ammonium lactate 7.5 g. 2.
3. Tyrosine 0.2 g.
3. FeP04 0.8 g.
Actlnomycetes. Preparation
(1) Dissolve 2 and 3 in
Reference: Waksman (1920 p. 18).
1.
phenolphthalein Constituents:
(2) Make neutral to
Water 1000.0 cc.
with NaOH. 1.
Preparation
isms found in water.
(1) Dissolve 2 and 3 in
1.
Reference: Dolt (1908 p. 620), Tanner
(2) Inoculate with soil.
(1919 p. 65).
Sterilization: Not specified.
412. Kendall, Walker and Day's Ammo- Use: To study denitrification by soil forms,
nium Asparagin Solution Bacillus pyocyaneus.
415. Blanchetiere's Asparagin Salt Solution 418. Uschinsky's Asparagin Salt Solution
Constituents: (Giltner)
1. Distilled water 900.0 cc. Constituents:
2. NaCl 5.0 g. 1. Distilled water 1000.0 cc.
3. Na2HP04 1.0 g. 2. Asparagin 34g
4. Potassium biphosphate 1-0 g. 3. NaCl 5.0 g.
5. Asparagin 3.0 g. 4. MgS04 0.2 g.
Preparation 5. CaCh 0.1 g.
(1) Dissolve 2, 3, 4 and 5 in enough water 6. KH2PO4 1.0 g.
to make 900.0 cc. 7. FeS04 trace
(2) Distribute in 450.0 cc. lots in liter Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
flasks. in 1.
Sterilization: Sterilize for 15 minutes at Sterilization: Not specified.
110 to 112C. Use: General culture medium.
Use: To study asparagin decomposition by Variants
Bacillus fluorescens liquefaciens (Flugge). (a) Gottheil did not specify the type of
Reference: Blanchetiere (1917 p. 294). phosphate used and used 0.3 g. MgS04
with 10.0 g. asparagin.
416. Park, Williams and Krumwiede's
(b) Linde used 0.01 g. Fe2Cl6 instead
Asparagin Salt Solution
of FeS04, used 0.3 g. MgS04-7H20,
Constituents: 0.1 g. NaCI, and did not specify the
1. Water 1000.0 cc. amount of asparagin added. H used
2. Asparagin 6.0 g. the solution with an alkaline reaction
3. Sodium phosphate (ortho).. 2.0 g. for the cultivation of Cladothrix.
4. NaCl 5.0 g. The addition of glucose, sucrose or
Preparation glycerol would have increased the
(1) Dissolve 2, 3 and 4 in 1. amount of growth.
(2) Ifnecessary make alkaline to litmus References: Giltner (1921 p. 369), Gottheil
by the addition of NaOH. (1901 p. 432), Linde (1913 p. 386).
(3) Tube in small quantities.
(4) Test for color production with. B. 419. Henneberg's Asparagin Salt Solution
pj/ocyaneus Constituents
Sterilization:Method not given. 1. Distilled water 1000.0 cc.
Use Culture medium for disinfection tests.
: 2. Asparagin (0.075 to
Reference: Park, Williams and Krum- 0.9%) 0.75 to 9.0 g.
wiede (1924 p. 122). 3. K2HPO4 (0.05 to
1-5%) 0.5 to 15.0 g.
417. Bokorny's Asparagin Salt Solution
4. MgS04 (0.02 to
Constituents 0.6%) 0.2 to 6.0 g.
1- Water 1000.0 cc. Preparation: (1) Dissolve 2, 3 and 4 in 1.
2. Asparagin (0.5%) 5.0 g. Sterilization: Not specified.
3. KH2PO4 (0.1%) 1.0 g. Use: Cultivation of yeast.
4. MgS04 (0.03%) 0.3 g. Variants
5. CaCIo trace (a) Bokorny used 25.0 g. asparagin, 5.0 g.
6. K2SO4 (0.1%) 1.0 g. KH2PO4 and 1.0 g. MgS04.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 (b) Evans used 5.0 g. asparagin, 1.0 g.
in 1.
MgS04 and 2.0 g. K2HPO4.
Sterilization: Not specified. (c) Heinemann and Tanner used 2.0 g.
Use: To study effect of metallic salts on asparagin, 1.0 g. MgS04 and 1.0 g.
yeast growth. The author reported that K2HPO4.
4.0% of K2SO4 did not inhibit the develop- (d) Tanner used 10.0 g. asparagin, 2.0 g.
ment of the yeasts. MgS04 and 1.0 g. KH2PO4 for pig-
Variants: The author used 0.5, 1.0, 2 or ment production by Ps. pyoctjaneus
4.0% K2SO4. and Ps. fluorescens liquefaciens.
Reference: Bokorny (1912 p. 122), (e) Boehncke used 2.0% asparagin, 0.5%
120 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Preparation: Dissolve 5, 6, 7
NaCl and 0.05% MgS04 to determine (1) 2, 3, 4,
421. Saltet's Glucose Asparagin Solution before sterilization and neutral after
sterilization.
Constituents Sterilization: Autoclave at 112 for 40
1. Distilled water 1000.0 cc.
minutes.
NaCl 12.0 g.
2.
Use: To study asparagin decomposition by
3. KCl 10 g.
Bacillus fluorescens liquefaciens (Flugge).
Na2HP04 0.40mg.
4.
Good growth when NH4CI is added but
5. Glucose 0.5 g.
produced.
no pigment is
6. Asparagin 1-0 g-
Variants: The author specified that 5.0 g.
7. Na2S04 varying amounts
NH4CI might be added.
Preparation:
Reference: Blanchetiere (1917 p. 301).
(1) Dissolve 2, 3, 4, 5 and 6
in 1.
8. Asparagin 1-0 g. in 1.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 (5) Pour the cool alcohol from the sugar
in 1.
and wash the sugar with absolute
Sterilization: Not specified. alcohol and then with ether.
CULTUEE MEDIA FOR CULTIVATION OF MICROORGANISMS 123
(6) Dry at 60C. until all traces of damp- Use: Cultivation of chromogenic bacteria.
ness of alcohol or ether have dis- The author reported that the chromogenic
appeared. bacteria developed rapidly with good
(7) Prepare a 10.0% solution of (6) in color production on this medium.
distilled water. Variants: (1) Bezangon used 2.5 g. MgS04,
(8) Mix about 10.0% of (7) with 10.0% 1.5 g. potassium phosphate, 4.0 g. aspara-
of (1). gin and 15.0 g. glycerol. This solution
(9) Distribute in 150.0 cc. lots in fer- was used for the cultivation of tubercle
mentation flasks. bacilli.
Sterilization: Sterilize in streaming steam. Reference: Sullivan (1905-06 p. 116),
Use: To study fermentation by yeast. Bezangon (1920 p. 547).
Reference: Korff (1898 p. 532).
437. Schweinitz and Dorset's Glycerol
434. Zikes' Magnesium Sucrose Asparagin Asparagin Solution (Goris)
Solution
Constituents
Constituents 1. Water 1000.0 cc.
1. Water 1000.0 cc. 2. Glycerol 70.0 g.
2. Asparagin 10.0 g. 3. KH2PO4 1.0 g.
3. K2SO4 5.0 g. 4. Ammonium phosphate 10.0 g.
4. MgS04 2.5 g. 5. NaCl 10.0 g.
5. Saccharose 75.0 g. 6. Asparagin 2.0 g.
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 7. MgS04 2.0 g.
Sterilization: Not specified. Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
Use: To study perithecium formation by in 1.
Use: To study growth requirements for (2) Adjust medium to different pH from
yeasts. Author reported that yeast de- 4.4 to 8.7 with phosphates and
veloped very well in this medium. carbonates.
Reference: Chrzaszcz (1904 p. 149). (3) Tube in 10-12 cc. lots.
Sterilization: Sterilize at 15 pounds for
436. Sullivan's Glycerol Asparagin
15 minutes.
Solution
Use: To study effect of reaction (pH) on
Constituents: metabolism of actinomycetes.
1. Water 1000.0 cc. Reference: Waksman and Joffe (1923 p. 44).
2. Asparagin 10.0 g.
439. Hadley's Glycerol Asparagin Solution
3. K2HPO4 1.0 g.
Nos. Ill and IV
4. MgS04 0.2 g.
5. Glycerol 20.0 g. Constituents
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Ammonium phosphate 10.0 g.
124 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
3. Asparagin 10.0 g.
4. CaCl2 0.1 g.
5. Glycerin 40.0 g.
6. KNO3 0.3 g.
7. K2HPO4 0.3 g.
8. NaCl 6.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
and 8 in 1.
Constituents
1. Distilled water 1000.0 cc.
2. KH2PO4 15.0 g.
3. CaCU 10 g.
4. MgS04 3.0 g.
5. NaCl 1.0 g.
6. CaCh 1-0 g.
7. FesCle trace
He diluted 200.0 cc. of this solution
Constituents
1. Distilled water 200.0 cc.
2. NaCl 8.0 g.
3. K2HPO4 2.0 g.
4. Ammonium lactate 6.0 g.
5. Sodium asparaginate 4.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
in 1.
References: van Delden (1903-04 p. S3), 450. Kuntze's Citrate Asparagin Solution
Tanner (1919 p. 64), Giltner (1921 p. 377).
Constituents
1. Distilled water 1000.0 cc.
448. Schroeder and Junger's Lactate
Asparagin Solution
2. KNO3 2.0 g.
3. Asparagin 1-0 g.
Constituents 4. MgS04 2.0 g.
1. Water 1000.0 cc. 5. Citric acid 5.0 g.
2. Asparagin 4.0 g. 6. KH2PO4 2.0 g.
3. Sodium lactate 6.0 g. 7. CaCl2 0.2 g.
4. Sodium phosphate (Ortho) 2.0 g. 8. Iron chloride several drops
5. NaCl 5.0 g. Preparation
Preparation (1) Dissolve 2 and 3 in part of 1.
(1) Powder 2, 3, and 5 separately.
4
(2) Dissolve 4, 5, 6, 7 and 8 in the re-
(2) Dissolve the powdered salts sepa- mainder of 1.
rately in 50 to 100.0 cc. of water.
(3) Neutralize (2) with KOH during the
(3) Mix the solutions and add the re- heating.
mainder of the liter of water.
(4) Mix (3) and (1).
(4) Add NaOH to make slightly alkaline
(5) Distribute into 100.0 cc. lots in com-
to litmus. bustion flasks.
(5) Filter. Sterilization: Method not given.
(6) Tube. use: To study morphology and physiology
Sterilization: Sterilize on 2 consecutive of denitrifying organisms Bacillus deni-
days in the Arnold sterilizer. trificans agilis (Ampola and Garino) and
Use: Cultivation of Bacillus pyogenes and Bacillus oxalaticus (Zopf).
chromogenes. The author reported that Variants
a green band of pigment was produced (a) Wojtkiewicz used 3.0 g. KNO3, 7.6
after 24 hours. The band gradually in- potassium citrate instead of citric
creased until the whole tube was light acid, used 0.2 g. CaCla and specified
green. Non chromogenic organisms were a trace of FeClo.
inhibited. (b) Arnd and Harvey specified a trace of
Variants: Harvey used 2.0 g. of Na2HP04. FeCl2.
References: Schroeder and Junger (1912 References: Kuntze (1904 p. 3), Smith
p. 601), Harvey (1921-22 p. 102). (1905 p. 198), Wojtkiewicz (1914 p. 258),
Arnd (1916 p. 567), Harvey (1921-22
449. Remy's Organic Acid Asparagin p. 102).
Solution
and Aberson's Citrate
451. Giltay
Constituents:
Asparagin Solution (Tanner)
1. Distilled water 1000.0 cc.
2. Asparagin 6.0 g. Constituents:
3. Oxalic acid 0.5 g. 1. Distilled water 1000.0 cc.
4. Lactic acid 0.15 g. 2. KNO3 2.0 g
5. Citric acid 0.15 g. 3. MgS04 2.0 g
6. Na^HPO^ 5.0 g. 4. Citric acid 5.0 g
7. MgS04 2.5 g. 5. K2HPO4 2.0 g
8. K2SO4 1.25 g. 6. CaCl2 0.2 g
9. NaCl 2.0 g. 7. NaaCOs 4.25 g
Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7, 8. Asparagin 1.0 g
8 and 9 in 1. Preparation
Sterilization: Not specified. (1) Dissolve 2 and 8 in a little distilled
Use: Isolation of typhoid bacilli. water.
Reference: Thoinot and Masselin (1902 (2) Dissolved 3, 4, 5, 6 and 7 in distilled
p. 337). water.
128 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents:
1. Distilled water 1000.0 cc.
2. Citric acid 7.0 g.
3. Asparagin 10.0 g.
4. K2HPO4 2.0 g.
5. NasCOs (crystalline) 2.5 g.
6. MgS04 0.4 g.
7. CaCU 0.01 g.
Preparation
(1) Neutralize 7.0 g. citric acid with pure
KOH.
(2) Dissolve (1), 3, 4, 5, 6 and 7 in 1.
6. Alcohol
8. (NH4)2C03 1.0 g.
(3) To each 10.0 cc. lot add 5, 10, 15 or 12. KH2PO4 0.5 g.
8 in 1. 6. NaCl 2.0 g.
Sterilization: Not specified. 7. Levulose 40.0 g.
Use: To study fermentation by Yihrio Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
cholerae asiaticae. in 1.
(j) Roux and Rochaix used 3.0 to 4.0 g. 3. Ammonium phosphate 10.0 g.
asparagin instead of sodium asparag- 4. Asparagin 10.0 g.
inate. 5. reP04 0.2 g.
(k) Besson used 30.0 g. glycerol, 5.0 g. 6. Glycerin 40.0 g.
NaCl, 0.2 g. MgS04, 2.0 g. K2HPO4, 7. MgS04 0.3 g.
6.0 g. ammonium lactate and 3.0 g. 8. K2HPO4 3.0 g.
asparagin. 9. NaCl 1.0 g.
(2) Add 1.0 cc. of N/100 NaOH to (1) References: Zipfel (1912 p. 74), Barthel
after sterilization. (1921 p. 85).
5. Glycocoll 2.0 g.
Dicyandiamide
Perotti's Glucose
6. Aspartic acid 2.0 g.
483
Solution
7. Glucose 2.0 g.
Dicyandiamide
Kappen's Glucose
8. Scombrine sulphate 2.0 g.
484
Solution
9. Glycerol 40.0 g.
Bs. Nitrogen supplied as ferrocyanamide.
Preparation
Doryland's Basal Ferrocyanide Salt
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9 in 1.
Solution 485
(2) Add 4.0 cc. of N/100 NaOH to (1)
Bokorny's Sucrose Ferrocyanide
after sterilization.
Solution 486
Sterilization: Method not given.
A2.'*Organic nitrogen supplied as urea.
Use: Cultivation of Koch's bacilli.
Bi. Incomplete or basal solutions, em-
References Armand-Delille,
: Mayer,
ployed with additional nutrients.
Schaeffer and Terroine (1913 p. 273).
Stutzer and Hartleb's Basal Urea
Solution 487
47Q. Zipfel's Tryptophane Asparagin Urea Salt Solution. 488
Beijerinck's Basal
Solution Waksman's Basal Urea Salt Solu-
tion 489
Constituents: Christensen's Basal Urea Salt Solu-
1. Distilled water 1000.0 cc. 490
tion
2. Asparagin Bj. Complete media.
3. Ammonium lactate 5.0 g.
Ci. Containing no additional organic com-
4. K,HP04 2.0 g.
pounds.
5. MgS04 0.2 g.
6. Tryptophane (0.1%) 1.0 g. *See also A3, A4, As and As on page 136.
136 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Distribute 200.0 cc. of 1 in a one liter distilled water, using phenolphthalein
flask. as indicator.
(3) Inoculate with 0.2 g. of garden earth. (2) Add to HCl the following salts:
Sterilization: Not specified. MgS04 0.5 g.
Use Cultivation of dicyandiamide bacteria
: Fe2(S04)3 0.01 g.
from the soil. CaO 0.01 g.
Variants: The author gives the following MnS04 0.01 g.
solution: K6Fe2(CN)i2, amount not given.
1. Water 1000.0 cc. (3) Standardize (2) against silicate solu-
2. Potassium phosphate 1.0 g. tion so that 1.0 cc. is equivalent to
Use: Cultivation of Schizomycetes. Au- (e) Sohngen used 0.05% K2HPO4 and
thor reported that mold spores did not 3.0% urea in the basic solution and
develop in this medium. Schizomycetes added 1.0% of calcium malate or cal-
developed producing lactic acid. The cium citrate to study urea decomposi-
acid produced may decompose the tion by bacteria from the soil.
K4Fe(CN)o. Potassium ferrocyanide was (f) Sohngen used a 0.5% urea and 0.05%
a suitable nitrogen source for schizomy- K2HPO4 solution without additions
cetes but not for molds. to cultivate Bacillus erythrogenes.
Variants Bokorny also used the following
: (g) Percival used 5.0% urea and 0.25%
solution: K2HPO4 in the basic solution and
1. Water 1000.0 cc. added 1.0% (NH4)2S04 to enrich
2. Sucrose 20.0 g urea splitting organisms found in the
3. Potassium ferrocyanide . . 2.0 g soil and in urine.
4. K2HPO4 2.0 g (h) Giltner used 5.0% urea and 0.05%
5. MgS04 0.052 g K2HPO4 in the basic solution and
6.CaCl2 0.012 g added 0.5 to 1.0% of one of the
Reference: Bokorny (1917 pp. 343, 344). following:
ammonium malate
487. Stutzer and Hartleb's Basal Urea calcium malate
Solution calcium citrate
Constituents: ammonium citrate
1. Water . 1000.0 cc. He used the media to show urea
2. Urea . 20.0 g. decomposition.
3. Glycerin . 10.0 g. References: Stutzer and Hartleb (1897
4. Potassium phosphate. 1.0 g. p. 404), Sohngen (1909 pp. 91, 93, 94, 95),
(b) Lohnis used 50.0 g. urea and 0.5 g. Sterilization: Not specified.
K2HPO4 in the basic solution, and Use: To study urea decomposition by soil
added 0.5 to 1.0% ammonium or cal- forms. Ammonia formation was appar-
cium malate, or calcium citrate or ent after 14 days when humus was added
tartrate. and after 5 days with glucose. If humua
(c) Percival used 2.5% KH2PO4 and 5.0% or glucose were not added, there was
urea and added 1.0% sodium acetate. apparently no ammonia formation.
References: Beijerinck (1901 p. 37), Lohnis Added nutrients: One of the following ma-
(1913 p. 108), Percival (1920 p. 225). terials were added:
(a) Humus acid small amount. Add
489. Waksman's Basal Urea Salt Solution
NaOH
sufficient dilute to keep the
Constituents: humus acid in solution.
1. Water 1000.0 cc. (b) Potassium salt of humus acid 2.0%.
2. Glycerol 30.0 g. (c) Glucose 1.0%.
3. K2HPO4 1.0 g. (d) "Buchenrohhumas" 0.1%.
4. KCl 0.5 g. (e) Xylose 1.0%.
5. MgS04 0.5 g. (f) Sodium formate 0.2%.
6. FeS04 0.01 g. (g) Meat extract 2.0%.
7. Urea 5.0 g. (h) Potassium humate 0.1% (see (8)
Preparation below),
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (i) Humin 0.2% (see (10) below).
(2) Add 2.0 g. of one of the added nu- Materials in (h) and (i) prepared as
trients. follows:
(3) Tube in 10 to 12 cc. quantities. (1) Dissolve 300.0 g. saccharose in
Sterilization: Sterilize at 15 pounds for 420.0 cc. water.
15 minutes. (2) Add 15.0 g. concentrated H2SO4
Use: To study metabolism of Actinomij- to (1).
cetes. (3) Place on a boiling water bath for
Added nutrients: Waksman added 2.0 g. 7 or 8 hours, adding the water lost
of one of the following nitrogen sources: by evaporation.
NaN03 (NH4).S04 (4) After completing the heating a
NaN02 (NH4)2C03 voluminous sediment forms at the
Reference: Waksman (1920 p. 3). bottom.
(5) Filter and carefully wash.
490. Christensen's Basal Urea Salt with an
(6) Treat the precipitate
Solution
excess of 5.0% KOH solution. A
Constituents: portion of the humus acid is dis-
Constituents :
3. MgSOi 0.5 g.
4. NaCl 2.0 g.
1. Water (tap) 1000.0 cc.
5. Methyl amine 0.5 g.
2. Uric acid 3.0 g.
Preparation :
3. K2HPO4 0.5 g.
(1) Dissolve 2, 3, 4 and 5 in 1.
Preparation :
(2) Adjustment of reaction not given.
(1) Dissolve 2 and 3 in 1.
(3) Add the usual amount of sterile
(2) Distribute in small Erlenmeyer flasks.
Sterilization: Not specified.
MgCOs to the sterile fluid.
Sterilization: Sterilize by heating at 115C.
Use: Cultivation of B. ^Morescens and uric
for 30 minutes.
acid bacteria.
Use: To
study nitrification by nitrite
Reference: Lohnis (1913 p. 96).
formers. The methyl amine or dimethyl
503. Bokorny's Amine Solution amine was not oxidized.
Variants: The author used 0.5 g. dimethyl-
Constituents :
for growth of molds. Molds grew readily Preparation: (1) Dissolve 2 and 3 in 1.
Variants: The author used the following Use : To study denitrification by soil forms,
amines and amides instead of methyl Bacillus nitroxus.
amine hydrochloride: Reference: Beijerinck and Minkman (1910
used had the following composition: (1) Prepare a 0.66% solution of hippuric
1. Water 1000.0 cc. acid.
2. Trimethyl amine (2) Prepare a 5.0% solution of one of the
acetic acid 5.0 g. added nutrients.
3. Sucrose 50.0 g. (3) Mix equal volumes of (1) and (2).
4. K2HPO4 2.0 g. Sterilization: Not specified.
5. MgSOi 0.2 g. Use: Cultivation of Monilia sitophila
6. CaCl2 0.2 g. (Mont.) Sacc.Maltose, glucose, lactose,
H3PO4
7. 0.0 or 1.0% sucrose and glycerol was the order of
Trimethyl amine served as a nitrogen vigor of growth for the carbon sources
source for bacteria and without the studied.
H3PO4 Schizomycetes developed then Added nutrients: One of the following
the molds. In the presence of H3PO1 carbon sources was employed:
molds developed at once. maltose glucose
Reference: Bokorny (1917 pp. 337, 338). lactose glycerol
sucrose
504. Omeliansky's Amine Solution
Variants: No additional carbon source was
Constituents : added. No growth occurred.
1. Water 1000.0 cc. Reference: Went (1901 p. 593).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 143
507. Stapp's Hippurate Solution Use: To study the cycle of the phosphate
ion in the soil. Author used Bacillus
Constituents:
mycoides, Bacillus subtilis, Bacillus pro-
1. Water 1000.0 cc.
teus vulgaris. Amount of phosphoric
2. KH2PO4 1.25 g.
anhydride formed by each organism was
3. MgSOi 0.625 g.
about the same after 60 days. If the
4. Sodium hippurate 3.1 g.
Preparation
NH4NOJ be omitted the nitrogen con-
tent is too low to give good growth.
(1) Dissolve 2, 3 and 4 in 1.
Variants: Author omitted the NH4NOS
(2) Distribute in 50.0 cc. lots in 200.0 cc.
and used only 0.8 g. NaCI instead of 1.0 g.
Erlenmeyer flasks.
Reference: Stoklasa (1911 pp. 423, 424).
Sterilization: Not specified.
Use: Isolation of uric acid splitting bac-
510. Stoklasa's Nucleic Acid Solution
teria from feces and soil. Bac. cobayae,
Bac. capri, Bad., guano, Bac. musculi, Constituents:
Bac. hollandicus. 1. Distilled water 8000.0 cc.
Reference: Stapp (1920 p. 4). 2. K2SO4 8.0 g.
3. MgCli 4.0 g.
508. Lohnis' Hippurate Solution
4. NaCl 1.0 g.
Constituents: 5. Al2(S04)3 + FeS04 0.12 g.
1. Water (tap) 1000.0 cc. 6. Nuclein acid 8.0 g.
2. Sodium hippurate 3.0 g. 7. Glucose 80.0 g.
3. K2HPO4 0.5 g. Preparation:
Preparation (1) Prepare nucleic acid from j^east by
(1) Dissolve 2 and 3 in 1. Herlant's method (method not given)
(2) Distribute in small Erlenmeyer flasks. (2) Dissolve 2, 3, 4 and 5 in 1.
Sterilization: Not specified. (3) Distribute in 250.0 cc. lots in large
Use: Cultivation of hippuric acid bacteria. Erlenmeyer flasks.
Author reported that fermentation took (4) Add 0.25 g. nucleic acid and 2.5 g.
place rather slowly when the medium was d-glucose to each flask.
inoculated with manure. (5) Heat at 45 to 50C. for 20 hours.
Reference: Lohnis (1913 p. 96). Sterilization: Method not given.
Use: To study the cycle of the phosphate
509. Stoklasa's Glucose Lecithin ion in the soil. Author used Bacillus
Solution mycoides, Bacillus subtilis, Bacillus
Constituents: proteus vulgaris. Bacillus mycoides util-
1. Distilled water 8000.0 cc ized the largest amount of phosphorous.
2. NH4NO3 20.0 Reference: Stoklasa (1911 p. 428).
g
3. K2SO4 8.0 g
511. Went's Basal Creatin Solution
4. MgClj 4.0 g
5. NaCl 1.0 g Constituents
6. Al2(S04)3 + FeS04 0.12 g 1. Water 1000.0 cc.
7. d-glucose 80.0 2. Creatin (0.66%) 6.6 g.
8. lecithin 8.0 Preparation
Preparation (1) Prepare a 0.66% creatin solution.
(1) Dissolve 2, 3, 4, 5, 6 and 7 in 1. (2) Prepare a 5.0% solution of one of the
(2) Distribute into large 2 liter Erlen- added carbohydrates.
meyer flasks in 250.0 cc. lots. (3) Mix equal volumes of (1) and (2).
(3) Adjustment of reaction not specified. Sterilization: Not specified.
(4) After sterilization add 0.25 g. leci- Use: Cultivation of Monilia sitophila
thin to each flask. Heat at 45 to (Mont.) Sacc. Maltose, glucose, lactose,
50 for 20 hours. sucrose and glycerol was the order of
Sterilization: Sterilize thoroughly (method vigor of growth for the carbon sources
not given). studied.
144 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Added nutrients: One of the following car- Sterilization: Sterilize in autoclave, time
bon sources was employed: not given.
maltose sucrose Use: To study acid proofness of B. tuber-
glucose glycerol culosis. On 19th day rods and diplococ-
lactose coids were non acid proof. No growth
Variants: The author used the basic solu- using caffein instead of theobromin.
tion withoutany added materials. Reference: Wherry (1913 p. 151).
Reference: Went (1901 p. 593).
515. Bokorny's Picric Acid Solution
512. Stutzer's Glucose Creatin Solution
Constituents:
Constituents: 1. Water 1000.0 cc.
1. Water 1000.0 cc. 2. Ca(N03) 2 (0.02%) 0.2 g.
2. Creatin 2.5 g. 3. KH2PO4 (0.02%) 0.2 g.
3. K2HPO4 1.0 g. 4. MgS04 (0.02%) 0.2 g.
4. KNO3 3.0 g. 5. Picric acid (0.5%) 5.0 g.
Preparation Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
Constituents 3 days.
1. Water References: Committee A. P. H. A. (1905
2. Cenovis p. 110), Heinemann (1905 p. 25), Buhlert
Reference: Lichtenstein (1923 p. 390). Use: Differentiation of Bad. coli and Bad.
aerogenes. The author reported that
519. Dunham's Peptone Solution (Committee 1-100,000 crystal violet prevented growth
A. P. H. A.) of Bod. coli, while Bad. aerogenes grew
Constituents: heavily. Decreasing the concentration
1. Water (tap) 1000.0 cc. of peptone to 0.5% increased markedly
2. Peptone 10.0 g. the inhibitory action of the dye.
Preparation Reference: Levine (1911 p. 22).
(3) Make up the loss due to evaporation 2. Dahlia (1.0% soln.) 5.0 cc.
Use: General culture medium. Indol test. (4) Add 0.05 cc. of 1.0% dahlia solution
Variants to each tube.
(a) Buhlert and Fickendey used 1.5% Sterilization: Not specified.
(1912 p. 23), Lohnis (1913, p. 44), Sears 527. Brussoff's Peptone Solution
(1916 p. 110), Roddy (1917 p. 42), Besson Constituents:
(1920 p. 29), Myers (1920 p. 242), Bezan- 1. Distilled water 1000.0 cc.
Qon (1920 p. Ill), Percival (1920 p. 113), 2. Peptone (Witte) 10.0 g.
Buchan Norton and Sawyer
(1910 p. 108), 3. Iron peptone (Merck) 1.0 g.
(1921 p. 473), Harvey (1921-22 p. 101), 4. NaCl 10.0 g.
Giltner (1921 p. 42), Abbott (1921 p. 140), Preparation
Dopter and Sacquepee (1921 p. 119), (1) Dissolve 2, 3 and 4 in 1 by heating in
Bitfield (1922 p. 118), Stitt (1923 p. 35), streaming steam for 30 minutes.
Park, and Krumwiede (1924
Williams Filter.
(2)
p. 120), Cunningham (1920 p. 18), Bristol Adjustment of reaction not specified.
(3)
(1925 p. 467), Almy and James (1926 Sterilization: Sterilizein the autoclave
p. 323). under 1.5 atmospheres pressure for 10
minutes.
525. Huss' Peptone Solution
Use: Cultivation of iron bacteria (sludge
Constituents forms), Ferribacterium duplex.
1. Water 1000.0 cc. Reference: Brussoff (1918 p. 196).
2. Peptone 10.0 g.
3. NaCl 5.0 g. 528. Redfield's Peptone Solution (Myers)
Preparation: (1) Dissolve 2 and 3 in 1.
Constituents
Sterilization: Not specified.
1. Water 1000.0 cc.
Use: To study indol production by aroma 2. Peptone (Witte or Difco) . . 300.0 g.
producing bacteria. Bacillus esterifi-
3. NaCl 75.0 g.
cans, Maassen, and Pseudomonas Tri- Preparation
folii. Reaction for indol was negative Boil 2 in 700.0 cc. tap water and
(1)
wlien using H2SO4 and several drops of 75.0 g. NaCl until as much of peptone
1.0% KNO2 as a test for indol. as possible has gone into solution.
Variants: Brussoff used this solution to (RedfieldusedKCl.)
cultivate iron bacteria from sludge,
Cool rapidly and make up to 1 liter.
(2)
Ferribacterium duplex. A yellow mem- Heat to boiling in a flask, plug and
(3)
brane formed after 3 days. cool rapidly.
Reference: Huss (1907 p. 61), Brussoff
(4) Add 0.5% NaCl (to clear).
(1918 p. 195). cold thru paper.
(5) Filter
(6) Distribute in 150.0 cc. Erlenmeyer
526. Nencki's Peptone Solution flasks in 10.0 cc. lots.
Constituents Sterilization: Sterilize at 15 pounds for
1. Water 900.0 cc. 15 minutes.
2. Peptone 100.0 g. Use: Detection of H2S by bacteria from
3. NaCl 20.0 g. animal feces. Used lead acetate paper
Preparation to detect HoS production.
(1) Dissolve 100.0 g. peptone in 900.0 cc. Reference: Myers (1920 p. 232-235).
water.
529. Berman and Rettger's Peptone
(2) Add 20.0 g. NaCl to (1).
Solution
(3) Filter.
(4) Distribute into test tubes. Constituents:
Sterilization: Sterilize in the autoclave. 1. Water 1000.0 cc.
Sterilization: Not specified. the rosolic acid was the dye that inhibited
Use: Enrichment medium. The inositol the gram positive rods.
fermenters were suppressed. Variants: The author prepared a stock
Reference: Harvey (1921-22 p. 91). china blue and rosolic acid solution by
dissolving 10.0 g. Grubler's china blue
534. Smith's Rosolic Acid Peptone Solution and 2.0 g. Grubler's rosolic acid in
Constituents 100.0 cc. of 50.0% alcohol. This mixture
1. Water 1000.0 cc. of dyes (0.25%) was added to the medium
2. NaCl 5.0 g. instead of just china blue solution alone.
3. Peptone 10.0 g. Reference: Bronfenbrenner (1920 p. 184).
4. Rosolic acid (0.5% soln. in
80.0% alcohol) 2.0 cc. 536. Diedudonne's Nitrate Peptone
Preparation Solution
(1) Dissolve 2 and 3 in 1.
Constituents:
(2) Filter.
Water 1000.0 cc.
(3) Prepare a 0.5% rosolic acid solution 1.
Use: To detect acid and alkali production. (2) Add 0.01% KNO3 to (1).
(1) Conn and Breed used 0.2 or 2.0 g. 539. Enlow's Basal Agar Peptone
and added 10.0 g. glucose.
nitrate Solution
(m) Conn and Breed used 0.2 g. KNO3 Constituents
with 2.0 or 5.0 g. peptone, 1. Water (tap or distilled) 2000.0 cc.
(n) Conn and Breed used 0.2 g. KNO3, 2. Peptone 10.0 g.
2.0 or 5.0 g. peptone and added K2HPO4
3. 17.0 g.
10.0 g. glucose. 4. Agar 2.0 g.
References: Diedudonne (1894-95 p. 510), 5. Indicator (Brom thymol blue)
.
Grimbert (1898 p. 385), Maassen (1902 Preparation
p. 28), Heinemann (1905 p. 131), Com- (1) Dissolve 2, 3 and 4 in 1800.0 cc.
mittee A. P. H. A. (1905 p. 109), Killer water in the Arnold sterilizer by
(1913 p. 521), Rogers, Clark and Davis heating for 40 minutes or autoclave
(1914 p. 415), Committee S. A. B. (1918 for 10 minutes or autoclave for 10
p. 116), Tanner (1919 p. 90), Conn and minutes at 15 pounds pressure.
Breed (1919 pp. 273-275), Tanner (1919 (2) Filter through paper while hot.
p. 45), Ball (1919 p. 76), Giltner (1921 (3) Dilute to 2000.0 cc. by the addition
p. 42), Harvey (1921-22 pp. 102, 108), of hot distilled water.
Park, Williams and Krumwiede (1924 (4) Adjust the hydrogen ion concentra-
p. 124). tion as desired.
(5) Add 0.5% of one of the added nu-
537. Harvey's Nitrate Peptone Solution trients.
(6) Add any desired indicator. (The
Constituents: author added 3 drops of Brom thymol
1. Distilled water 1000.0 cc. blue to each 500.0 cc. of medium.
2. NaNO, 1.0 g. The indicator solution was prepared
3. Peptone 10.0 g. by grinding 0.1 gram Brom thymol
Preparation: (1) Dissolve 2 and 3 in 1. Blue and 3.2 cc. of N/20 NaOH in an
Sterilization: Not specified. agate mortar. When solution of the
Use: Study of nitrate reduction. dye was complete, add 3.0 cc. of dis-
Reference: Harvey (1921-22 p. 107). tilled water.
(7) Tube.
538. Cohen and Clark's Phosphate Peptone Sterilization: Sterilize for 30 minutes in
Solution the Arnold sterilizer on each of 3 succes-
sive days.
Constituents:
Use: Used as a basic sugar free medium.
1. Water 1000.0 cc.
Variants The author used 14.5 g. Na2HP04
:
'
Reference: Cohen and Clark (1919 p. 410). (2) Make alkaline with NsaCO,.
152 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
NaCl 5.0 g.
paratyphoid bacilli. Indol was not pro- 4.
Preparation
duced in this medium.
(1) Extract 15.0 g. of 2 for two weeks with
Reference: NicoUe, Raphael and Debains
ether, two weeks with alcohol,
for
(1917 p. 380).
two weeks with acetone and 10 days
543. Levine's Boric Acid Peptone Solution with petroleum ether, respectively,
in a Soxhlet extractor, the successive
Constituents
1000.0 cc. extractions occurring at intervals of
1. Water
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 153
about 12 minutes. These extractions Added nutrients : The author added 100.0 g.
to be continued for 6 hours a day and of lactose or 100.0 g. of calcium lactate.
6 days per week. Reference: Klecki (1896 p. 255).
(2) Dissolve residue from the extraction
549. Pergola's Nitrate Peptone Solution
in 1.
5. MgS04 .'
0.275 g.
551. Bleisch's Nitrate Peptone Solution
6. (NH4)2S04 0.115 g.
Preparation Constituents:
(1) Dissolve 2, 3, 4, 5, 6 and 100.0 g. of 1. Distilled water 1000.0 cc.
one of the added nutrients in 1. 2. Peptone (Witte) 20.0 g.
(6) Shake thoroly, removing the stopper 559. Fremlin's Ammonium Sulphate
occasionally. Peptone Solution
(7) Place a paper around the flask and Constituents:
place in the incubator at 37C. for
1. Water 1000.0 cc.
24 hours.
2. (NH4)2S04 1.0 g.
(8) Shake thoroly. 3. Potassium phosphate. 1.0 g.
(9) Filter thru a sterile moistened filter
4. MgCOa 10.0 g.
paper. Peptone
5. 0.1 to 0.5 g.
Sterilization: Sterilize for 45 minutes
Preparation
(method not given).
(1) Dissolve 2 and 3 in 1.
Use: Study of indol production. Culture
(2) Dissolve 4 in the remainder of 1.
medium used in water analysis.
and (2) under aseptic
(3) Mi.x sterile (1)
Reference: Gersbach (1922 p. 146).
conditions and add from 0.1 to 0.5 g.
557. Kappen's Peptone Solution peptone.
Sterilization: Method not given.
Constituents
Use: To study nitrogen o.xidation by
1. Water 1000.0 cc.
nitroso bacteria.
2. Peptone (0.5%) 5.0 g.
Reference: Fremlin (1903 p. 368).
3. K2HPO4I
4. NaCl I 560. Mortensen's Peptone Solution
,- Tv,r CI/-. } usual amounts
5. MgS04 Constituents:
6. FeS04 J 1. Water 1000.0 cc.
Preparation: Prepare a 0.5% solution
(1) 2. Peptone (Witte (1.0%)) .... 10.0 g.
of peptone in water containing the usual 3. Cobalt salts
amounts of K2HPO4, NaCl, MgSOi, and Preparation
FeS04 (amounts not given). (1) Dissolve 2 in 1.
Sterilization: Method not given. (2) Add cobalt salts in varying amounts
Use: To study decomposition of peptone from 4 to 1/32%.
by cyanamide decomposing bacteria. Sterilization: Not specified.
Reference: Kappen (1909 p. 395). Use: To study the effect of cobalt salts on
Aspergillus niger. The author reported
558. Percival's Ammonium Sulphate
that the toxic properties of cobalt were
Peptone Solution
different in different media. A 0.5%
Constituents: cobalt chloride solution in a liquid
1. Water 1000.0 cc. medium was as toxic as 1.0% cobalt
2. (NH4)2S04 2.0 g. chloride in a gelatin medium.
3. K2HPO4 1.0 g. Reference: Mortensen (1909 p. 523).
4. NaCl 2.0 g.
5. MgS04 0.5 g. 561. Kligler's Nitrate Peptone Solution
6. FeS04 0.4 g. Constituents:
7. MgC03 10.0 g. 1. Distilled water 1000.0 cc.
8. Peptone (0.01%) 0.1 g. 2. Peptone 10.0 g.
Preparation 3. K2HPO4.. 0.2 g.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. 4. MgS04 0.1 g.
(2) Distribute in 50.0 cc. into conical 5. NaCl 0.2 g.
flasks. 6. KNO3 0.2 g.
(3) Add 0.5 g. of basic MgCOj to each Preparation: (1) Dissolve 2, 3, 4, 5 and 6
flask. in 1.
Use: Cultivation of bacteria from the toma and Cladothrix natans in the
nodules of legumes. following solution.
Reference: Percival (1920 p. 204). 1. Water 1000.0 cc.
2. MgS04 1.0 g.
563. Capaldi and Proskauer's Peptone 3. K2HPO4 0.25 g.
Solution Peptone 1.25 g.
4.
(4) Adjust (1) to desired pH. References: Robinson (1915 p. 407), Harvey
(5) Add 5.0 cc. of (3) and 1.2 cc. of (2) (1921-22 pp. 101, 109).
to (4) for every 100.0 cc. of (4).
568. Capaldi and Proskauer's Basal Peptone
(6) Add one of the added nutrients.
Solution
Sterilization: Autoclave at 10 pounds pres-
sure for 10 minutes. Constituents:
Use: To determine fermentation of sugars. 1. Water 1000.0 cc.
Pink color indicates alkali formation. 2. Peptone (Witte) (0.5
Acid indicated by first a bright green and or 2.0%) 5.0 or 20.0 g.
then changing to deep blue. Preparation: Dissolve 2 and 0.1% of one
(1)
Added nutrients: Author suggested the use of the added nutrients in 1.
of any carbohydrate or fermentable ma- Sterilization: Not specified.
terial. The carbohydrate solution may Use: To determine fermentation of sugars
be sterilized separately and added aseptic- by the colon-typhoid group. Different
ally to sterile (5), or the sugar can be investigators added various materials to
added to sterilized (5) and the mixture the basic solution, or a modification of it,
be heated a second time in the autoclave and used the media for a variety of
at 10 pounds for 10 minutes. purposes.
Reference: Morishima (1920 p. 43). Added nutrients and variants:
(a) The authors added 0.1% of one of
567. Robinson's Basal Azolitmin Peptone
the following materials:
Solution
glucose raffinose
Constituents: levulose dextrin
1. Water 1000.0 cc. mannose inulin
2. Peptone (Witte) 10.0 g. galactose mannitol
3. Azolitmin 0.5 g. sorbose adonitol
Preparation lactose sorbitol
(1) Prepare 500.0 cc. of a 2.0% solution of maltose isodulcitol
Witte' s peptone. melibiose erythritol
(2) Adjust to a reaction of -f-0.6 to trehalose dulcitol
phenolphthalein. They reported growth poor with a
(3) Prepare 500.0 cc. of a solution con- M.L.D. = 1.0-f- cc.
taining 2.0% of an added nutrient (b) Matzuschita used 10.0 g. Koch's
(sugar). meat peptone per liter water as a
(4) Mix sterile (2) and (3) in equal basic solution and added one of the
amounts, and add 0.05% azolitmin. following materials or combinations:
(5) Tube in sterile tubes. NaCl 0.5, 0.7 to 10.5%.
Sterilization: Sterilize (2) in the autoclave. NaCl and glucose 0.5 to 50.0%.
5.0 g.
Filter (3) thru Berkefeld candles into NaCl 5.0 g., glucose 2.0% and from 0.2
sterile flasks. Incubate 24 hours to to 15.0% Na.COs. Add the NajCOs
insure sterility. to the neutral medium.
Use To determine fermentation by typhoid
: NaCl 5.0 g., glucose 2.0%, glycerol
and paratyphoid bacilli. 6.0%.
Added nutrients and variants NaCl 5.0 g., glucose, 2.0% and from 0.1
(a) Robinson used 10.0 g. of any desired to 0.4% HCl. Add the HCl to the
sugar. neutral medium.
(b) Harvey used 2.0 to 10.0 g. peptone, No additions to the basic solution.
50.0 cc. of litmus solution and 10.0 g. These media were used to cultivate
of any desired carbon source or fer- Clostridium butyricum, Bacillus
mentable material. oedematis maligni, Bacillus anthracis
(c) Harvey used 20.0 g. peptone, 50.0 cc. sym-ptomaiici, Bacillus sporogenes,
of litmus solution and 10.0 g. of any Bacillus botulinus.
carbon source or fermentable ma- (c) Revis did not specify the exact com-
terials per 850.0 cc. of distilled water. position of the basic solution (pep-
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 159
t:)ne water) and added one of the added to lactic acid until a pH = 7.0
following materials: is reached. In case of phos-
the
mucic acid (sodium salt) 1-0% phates, mono and di-sodium phos-
saccharic acid (potassium salt). . 1.0% phates are mixed in the proper
gluconic acid (potassium salt)... 0.5% proportions to give a pH of approxi-
The media were used to study the mately 7.0. NaCl is used also in
fermentation of acids by the colon 0.4M and MgCU in O.IM concentra-
groip. The author reported that tions. The media were used to show
with 1.0% of the acid sodium salt of the effect of salts on the growth of
mucic acid no gas was produced with B. coli. The authors reported that
B. coll or B. lactis aerogenes, but generally the order of growth accele-
slightly more acid was produced. ration of B. coli by these salts was
With the normal sodium salt and in approximately the same as their
the presence of Mg(0H)2 acid and order in the lyotropic series,
gas were produced by B. coli and B. (f) Hotchkiss used 10.0 g. peptone per
lactis aerogenes. liter in the basic solution and added
Same thing was true with saccharic a number of salts to study the effect
acid. (Acid and gas produced when of various cations on a communis
medium was alkaline.) Gluconic type of B. coli. He used the follow-
acid was attacked when present as ing salts and reported that the
the potassium salt orwhen alkalinity concentrations here indicated stimu-
was increased to 1.0 cc. N/1 NaOH lated growth. NaCl, KCl, CaCU and
per 100.0 cc, or when the acid itself MgCl2 may be added to the peptone
was employed. before sterilization. Other salt solu-
(d) Davis and Ferry cultivated Bad. tions were made up and stored until
diphtheriae and studied toxin pro- sterile and added under aseptic
duction using 20.0 g. of peptone in the conditions to the sterile peptone
basic solution and adding one of the solution. pH for all solutions except
following materials: NH4CI were from 6.6 to 7.0. NH4CI
glycocoll 0.75 g solution pH = 6.0 to 6.4.
leucine 30.0 g NaCI 0.25 molar concentration in the
histidine dichloride 0.5 g media.
glutaminic acid hydrochloride. 2.5 g KCl 0.25 molar concentration in the
tyrosine 20.0 g media.
cystine 0.5 g NH4CI 0.25 molar concentration in the
tryptophane 0.6 g media.
sodium asparaginate 1.5 g CaCU 0.05 molar concentration in the
creatine 0.2g + creatinine 0.15g. media.
xanthin 0.05 g.+ hypoxanthine 0.05 g MgClo 0.05 molar concentration in the
glucose amine hydrochloride . . 2.0 g media.
(e) Holm and Sherman used 10.0 g. of SrCl2 0.025 molar concentration in the
peptone per liter in their basic solu- media.
tion and added one of the following BaCl2 0.05 molar concentration in the
salts in sufficient quantities to make media.
the medium 0.2 molar for each salt. TiCl2 molar concentration in
0.0005
NaCl Na citrate the media.
NaXOs Na fluoride NiCl2 0.0001 to 0.00005 molar concen-
NaHP04 KCl tration in the media.
Nal CaCl2 PbCla 0.0005 to 0.00005 molar concen-
Na2S04 MgClo tration in the media.
Na lactate FeClj SnCl2 0.00005 to 0.000005 molar con-
Na o.xalate NH4CI centration in the media.
Na acetate ZnCl2 0.00005 to 0.00001 molar con-
In case of sodium lactate, NaOH is centration in the media.
160 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Sterilization: Sterilize (3) in the autoclave. (2) Add 1.0% of one of the added nu-
Sterilize (4) at 105C. for 10 minutes. trients (see sterilization).
Sterilize the tubes at 105C. for 10 Sterilization: Sterilize for 30 minutes on
minutes or at 100 on each of 2 successive each of 2 successive days. On the third
days for 20 minutes. day add 1.0% of one of the added nutri-
Use: To observe fermentation by bacteria. ents, and sterilize for 15 minutes.
Added nutrients: The author did not Use: To study decomposition of sugars.
specify the sugars used. One per cent of Other investigators used variants of the
any desired carbon source may be em- medium to cultivate sludge forms and to
ployed. study proteolysis.
Reference: Harvey (1921-22 p. 108). Added nutrients and variants:
(a) Mendel added 1.0%, of one of the
572. Abba's Basal Phenolphthalein Peptone following:
Solution glucose lactose
Constituents sucrose maltose
1. Water 1000.0 cc. (b) Rettger, Berman and Sturges studied
2. Peptone 100.0 g. proteolysis used 5.0 g. NaCl and 2.5,
3. NaCl 50.0 g. 5.0 or 20.0 g. of Witte's peptone in
4. Phenolphthalein (1.0% alco- the basic solution and adding the
holic soln.) 0.5 cc. following materials individually or in
Preparation combination:
(1) Dissolve 2, 3 and 200.0 g. of one of the glucose 10.0 g.
added nutrients in 1. beef extract 5.0 g.
(2) Boil in the steamer for 30 minutes at (NH4)2S04 2.5 g.
100C. (c) Brussoff used 10.0 g. NaCl in the
(3) Filter. basic solution. He used the media
(4) Distribute into 100.0 cc. lots into to cultivate Ferribacterium duplex,
sterile containers. and other iron bacteria from sludge.
(5) After sterilization add 0.5 cc. of He added one of the following ma-
phenolphthalein solution and Na2C03 terials to the basic solution.
solution until the solution is perma- Iron ammonium citrate 0.5 g.
nently red (see use). K,C03 1.0 g.
Sterilization: Not specified. K2SO4 1.0 g.
Use: Isolation of Bacillus coli communis Potassium acetate 1.0 g.
from water. About 1 liter of the water Potassium citrate 1.0 g.
under investigation is added to each flask References: Mendel (1911 p. 297), Brussoff
of medium. Bacillus coli communis (1918 pp. 195, 208) Rettger,, Burman and
decolorized the solution after from 12 to Sturges (1916 p. 20-22).
24 hours (using glucose). Using lactose
574. Nicolle, Raphael and Debains' Basal
B. coli caused decolorization after about
Peptone Solution
8 to 24 hours. Typhoid bacilli require a
greater length of time. Constituents:
Added nutrients The author added 200.0
: g. 1. Distilled water 1000.0 cc.
of lactose or glucose. 2. Peptone (Defresne) 1.0 g.
Reference: Abba (1896 p. 13), (1896 p. 224), 3. Soda (normal) 20.0 cc.
(1895 ^176). Preparation
(1) Dissolve 2, 3 and one of the added
573. Mendel's Basal Peptone Solution
nutrients in 1.
Constituents: (2) Distribute in test tubes containing
1. Distilled water 1000.0 cc. fermentation tubes.
2. Peptone (Witte) 10.0 g. Sterilization: Sterilize at 110 for 15
3. NaCl 5.0 g. minutes.
Preparation Use: To study the fermentation of sugars
(1) Dissolve 2 and 3 in 1. by typhoid and paratyphoid bacilli.
162 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Authors report that glucose may or may Use: To study fermentation by meningo-
not be fermented, lactose never. cocci.
Added nutrients: Authors added 1.0% of Added nutrients: The author added 10.0 g.
glucose or lactose. of one of the following:
Reference: Nicolle, Raphael and Debains glucose sucrose
(1917 p. 379). galactose mannitol
levulose dulcitol
575. Robinson and Rettger's Basal Opsine
lactose inulin
Solution
maltose dextrin
Constituents: Reference: Elser and Huntoon (1909
1. Water 1000.0 cc. p. 404).
2. Opsine 10.0 g.
577. Clark and Lubs' Basal Phosphate
3. NaCl 5.0 g.
Peptone Solution
4. Glycerol 50.0 g.
Preparation : Constituents:
(1) Dissolve 2, 3 and one of the added 1. Distilled water 1000.0 cc.
nutrients in 1 by heating. 2. Peptone (Witte) 5.0 g.
(2) Adjust the reaction may be used 3. K2HPO4 (c.p.) 5.0 g.
either slightly acid or basic to litmus. Preparation
(3) Boil over a flame a few minutes. (1) Dissolve 2, 3 and one of the added
(4) Filter and tube. nutrients in 800.0 cc. distilled water
Sterilization: Sterilize at 12 pounds extra by heating for 20 minutes over a
pressure for 15 minutes. steam bath. Stir occasionally.
Use: General culture medium. Opsine is (2) Filter thru a Schleicher and SchuU
a protein free trypsin, erepsin and pepsin No. 588 folded filter.
digest of certain proteins. The medium (3) Cool to 20C. and make up e.xactly
supported the growth of some pathogenic to 1000.0 cc.
forms. (4) Tube in 10.0 cc. quantities in clean
Added nutrients: The authors added 5.0% sterile test tubes.
glycerol or 1.0% glucose. by the intermittent
Sterilization: Sterilize
Reference: Robinson and Rettger (1918 method.
p. 212). Use: Used to study fermentation of sugars.
Added nutrients and variants:
576. Elser and Huntoon's Basal Nahrstoflf-
(a) Clark and Lubs added 5.0 g. c.p.
Heyden Solution
glucose.
Constituents: (b) Levine added 5.0 g. of one of the
1. Distilled water 1000.0 cc. following materials:
2. Nahrstoff Heyden. . . . 10.0 g. fructose raffinose
3. NaCl 5.0 g. galactose mannitol
4. Litmus (Merck's maltose glycerol
soln) 5.0 to 7.5 cc. lactose salicin
Preparation : sucrose dextrin
(1) Dissolve 2 and 3 in 1. The media were sterilized in the
(2) Add 5.0 to 7.5 cc. of a watery solution autoclave.
ofMerck's highly sensitized litmus. (c) Rogers, Clark and Evans used 10.0 g.
(3) Prepare a 10.0% solution of one of Witte's peptone instead of 5.0 g. and
the added nutrients in distilled water. added 10.0 g. of one of the following:
(4) Mix sterile (2) and sterile (3). glucose inulin
(5) Tube in sterile tubes. lactose mannitol
(6) Incubate for 3 days to detect acci- sucrose dulcitol
dental contamination. raffinose adonitol
Sterilization: Sterilize (2) in the usual melibiose glycerol
manner, method not given. Sterilize arabinose
(3) by heating at 100C. for 10 minutes. The media was tubed in 10.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 163
quantities and sterilized by the inter- Rettger (1917 p. 165), Kligler (1918 p. 467),
mittent method in flowing steam. Tanner (1919 p. 47), Winslow, Rothberg
(d) Levine used Digestive Ferments and Parsons (1920 p. 151), Levine (1921
Co. peptone instead of Witte's, and p. 117), AyeTs, Rupp and Mudge (1921
added 5.0 g. of glucose. The medium p. 258), Giltner (1921 p. 383), Cunning-
was sterilized in the autoclave. ham (1924 p. 88), Wilson and Blair (1925
(e) Burton and Rettger added 5.0 g. p. 112), Committee A. P. H. A. (1925
glucose to the basic solution. The p. 111).
reaction was adjusted to neutral to
578. Grimbert's Basal Peptone Solution
litmus (pH = 7.0 or +1.0). Method of
sterilization not specified. Constituents:
(f) Kligler used 10.0 g. of peptone in- 1. Water 1000.0 cc.
stead of 5.0 g. Witte's peptone, added 2. Peptone (dry) 20.0 g.
5.0 g. NaCl, 1.0 g. of glucose and 3. CaCOs
added any organic test material in Preparation
any desired quantity. The ma- (1) Dissolve 2 and one of the added nu-
terials used were added to test their trients in 1.
antiseptic ability. Sterilize the basic (2) Add a sufficient quantity of CaCOa.
solution with the glucose in the auto- Sterilization: Not specified.
clave. Then add one of the ma- Use: Determine fermentation of Fried-
terialsunder aseptic conditions. lander's pneumobacillus.
(g) Winslow, Rothberg and Parsons Added nutrients and variants:
added 27.0 cc. of a 0.04% alcoholic (a) The author added 30.0 g. of any
solution of brom cresol purple and fermentable sugar.
5.0 g. of any carbon source or fer- (b) Pottevin used 10.0 g. peptone, 12.0 g.
mentable material. The basic solu- of CaCOa in the basic solution to
tion was adjusted to pH = 7.4, brom study the lactic fermentation by
cresol purple added and autoclaved lactic acid forming organisms. He
at 15 pounds pressure for 15 minutes. added one of the following:
The sterile added nutrient was added lactose 8.86 g
under aseptic conditions. sucrose 10.1 g
(h) Ayers, Rupp and Mudge used 40.0 g. maltose 11.4 g
of Bacto peptone instead of 5.0 g. glucose 10.0 g
Witte's peptone and added 2.0 g. Invert sugar 11.2 g
glucose. Method of sterilization not galactose 9.2 g
specified, mannose 8.96 g
(i) Cunningham autoclaved the K2HPO4, mannitol g 10.0
peptone and water to 30 pounds dulcitol g 10.0
pressure, filtered, made up to glycerol 10.0 g
1000.0 cc, distributed in 5.0 cc. malic acid 10.0 g
quantities and autoclaved at 22.5 Reference: Grimbert (1895 p. 843), Pot-
pounds pressure, tevin (1898 p. 54).
(j) Wilson and Blair made up the basic
579. Kendall, Walker and Day's Basal
solution with 5.0 g. glucose in double
Peptone Salt Solution
strength. They used the medium to
enrich streptococci in water analyses. Constituents:
Method of sterilization not given. 1. Redistilled water 1000.0 cc.
An equal volume of water under 2. Peptone (Fairchild) 5.0 g.
investigation was added to the 3. Na2HP04 2.0 g.
medium. 4. NaCl 5.0 g.
References: Clark and Lubs (1915 p. 169), Preparation
Levine (1916 pp. 160, 161), Committee (1) Extract 5.0 g. of Fairchild's peptone
A. P. H. A. (1917 p. 107), Rogers, Clark for 2 weeks with ether, 2 weeks with
and Lubs (1918 p. 234), Burton and alcohol, 2 weeks with acetone and 10
164 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
hours a day for 6 days per week. (b) lactose, 2.0 to 10.0 g.
(2) Dissolve the residue from (1), 3, 4 (c) glucose, 2.0 to 10.0 g. + beef extract
and one of the added nutrients in 1. 2.5 g.
(3) Distribute in 100.0 cc. lots. (d) lactose, 2.0 to 10.0 g. + beef e.xtract
Sterilization: Not specified. 2.5 g.
Use: To study lipolytic and proteolytic Reference: Berman and Rettger (1918
activity of tubercle bacilli. To deter- p. 392).
(2) Add one of the combinations listed Use: Cultivation of organisms found in
under added nutrients to (1). cheese.
Sterilization: Not specified. Added nutrients: The authors added 0.5%
Use: To study nitrogen metabolism of of glucose or galactose.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 165
Reference: Boekhout and Ott de Vries acids by organisms. The author used 51
(1918 p. 134). organisms and reported that 41 organisms
developed in the presence of malic acid
583. Will's Basal Peptone Salt Solution and 38 in the presence of citric and
Constituents: fumaric acids. These acids allowed the
1. Distilled water 1000.0 cc. development of the greatest number of
2. CaHP04 0.5 g. organisms.
3. KH2PO4 4.55 g. Added nutrients: One tenth equivalent
4. Peptone (Witte) 20.0 g. weight of one of the following acids was
Preparation added:
(1) Dissolve 2, 3 and 4 in 1. formic acid malonic acid
(2) Add one of the added nutrients. acetic acid tartaric acid
Sterilization: Not specified. propionic acid fumaric acid
Use: Cultivation of non-spore forming glycolic acid malic acid
yeast. lactic acid "trikarballysaure"
Added nutrients and variants: butyric acid citric acid
(a) The author added one of the glyceric acid aconitic acid
following: "Oralsaure" mucic acid
sucrose 5.0% malic acid quinic acid
organic acid, carbohydrate or alcohol succinic acid mandelic acid
(b) Grosbiisch added one of the fol- Reference: Maassen (1895-6 p. 341).
lowing:
585. Calmette, Massol and Breton's Basal
alcohol 5.0%
Peptone Salt Solution
formic acid 0.1%
acetic acid 0.2% Constituents:
tartaric acid . .-
0.3% 1. Water lOOO.O cc.
lactic acid 4.0% 2. NaaCOa 1.0 g.
citric acid 8.0% 3. FeS04 0.04 g.
malic acid 10.0% 4. MgS04 0.05 g.
succinic acid 11.0% 5. K0HPO4 1.05 g.
The amounts of acids given was the 6. NaCl 8.5 g.
largest amount at which Torida 7. Peptone 10.0 g.
rubefaciens developed. Preparation
Reference: Will (1908 p. 387), (1912 p. 3), (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
or glucose.
cultivated the causative acids slightly alkaline by the addi-
(b) Stormer
organism of water rust in flax (Plec- tion of NazCOs).
tridium pectinovorum) using 5.0 g. (4) Add 2.0 g.CaCOs to each flask.
KH2PO4, 2.5 g. MgS04 and 5.0 g. of Sterilization: Method not given.
peptone in the basic solution. One Use: To study nitrogen assimilation by
Bacillus megatherium (Alinit bacteria).
of the following materials was added
starch 5.0 to 10.0 g Authors reported that organisms showed
glucose 5.0 to 10.0 g greater development in medium contain-
1-0% 3. CaCU 01 g-
glycerol
mannitol 1-0% 4. MgS04 0.1 g.
5. FeCl3 trace
He reported no growth using tartaric
NaCl trace
acid. Citric acid also inhibited most 6.
7. Peptone 10-0 g.
yeast.
Stormer Preparation
References: Hansen (1899 p. 6),
(1) Dissolve 2, 3, 4, 5, 6
and 7 in 1.
(1904 p. 177), Buromsky (1914 p. 532).
(2) Add one of the
combinations given
587. Stoklasa and Vitek's Basal Peptone under added nutrients.
Salt Solution Distribute in 5.0 cc. quantities in
(3)
tubes.
Constituents:
1000.0 cc. Sterilization: Not specified.
1. Water
Na2HP04 0.25 g. Use: To study nitrate reduction by myco-
2.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 167
Constituents:
6. K0HPO4 2.0 g.
7. Glucose 2.0 g.
1. Water tap 1000.0 cc.
8. Phenol red (0.02% soln.) ... 2.0 g.
2. Glucose (4.0%) 40.0 g.
Preparation
3. Peptone (0.1%) 1.0 g.
(1) Dissolve 2 in 1000.0 cc. of 1.
4. K2HPO4 (0.05%) 0.5 g.
(2) Dissolve 3, 4, 5, 6, 7 and 8 in 1000.0 cc.
Preparation: (1) Dissolve 2, 3 and 4 in 1.
of 1.
Sterilization: Not specified.
(3) Mix (1) and (2).
Use: Cultivation of aerobacter and amylo-
(4) Add NaOH until the medium is red
bacter. Tiie medium was inoculated
(pH = 7.8).
with 10.0% soil. Similar media were used
Sterilization: Sterilize in autoclave at 10
to study the fermentation of glucose, for
pounds for 10 minutes.
methyl red test.
Use: To study the constituents essential
Variants
for the growth of streptococci and pneu-
(a) Rogers, Clark and Evans prepared
mococci. The author reported this as a
the medium as follows:
poor medium.
(1) Dissolve 10.0 g. glucose, Merck's
Reference: Mueller (1922 pp. 317, 325).
highest qi\ality, 10.0 g. Witte's pep-
tone and 5.0 g. K2HPO4 in 800.0
593. Schukow's Basal Glucose Peptone Salt
cc. distilled water, heating over the
Solution
steam for 20 minutes, and occa-
sionally stirring. Constituents:
medium as in variant (a), but omitted (2) Add 9.0 to 10.0% of one of the added
the K2HPO4 with or without the nutrients to (1).
addition of H3PO4 until the solution (3) Adjustment of reaction not specified.
(c) Clark and Lubs used the same (sealed with H2SO4).
medium as in variant (a) but used 5.0 Sterilization: Sterilize in a steamer.
Use: Cultivation of lactic acid bacteria. ascitic fluid might be added. Pneu-
Reference: Henneberg (1903 p. 8). mococci grew very well without the
ascitic fluid however.
595. Harvey's Glucose Peptone Solution (b) Stewart and Stitt, determined the
Constituents: Voges Proskauer reaction using 5.0 g.
1. Distilled water 960.0 cc. NaCl, 20.0 g. glucose and 10.0 g.
2. Peptone 10.0 g. Witte's peptone per liter.
3. NaCl 5.0 g. (c) Meyers studied H2S production using
4. Glucose 10.0 g. 30.0 g. of Witte's, Difco or Fairchild's
5. Litmus solution 40.0 cc. g. glucose, 5.0 g. NaCl,
peptone, 5.0
Preparation and 0.0 or 5.0 g. sucrose per liter.
(1) Dissolve 2 and 3 in 1. (d) Klimmer used 10.0 g. peptone, 5.0 g.
(2) Filter sterile (1) and adjust the NaCl and 10.0 g. of glucose per liter.
reaction if necessary. References: Eijkman (1904 p. 745), Truche
(3) Dissolve 10.0 g. glucose in 40.0 cc. of and Cotoni (1911 p. 480), Stewart (1917-18
litmus solution. p. 294), Myers (1920 p. 242), Stitt (1923
(4) Mix (2) and (3). p. 36), Klimmer (1923 p. 221).
(5) Filter thru paper.
597. Matzuschita's Glucose Peptone
(6) Steam 10 minutes.
Solution
(7) Tube sterile (6) into sterile test tubes.
Sterilization: Sterilize (1) in the autoclave. Constituents:
Sterilize (6) by thru a candle.
filtering 1. Water 1000.0 cc.
Incubate to test sterility before use. 2. Meat peptone
Use: General culture medium. (Koch) 10.0 g.
Reference: Harvey (1921-22 p. 109). 3. Gum arable 50.0 to 300.0 g.
4. NaCl 5.0 g.
596. Eijkman's Glucose Peptone Solution 5. Glucose 20.0 g.
Constituents: Preparation:
1. Water 1000.0 cc. (1) Dissolve 2 in 1.
Variants
598. Gosio's Glucose Peptone Solution
(a) Truche and Cotoni enriched pneumo-
cocci in the following solution: Constituents:
1. Water 1000.0 cc. 1. Distilled water 3000.0 cc.
2. Peptone (Chapoteaut) 40.0 g. 2. Peptone 30.0 g.
3. NaCl 5.0 g. 3. Dextrose 138.0 to 150.0 g.
Constituents
1. Water 1000.0 cc.
2. Peptone 20.0 g.
3. Glucose 5.0 g.
4. NaCl 50 g.
5. CaCOs
Preparation
(1) Dissolve 2 and 4 in 1.
Constituents
1. Water 00.0 cc
2. NaCl
3. KCl
4. CaCls
5. Sodium bicarbonate.
6. Glucose
7. Peptone (1.0 or
2.0%) 10.0 or 20.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7
in 1.
Constituents
1. Distilled water 1000.0 cc.
2. Lactose 300.0 g.
3. Peptone
Preparation
(1) Distribute distilled water in 25.0 cc.
quantities in small flasks that have
been sterilized at 140C.
(2) Add 5.0 g. of lactoseand a small
amount (exact amount not given) of
peptone to each flask.
Sterilization: Sterilize in flowing steam on
3 successive days for ^ to 1 hour.
Use: To studj^ fermentation by B. lactis.
Reference: Baginsky (1888 pp. 434-462).
(1) Dissolve 75.0 g. peptone and 50.0 g. Reference: Fischer and Andersen (1912
NaCl in 600.0 cc. water. p. 289).
(2) Filter.
(3) Dissolve 75.0 g. lactose in 500.0 cc. 611. Baginsky's Lactose Peptone Salt
made according to
litmus solution, Solution
Kubel and Tiemann with Kahlbaum's Constituents:
litmus (method or reference not
1 Distilled water 750.0 cc.
given). 2. Lactose 36.0 g.
(4) Boil (3) for 15 minutes. 3. Peptqne (siccum) 8.0 g.
(5) Filter. 4. K2HPO4 1-6 g.
(6) Add NaOH or soda solution to neu- 5. CaCl, 0.15 g.
tralize if necessary. MgS04 0.3 g.
6.
(7) Mix sterile (2) and (6) when ready 7. CaCOs 0.02 g.
for use. Preparation
(8) Tube. (1) Dissolve 2, 3, 4, 5, 6 and 7 in 1.
Sterilization: Method of sterilization of
Erlenmeyer
(2) Distribute into sterile
(2), (6) or (8) not given. flasks.
Use: Presumptive test for B. coli in water Sterilization: Sterilize in flowing steam for
analysis. Ten under in-
parts water hour.
4 days for | to 1
vestigation were added to one part of Use: To study fermentation by B. lactis.
medium. B. coli turned the medium red. Reference: Baginsky (1888 pp. 434-462).
Reference: Olszewski and Kohler (1922
p. 305). Sucrose
612. Bendick's Phenolphthalein
609. Bacto Neutral Red Medium Solution
(Dehydrated) Constituents:
Constituents: 1. Water 1000.0 cc.
Use: Detection of the cholera vibrio. The Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
cholera vibrio decolorized the medium. Sterilization: Not specified.
References: Bendick (1911 p. 907), (1912 Use: To study the effect of constituents of
p. 536), Park, Williams and Krumwiede the medium on the growth of yeast.
(1924 p. 130), Abbott (1921 p. 570). Variants: Bokorny gives the following
variants:
613. Smith's Sucrose Peptone Solution
(a) 50.0 g. of sucrose instead of 200.0 g.
(Owen)
(b) 2.0 g. of MgS04 instead of 1.0 g. and
Constituents added 0.1 g.H2S04.
1. Water 1000.0 cc. (c) Added 0.1 g. (or more) of H3PO4 or
2. Sucrose 100.0 or 200.0 g. lactic acid.
3. KCl 5.0 g. (d) Added 0.1 g. of HF or formaldehyde
4. Na2HP04 2.0 g. (e) Added 1.0 g. of formaldehyde, NaCl
5. Peptone 1-0 g. CaCl2 or caffeine.
Preparation (f) Used 1.0 g. peptone, 1.0 or 10.0 g
(1) Dissolve 2, 3, 4 and 5 in 1. sucrose, 1.0 g. KH.2PO4 and 0.5 g
(2) Distribute in 200.0 cc. quantities in MgS04 instead of amounts indicated
300.0 cc. Erlenmeyer flasks. in original medium and added 0.5 g
(3) After final sterilization adjust to pH CaCl2 and a trace of FeCU.
values varying from pH = 6.7 to 8.5 (g) Used 1.0 g. peptone, 1.0 or 10.0 g
by the addition of normal NaOH sucrose 1.0 g. KH2PO4 instead of
and H2SO4. The medium should still amounts indicated, omitted the
be warm. MgS04 and added 0.5 g. K2SO4, a
Sterilization: Sterilize on each of 3 suc- trace of FeCl2 and 0.5 g. of KCl or
cessive days for 30 minutes. CaCl2.
Use: To study gum formation. Author (h) Used 50.0 g. sucrose, 10.0 g. peptone,
reported the optimum pH between 6.7 5.0g.KH2PO4 or NaH2P04 and 5.0 g.
and 7.0. of MgS04 instead of the amounts
Variants: Invertase may be added to invert given in the original medium and
the sugar. Levan formation was not added 0.0 or 5.0 g. Rb2S04.
aided. (i) Used 100.0 g. sucrose, 25.0 g. peptone,
Reference: Owen (1923 pp. 423, 431). 4.0 g. KH2PO4 instead of given
amounts,
614. Breaudat's Sucrose Peptone Solution
(j) Used 1.0 g. peptone, 10.0 g. sucrose,
Constituents: 1.0 g. KH2PO4, and 0.5 g. MgS04
1. Water 1000.0 cc. instead of the given amounts, and
2. Peptone (Chapoteaut) 10.0 g. added a trace of iron chloride with
3. Sucrose 30.0 g. 0.5 g. of CaCl2, 0.5 g. KCl or 0.5 g.
4. K2CO3 4.0 g. CaCl2 + 0.5g. K2SO4.
Preparation (k) Used 50.0 g. sucrose, 1.0 g. peptone,
(1) Dissolve 2,and 4 in 1.
3 0.5 g. MgS04, omitted the KH2PO4
(2) Distribute in two liter flasks. from the original medium and added
Sterilization: Method not given. a trace of iron chloride, a trace of
Use: Acetone production by J5aci7rMs viola- CaCla with 1.0 g. KH2PO4, 0.1 g.
cius acetonicus. Rb2S04 + 1.0 g. NaH2P04 or 1.0 g.
Reference: Breaudat (1906 p. 877). NaH2P04.
References: Bokorny (1902 p. 58), (July
615. Bokomy's Sucrose Peptone Solution (1903-04 p. 20),
1903), (1903-04 p. 16),
Constituents (1911 p. 182), (1912 p. 128), (1912 p. 12S).
1. Distilled water 1000.0 cc.
616. Mutchler's Sucrose Peptone Solution
2. Sucrose 200.0 g.
3. Peptone (meat) 5.0 g. Constituents:
4. KH2PO4 2.0 g. 1. Water 1000.0 cc.
5. MgS04 1.0 g. 2. Peptone 20.0 g
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 175
Magnusi. 7 and 8 in 1.
Constituents
CULTURE MEDIA FOR CULTIVATION OF MICRO ORG AXISMS 177
Variants: Author used 50.0 g. sucrose in- Use: Substitute for litmus milk.
stead of 20.0 g. and omitted the starch. Variants: Klimmer specified the use of
Reference: Behrens (1898 pp. 775-776). Witte's peptone and used 0.25 g. Kahl-
baum's azolitmin or added 1 or 2 drops of a
631, Dombrowski's Glucose Peptone saturated watery solution of China blue.
Solution He used the medium as a whey substitute.
Constituents References: Harvey (1921-22 p. 109), Klim-
1. Water 1000.0 cc. mer (1923 p. 208).
2. Peptone (Witte) 10.0 g.
634. Miiller, Thurgau and Osterwalder's
3. Glucose 50.0 g.
Malic Acid Peptone Solution
4. K2HPO4 2.0 g.
5. MgS04 10 g. Constituents:
6. NaCl 1.0 g. 1. Water 1000.0 cc.
Preparation 3. H2KPO4 10 g.
676
tion
C3. Ammonium salts of phosphoric acid
B2. Organic nitrogen other than amino
added. acids added.
Hauman's Pectin Peptone Solution. . 655
Ci. Containing urea.
Charpentier's Sucrose Peptone Solu- Urea Peptone Solution.. 677
Hiss' Basal
tion 656
Urea Peptone Solution
Killer's 678
B2. Inorganic nitrogen supplied as nitrate. Cunningham's Urea Peptone Solu-
Ci. Additional organic carbon supplied as tion 679
carbohydrates. C2. Containing hippuric acid.
Di. Monosaccharides added. Ayers and Rupp's Hippurate Peptone
Lustig's Nitrate Peptone Solution.. 657 Solution 680
Stoklasa's Nitrate Peptone Solution. 658 Ayers and Rupp's Pepsin Peptone
Stutzer's Nitrate Glucose Solution. . 659 Solution 681
Duchacek's Nitrate Peptone Solu- C3. Containing Bile Salts.
tion 660 MacConkey's Bile Salt Peptone Solu-
D2. Polysaccharides added. tion 682
Khouvine's Cellulose Nitrate Solu- Harrison and van der Leek's Bile Salt
tion 661 Peptone Solution 683
Omeliansky's Cellulose Peptone Solu-
tion (Khouvine) 662 648. Jones' Glucose Peptone Solution
C2. Additional Organic carbon supplied as Constituents:
alcohols or organic acids. 1. Water 1000.0 cc.
Maassen's Nitrate Peptone Solution. 663 2. NH4CI 5.0 g.
Wassermann's Nitrate Peptone Solu- 3. Glucose 5.0 g.
tion 664 4. Peptone 10.0 g.
Duchacek's Nitrate Tartaric Acid 5. Calcium lactate 10.0, 20.0, 30.0, 40.0,
Solution 665 50.0, 60.0, 70.0, 80.0, 90.0, or 100.0 g.
A2. Organic nitrogen present in addition to Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
Preparation :
acted as typhoid. Bacillus faecalis alcali-
ditch water genes produced an alkaline reaction.
(1) Dissolve 2, 3, and 4 in
("Grabenwasser"). Reference: Seitz (1912 p. 434).
6. NaCl 5.0 g.
The author reported no growth when
7. Peptone (Witte sice) 0.05 g.
arsenic was present in one part in 1500.
8. Azolitmin (Kahlbaum's) 0.25 g.
Reference: Charpentier (1915 p. 452).
. .
Preparation
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9
in 1. 657. Lustig's Nitrate Pptone Solution
(2) The reaction should be neutral.
Constituents
(3) The medium is bluish violet in
color.
Water 4000.0 cc.
Sterilization: Do not sterilize longer than 1.
(3) Mix 200.0 cc. (1) and 120.0 cc. of (2). Sterilization: Not specified.
(4) Dissolve 0.5 g. peptone, 7.5 g. cal- Use: Cultivation of Bacillus typhi ab-
cium carbonate and 5.72 g. calcium dominalis and Bact. coli commune.
nitrate in (3). Reference: Duchacek (1904 p. 162).
(5) Dilute to 2 liters.
661. Khouvine's Cellulose Nitrate Solution
(6) Distribute in 500.0 cc. lots.
Sterilization: Sterilize in the steamer for Constituents:
3 hours. 1. Water 1000.0 cc.
Use : To study nitrate reduction by Bacillus 2. K2HPO4 5.0 g.
megatherium, {Bacillvs Ellenbachii) or 3. KNO3 2.5 g.
"Alinit." The author reported that the 4. NaCl., 1.0 g.
nitrate was reduced about 8 to 10% after 5. Peptone (pancreatic) 1-0 g.
19 days. Using 1.0 g. of peptone + 6.0 g. 6. Cellulose
186 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents:
1. Distilled water 1000.0 cc.
2. Peptone 10.0 g.
3. Asparagin 5.0 g.
4. 5.0% litmus solution 10.0 cc.
Preparation
)0.0
(1) Dissolve 2, 3 and 4 in 1.
Constituents:
1. Distilled water 1000.0 cc.
2. KH2PO4 (0.3%) 3.0 g.
3. MgS04 (0.01%) 0.1 g.
4. Asparagin (0.3%) 3.0 g.
5. Peptone (1.0%) 10.0 g.
Preparation: (1) Dissolve 2, 3, 4, 5 and
5.0% of one of the added nutrients in 1.
Preparation
2. NaCl (0.5%) 5.0 g.
Tube.
4. MgS04 (0.02%) 0.2 g.
(2)
5. Tryptophane (0.03%) 0.3 g.
Sterilization: Sterilize in Arnold.
6. Peptone (Witte) (0.25%) ... 2.5 g.
Use: To sulphur metabolism of
study
Preparation: (1) Dissolve 2, 3, 4, 5 and 6
fluorescent bacteria, colon typhoid group
in 1.
and others.
Reference: Tanner (1917 p. 586).
Sterilization: Not specified.
Use: Indol production by Bad. pyocy-
aneus, Staphylococcus pyogenes, Bad.
673. Berthelot's Tyrosine Peptone Solution
pneumoniae, Bac. diphtheriae, potato
Constituents bacillus, disciformans and glanders bacil-
1. Water 1000.0 cc. lus. Author reported that the organ-
2. Peptone (Witte) 20.0 g. isms listed above gave positive reactions
3. Glucose 20.0 g. for indol when using the Salkowski
4. Tyrosine 10.0 g. (H2SO4 and nitrites) test. Ehrlich's test
Preparation: (1) Dissolve 2, 3 and 4 in 1. gave negative indol reaction for these
Sterilization: Not specified. organisms.
Use: Production of aromatic hydroxy acids Reference: Frieber (1921-22 p. 264).
and the study of phenol production by
676. Harvey's Tryptophane Peptone Solution
Bacillus phenologenes.
Variants: The author used the following Constituents:
variants to study phenol production: 1. Distilled water 1000.0 cc.
(a) 2.0 g. tyrosine in 1000.0 cc. of peptone 2. Ammonium lactate 5.0 g.
water. 3. Na2S04 2.0 g.
(b) 2.0 g. tyrosine and 10.0 g. glucose in 4. MgS04 0.2 g.
1000.0 cc. peptone water. 5. Peptone 30.0 g.
Reference: Berthelot (1909 p. 87), (1918 6. Tryptophane 1:1000 soln. 0.1, 0.3 or
Constituents: Constituents:
1. Water 1000.0 cc. 1. Distilled water 1000.0 cc.
2. Peptone 10.0 g. 2. Peptone (Park Davis) 10.0 g.
3. Urea 100.0 g. 3. Pepsin 5.0 g..
682. MacConkey's Bile Salt Peptone (f) Levine (1921) omitted the litmus,
Solution used 1.0% peptone, used ox bile or a
Constituents: 1.0% solution of dry ox bile instead
Water 1000.0 cc. of sodium taurocholate and used
1.
5.0 g. glucose or 5.0 g. lactose. Levine (1921 p. 110), Giltner (1921 p. 382),
(c) MacConkey (1908) specified the use Harvey (1921-22 pp. 89, 90, 109), Klimmer
of distilled water, Witte's peptone, (1923 p. 214), Cunningham (1924 p. 103).
used 2.5 cc. 1.0% neutral red
of a
683. Harrison and van der Leek's Bile Salt
solution instead litmus, added
of
Peptone Solution
0.3 g. CaCl2 and added one of the
following: Constituents:
glucose 5.0 g 1. Water 1000.0 cc.
Use: Presumptive test for colon group in A2. Bacterial Derivatives Added.
water analysis. Colon bacilli blacken Thjotta's Basal Bacterial Infusion
the medium. Peptone Solution 700
Variants A3. Fungus derivatives added.
(a) Authors used 0.05% aesculin, and Wiegert's Fungus Infusion Peptone
0.3% sodium taurocholate instead of Solution 701
amount given above. A4. Derivatives of other plants added.
(b) Authors used 0.25% iron citrate, and Bi. Worts, malt or other derivatives added.
0.25% sodium taurocholate. Will and Wanderscheck's Sulphur
(c) Giltner used 0.1 g. aesculin instead of Wort Solution 702
1.0 g. aesculin. Gottheil's Mannitol Wort Solution. 703 .
References: Harrison and van der Leek de Kruyff's Malt Extract Peptone
(1908 p. 312), (1909 pp. 549, 616, 622), Solution 704
Giltner (1921 p. 382). Zikes'Wort Peptone Solution 705
Peklo's Wort Peptone Solution 706
SUBGROUP I-C. SECTION 11
B2. Materials other than worts or malts
(b) The author omitted the Na2HP04 and Added nutrients: The author added 0.5 cc.
used 10.0 g. of peptone instead of of one of the following fats or oils to each
and added 10.0 g. of lactic acid
20.0 g. 50.0 cc. lot of the basic solution:
form of sodium lactate.
in the Sesame Chia seed
Reference: Sherman (1921 p. 129), (1921 Chaulmoogra Mustard seed
p. 383). Castor Okra seed
Corn Linseed
685. Cannon's Basal Yeast Peptone
Rape seed _ , fOpen kettle
Solution
Lumbang \ Steam refined
Constituents Cocoanut Butterfat
1. Tap water 1000.0 cc.
p frefined Cod liver
2. Peptone (Armours) 10.0 g. \ crude Mineral and 2%
1
3. Peptone (Difco) 10.0 g. ing yeast infusions and used them in-
Preparation stead of the solution obtained from step
(1) Add 10.0 g. yeast to 200.0 cc. distilled (7) as above in preparing media,
water, allow to stand about 1 hour, (a) This medium should contain a con-
shaking before filtering. siderable quantity of water soluble B
(2) Add 50.0 g. Lloyd's Reagent (Fuller's vitamin.
earth) and HCl to make solution (1) Take the filtrate from step (6) above
0.01normal HCl. and concentrate to 200.0 cc.
(3) Shake every ^ hour for four hours and (2) Add sufficient 95.0% alcohol to give
filter. a 79.0% alcohol solution.
(4) Make up filtrate to 1000.0 cc. (3) Allow to stand over night at 5C.
194 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Clarify by repeated filtering thru (5) When inoculating add a few drops of
paper, by addition of serum, or egg brom cresol purple.
white, or by the addition of 5.0 cc. Sterilization: Sterilize at 15pounds pres-
liquid Ferric oxychlorate, and then sure for 30 minutes in the autoclave.
filtering. Use: To determine fermentation of xylose.
(3) Add 1.0% peptone but no NaCI as the Reference: Fred and Peterson (1920 p. 540).
extract contains 50.0% NaCl.
697. Ventre's Basal Yeast Ash Peptone
Sterilization: Not specified.
Solution
Use: Meat infusion substitute for the
preparation of special media, Endoagar, Constituents
lactose litmus agar, etc. Do not heat 1. Water 1000.0 cc.
over 100C. or a brown color and a pre- 2. Glucose 170.0 g.
cipitate will develop. 3. Malt dust extract 2.0
Reference: Ickert (1918 p. 186). 4. Peptone (malto peptone).. 0.5
5. Yeast ashes 0.5 g.
695. Ickert's Yeast Infusion Peptone (NH4)2HP04
6. 1.0 g.
Solution
7. K0CO3 1.85 g.
Constituents: Preparation
1. Water 1000.0 cc. (1) Dissolve 2, 3, 4, 5 and 6 in 1.
2. Pressed yeast 30.0 g. (2) Add one of the added nutrients and
3. Peptone 10.0 g. 1.85g. of K.COsto (1).
4. NaCI 5.0 g. Sterilization: Sterilize by heating in the
Preparation: autoclave at 120C., by filtration thru a
(1) Suspend 30.0 g. (40.0 g. when pre- candle or by treating with H..SO4 (250 mg.
paring agar medium) of pressed yeast H2SO4 per liter) (details of method not
in 1 liter of water. given).
(2) Allow to infuse for one hour. Use: Cultivation of yeast.
(3) Boil for 2 hours in the steamer. Added nutrients and variants:
(4) Clarify by the addition of 5.0 cc. of (a) The author added one of the fol-
ferric oxychlorate while hot, and filter lowing:
twice thru a double filter. tartaric acid 4.0 g.
(5) Add 0.5% NaCI and 1.0% peptone. malic acid 5.0 g.
Sterilization: Not specified. citric acid 0.5 g.
Use: Meat substitute for the
infusion (b) Used the basic solution without the
preparation of special media, Endo, lit- addition of other materials.
mus lactose agar, etc. Do not heat over Reference: Ventre (1914 p. 198).
100 C. or a brown color and a precipi-
698. Henneberg's Glucose Yeast Infusion
tate will form.
Solution
Reference: Ickert (1918 p. 186).
Constituents:
696. Fred and Peterson's Yeast Infusion
1. Yeast infusion (1.0%) 1000.0 cc.
Peptone Solution
2. Peptone (1.0%) 10.0 g.
Constituents: 3. Glucose (5.0%) 50.0 g.
1. Fresh yeast water extract. 1000.0 cc. Preparation:
2. K2HPO4 5.0 g. (1) Prepare a 1.0% yeast infusion
3. Difco peptone 5.0 g. (method not given).
4. Xylose 20.0 g. (2) Dissolve 2 and 3 in (1).
5. Brom cresol purple Sterilization: Not specified.
196 CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS
Use: Cultivation of lactic acid bacteria. Added nutrients: The author added 1.0%
Reference: Henneberg (1903 p. 8). of one of the following materials:
lactose mannitol
699. Dietrich's Glycerol Yeast Infusion
raffinose inulin
Peptone Solution maltose salicin
Constituents: glucose sucrose
1. Water 1000.0 cc. Reference: Thjotta (1921 p. 770).
2. Peptone (Witte) (1.0%) .... 10.0 g.
701. Wiegert's Fungus Infusion Peptone
3. Yeast (dry 1.0%) 10.0 g.
Solution
4. Glycerol 3.0% 30.0 g.
Preparation Constituents:
(1) Add 1.0% dry yeast to a 1.0% Witte 1. Water 1000.0 cc.
peptone solution. 2. Fungus 250.0 g.
(2) Boil 2 or 3 times in the autoclave. 3. Peptone 10.0 g.
(5) Suspend a 24 hour growth of B. pro- Use: Study of hydrogen sulphide produc-
teus on agar plates, in normal saline tion by yeast. Authors reported that the
solution using 1.0 cc. for each plate. addition of peptone to beer wort con-
(6) Collect in a sterile centrifuge tube. taining sulphur did not increase the H2S
(7) Boil 5 minutes, centrifuge and pipette
production.
off the supernatent fluid. (Reaction Reference: Will and Wanderscheck (1906
pH = 7.6.) p. 308).
not change its value for promoting Preparation: (1) Dissolve 2, 3 and 4 in 1.
Use: General culture medium for organ- 707. Otabe's Wheat Infusion Peptone
isms found in the soil, on roots and Solution
rhizomes. Constituents:
Reference: Gottheil (1901 p. 432). 1. Distilled water 1600.0 cc.
2. Wheat 1.0 pound
704. de Kruyff's Malt Extract Peptone 3. Diastase 0.5 g.
Solution 4. Peptone 10.0 g.
Constituents: 5. NaCl 5.0 g.
1. Water 1000.0 cc. Preparation
2. Malt extract (Merck) 7.0 g. (1) Roast wheat (with or without husks)
3. Maltose 10.0 g. in an iron pan until it becomes
4. Glucose 10.0 g. brown.
5. Peptone 0.5 g. (2) Put 1 pound of (1) into 1600.0 cc. of
6. Asparagin 0.5 g. distilled water. Do not wash the
Preparation wheat.
(1) Dissolve 2, 3, 4, 5 and 6 in 1. (3) Boil in a Koch boiler for 30 minutes.
(2) After sterilization add H3PO4 so that (4) Strain thru a clean cloth.
100.0 cc. of the medium will neu- (5) Make up to 1000.0 cc. with distilled
tralize 3.0 cc. of N/1 NaOH. water if the volume is below this
Method not given.
Sterilization: quantity.
Use: Enrichment of yeast Saccharomyces (6) Add 0.5 g. of taka-diastase or ordi-
javanicus. nary diastase and shake the flask
Reference: de Kruyff (1908 p. 617). well.
(7) Keep at 30 to 40C. for 30 minutes.
705. Zikes' Wort Peptone Solution (8) Filter. The filtrate should be quite
transparent with a yellowish color,
Constituents:
nearly the same as meat broth. It
1. Wort 1000.0 cc.
has a sweet smell and of a slightly
2. Glucose (5.0%) 50.0 g.
alkaline reaction.
3. Peptone (1.0%) 10.0 g.
(9) Add 5.0 g. NaCl and 10.0 g. peptone
Preparation: (1) Dissolve 5.0% glucose and
albumin.
1.0% peptone in wort.
(10) Boil and filter.
Sterilization: Not specified.
(11) To prepare wheat agar add 15.0 g.
Use: Cultivation of Apiculatus yeast,
(in winter) and 20.0 g. (in summer) of
Torula alba, Torula Molischiana, MycO'
agar to the filtrate of (10). Prepare
derma cerevisiae, Blastoderma salmoni-
almost as in the same manner as in
color.
the preparation of meat agar (de-
Reference: Zikes (1911 p. 147).
tails not given).
Sterilization: Not specified.
706. Peklo's Wort Peptone Solution
Use: Substitute for meat infusion medium.
Constituents: May be used as a basis for special media.
1. Distilled water 200.0 cc. Reference: Otabe (1919 p. 576).
2. Beer wort 800.0 cc.
3. K2HPO4 708. Ayers and Mudge's Cabbage Infusion
15.0 g.
4. K2CO3 Peptone Solution
2.0 g.
5. MgS04 1.8 g. Constituent^
6. Peptone (Witte 0.5%) 5.0 g. 1. Water 1000.0 cc.
Preparation 2. Peptone (Difco) 20.0 g.
(1) Mix 1 and 2. 3. Cabbage 100.0 g.
(2) Dissolve 3, 4, 5 and 6 in 1. Preparation
Sterilization: Not specified. (1) Soak 100.0 g. finely minced cabbage in
Use: Cultivation of plant actinomyces. 300.0 cc. distilled water in ice box for
Reference: Peklo (1910 p. 551). 48 hours.
198 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Steam 30 minutes and filter. Preparation: (1) Dissolve 5.0% glucose and
(3) Distribute 2.0% Difco peptone solu- 1.0% peptone inmust.
tion into a series of flasks in 50.0 cc. Sterilization: Not specified.
quantities. Use: Cultivation Torula alba,
of yeasts,
(4) Add varying amounts of filtrate of Torula Molischiana, Mycoderma cerevisiae,
(2) 1.0 cc, 5.0 cc. and 50.0 cc. Blastoderma salmonicolor.
(5) Make each flask to 100.0 cc. volume Reference: Zikes (1911 p. 147).
with water.
(6) Adjust to pH = 7.2. 711. Henneberg's Prune Infusion Peptone
Steam and filter. Solution
(7)
(8) Tube. Constituents
Sterilization: Method not given. 1. Prune infusion (1.0%) 1000.0 cc.
Use: To study promoting sub-
growth 2. Peptone (1.0%) 10.0 g.
stances for streptococci. Author re- 3. Glucose (5.0%) 50.0 g.
ported that the more cabbage extract Preparation
added, the better the growth. (1) Method of preparation of 1.0% prune
Reference: Ayers and Mudge (1922 p. 455). infusion not given.
(2) Dissolve 2 and 3 in 1.
709. Kaufmann's Jequirity Seed Infusion Sterilization: Not specified.
Peptone Solution Use: Cultivation of lactic acid bacteria.
Reference: Henneberg (1903 p. 8).
Constituents:
1. Water 1000.0 cc. 712. Miquel's Wood Ash Peptone Solution
2. Jequirity seeds. 100.0 g. (Besson)
3. Peptone 20.0 g. Constituents:
4. NaCl 5.0 g. 1. Water 1000.0 cc.
Preparation 2. NaCl 5.0 g.
(1) Grind 100.0 g. Jequirity seeds in a 3. Peptone (Chapoteaut) 20.0 g.
mortar. 4. Wood ashes 0.1 g.
(2) The peeled or shelled seeds now weigh Preparation
about 80.0 g. (1) Dissolve 2 and 3 in 1 by heating.
(3) Add to 1000.0 cc. of water. (2) Add 0.1 g. of wood ashes to (1).
(4) Boil in a steam sterilizer for about (3) Boil.
2 hours. (4) Filter thru paper.
(5) Cool and filter.
(5) Add tartaric acid until the solution is
(6) Dissolve peptone in (5). neutral to litmus.
(7) Add 8 drops of concentrated soda (6) Boil 5 minutes.
solution to each 100.0 cc. of (6). (7) Filter.
(8) Distribute into test tubes. (8) Make up to 1000.0 cc.
Sterilization: Sterilize in the usual manner (9) Tube.
(Method not given). Sterilization: Sterilize at 113C.
Use: General culture medium. Bacillus
Use: General culture medium.
pyocyaneues and many other organisms Reference: Besson (1920 p. 30).
grew very well.
Variants: The author gave the following SUBGROUP I-C. SECTION 12
Constituents
4. NaCl 10.0 g.
Preparation
1. Distilled water 1000.0 cc.
(1) Boil 2.5 liters of water with 1250 kg.
2. Sheep brain 1000.0 cc.
finely chopped peptone
beef, 25.0 g.
3. Peptone 40.0 g.
and 10.0 g. NaCl streaming steam.
in
4. Dextrose. ^ . . 20.0 g.
Preparation (2) Make slightly alkaline by the addi-
tion of soda.
(1) Boil sheep brains with an equal
Sterilization: Digest in the steamer until
volume of distilled water.
the meat extract is germ free.
(2) Decant water (save) and press brains
thru a potato ricer.
Use: Tetanin production. When the me-
dium has cooled following digestion,
(3) Add 2.0% peptone and 0.1% glucose
inoculate and replace the air in the con-
to the water decanted from (2).
tainer by hydrogen.
(4) Mix (3) and the brain tissue after it
Reference: Kitasato and Weyl (1890 p. 405),
has been passed thru a potato ricer.
(5) Tube by punching thru the filling 718. Jablon and Pease's Liver Peptone
funnel with a glass rod, filling tubes Medium
about half full.
Constituents:
Sterilization : Sterilize intermittently in the
1. Distilled water 1000.0 cc.
Arnold sterilizer. Five daily runs of
2. Peptone 10.0 g.
30 minutes each are recommended.
Use: To enrich anaerobes. Growth indi-
3. NaCl 5.0 g.
4. Liver (rabbit, beef or human)
cated by turbidity and often gas pro-
Preparation
duction.
(1) Dissolve 2 and 3 in 1 by boiling
Reference: Hall (1920 p. 579).
30 minutes.
716. Hall and Peterson's Meat Mash (2) Neutralize to phenolphthalein.
Peptone Medium (3) Add 20.0 cc. of a normal NaOH solu-
Constituents
tion. The reaction must be a minus 2.
(4) Autoclave for 15 minutes at 115.
1. Water 1000.0 cc.
(5) Filter.
2. Ground meat 1.0 lb.
NaCl 5.0 g. (6) Tube in 10.0 cc. lots.
3.
Store sterile (2) as a stock solution. Use: Cultivation of lactobacilli. The au-
(3)
Dissolve 2 and 3 in 1 liter of distilled thors report that the 0.1% agar is not
(4)
water. essential but seems to favor the growth
Sterilization: Sterilize (2) and (5) by heat- of any desired fermentable carbohydrate.
ing for one hour at 100C. Variants: Lard and mineral oil were sub-
Use: Enrichment of V. cholera. stituted for butterfat.
Reference: Harvey (1921-22 p. 86). Reference: Sherman and Albus (1922 p. 17).
721. Weissenbach's Albumin Peptone 723. Weiss and Wilkes-Weiss' Egg Solution
Solution Constituents:
1. Water 200.0 cc.
Constituents:
Peptone (Difco) (1.0%) 2.0 g.
1. Distilled water 2.
Sodium phosphate (0.2%) ... 0.4 g.
2. Peptone (Chapoteaut) 4.0 g. 3.
Agar (0.2%) 0.4 g.
3. NaCl 0.5 g. 4.
Eggs 2
4. Glucose 0.2 g. 5.
(1) Dissolve 2 and 3 in 100.0 cc. dis- eggs with sterile precautions.
tilled water. (2) Dissolve 2, 3 and 4 in 1.
(2) Make slightly alkaline to litmus. (3) Emulsify (1) in (2).
(3) Autoclave at 120C. for 15 minutes. (4) Adjust to pH = 7.8 and bring slowly
(4) Filter and add 0.2 g. glucose to the
to a boil, stirring frequently.
filtrate. (5) Tube in spirochete tubes.
Sterilization: Sterilize in the Arnold
for
(5) Add slowly one volume of egg white
sterile
to three volumes distilled water. 30 minutes and seal with 1 cm. of
Shake but do not cause the mixture yellow petrolatum while the tubes are
to foam. still hot.
202 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Use: Cultivation of Spirochaeta pallida. ing the water, clot serum mixture
Reference: Weiss and Wilkes-Weiss (1924 (see (4) above) at 50C. for 20 minutes
p. 222). and then boiling 10 minutes instead of
5, boiled 5 minutes after the addition
724. Kelser's Blood Infusion Peptone
of the glacial acetic acid (see (9)
Solution
above), steamed the peptone NaCl
Constituents mixture (see (11) above) for 45
1. Distilled water 1000.0 cc. minutes instead of heating only until
2. Beef blood 500.0 g. solution was complete and adjusted
3. Peptone 15.0 g. the reaction to a definite pH value,
4. NaCl 7.5 g! faintly alkaline to litmus or 1.0%
Preparation acid to phenolphthalein. After ad-
(1) Allow beef blood to clot in covered justing the reaction the medium was
vessel. Let stand in refrigerator to steamed
for 30 minutes, filtered while
allow serum to separate from clot. hot thru wet thick filter paper and
(2) Remove clot and chop finely in tubed.
chopping machine. References: Kelser (1916 pp. 615, 616)^
(3) Mix serum and ground clot. Weigh. Harvey (1921-22 pp. 69, 76).
(4) Add two volumes of distilled water.
(5) Boil gently for 5 minutes. 725. Wiens' Blood Peptone Solution
(6) Filter thru cheese cloth. Take resi-
(Klimmer)
due and put thru fruit press to ex- Constituents:
tract as much fluid as possible, 1. Water 1000.0 cc.
lining presswith towel or heavy 2. Peptone (10.0%) 100.0 g.
cloth so that pulp does not pass thru. 3. Glucose (1.0%) 10.0 g.
(7) Discard the residue. 4. Blood.. 100.0 cc.
(8) Boil fluid, skimming off coagulated Preparation
proteins. (1) Prepare a 10.0% peptone solution in
(9) Add concentrated CH3COOH (0.5 cc. water.
per L) to cause flocculation and con- (2) Make slightly alkaline.
tinue to boil 5 minutes. (3) Dissolve 1.0% glucose in (2).
(10) Filter first thru absorbent cotton (4) Tube in 10.0 cc. quantities.
and two filter papers. (5) Add 1.0 cc. of blood to each tube.
(11) Ascertain volume and add 0.5% Sterilization: Not specified.
NaCl, and 1.0% peptone. Heat to Use: Cultivation of pneumococci.
get into solution. Reference: Klimmer (1923 p. 221).
(12) Neutralize with NaOH.
Sterilization: Sterilize in autoclave i hour 726. Kligler's Peptone Serum Blood
under 12 pounds pressure. Medium
Use: Nutrient medium for pathogenic Constituents
forms. The author reported that glucose 1. Saline 200.0 cc.
aided the growth of some organisms. 2. Serum (horse or rabbit) 100.0 cc.
Variants 3. Blood
(a) Kelser added 0.25% glucose. 4. Peptone (1.0%) 3.0 g.
(b) Harvey prepared a medium, boiling Preparation:
the water, clot and serum mixture (1) Dilute horse or rabbit serum 1:2 with
(see (5) above) for 10 minutes instead saline.
of 5, specified glacial acetic acid, (2) Adjust to pH 7.0.
steamed the peptone NaCl mixture (3) Add 1.0% peptone (1.0 cc. of a 10.0%
(see (11) above) for 45 minutes, and solution per 10.0 cc. medium).
adjusted the reaction to a definite (4) Distribute in 3 to 4.0 cc. lots in Was-
pH value or faintly alkaline to litmus, sermann tubes.
or 1.0% acid to phenolphthalein. (5) Add 0.1 cc. of blood. (For initial
(c) Harvey prepared a medium by heat- cultures, add infected blood. For
203
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
sufficient Kahlbaum's litmus to give (d) Park, Williams and Krumwiede pre-
the desired color. pared the medium as follows:
Sterilization: Sterilize for 15 minutes on (1) Dilute serum with 2 or 3 times its
volume of distilled water.
each of 3 or 4 successive days in streaming
the Arnold.
(2) Sterilize (1) in
steam.
Use: To study fermentation ability of (3) Prepare a 10.0% solution of pep-
pneumococci, gonococci, streptococci, etc. tone.
(4) Sterilize (3), method not given.
Added nutrients and variants:
(a) Buerger added 1.0% inulin. (5) Prepare a 10.0 or 20.0% solution
Ruediger added 5.0 g. NaCl, used of any desired carbon source or
(b)
20.0 g. Witte's peptone and prepared
fermentable material.
the medium as follows: (6) Heat (5) in small containers in the
Witte's Arnold sterilizer for 30 minutes on
(1) Dissolve 5.0 g. NaCl, 20.0 g.
each of 3 successive days. When
peptone and 2.0% inulin or other
carbohydrate in a liter of water. using inulin, sterilize in the auto-
Add 20.0 cc. litmus. clave.
(2)
Tube in 2.0 cc. lots. (7) Add, under aseptic conditions, suffi-
(3)
1 .0% peptone
cient (4) to (2) to give a
(4) Sterilize in the autoclave if using
inulin; using other carbohydrates
concentration.
sterilize intermittently. (8) Add, under aseptic conditions, suffi-
Collect beef serum without special cient (6) to (7) to give a 1.0% con-
(5)
precautions and dilute with an centration of carbon source. (Gen-
equal volume of water. erally 5.0% glycerol is employed
6. Lactose 75.0 g.
22 p. 89).
Preparation
733. Jackson and Melia's Bile Peptone (1) Dissolve 75.0 g. peptone and 50.0 g.
Solution NaCl in 500.0 cc. of bile by boiling.
1. Bile (sterile fresh ox) 1000.0 cc. (3) Dissolve 75.0 g. lactose in 600.0 cc.
Peptone 100 g- litmus solution prepared according to
2.
3. Lactose 10.0 g. Kubel and Tiemann with Kahlbaum's
Preparation litmus (method or reference not
given).
(1) Dissolve 2 and 3 in 1.
Boil (3) for 15 minutes.
(2) Tube in fermentation tubes.
(4)
Sterilization: Method of sterilization of ox (5) Filter and add NaOH or soda solu-
tion to neutralize if necessary.
bile or final medium not given.
Mix sterile (2) and sterile (5) before
Use Enrichment medium in water analysis.
:
(6)
bile in a liter of water instead of B. coli. Add 10 parts of the water under
1000.0 cc. of fresh bile. investigation to 1 part of the medium.
(4) Add 6.0 cc. of glacial acetic acid (2) Inoculate with a culture of B. coli.
(10) Add the whites of 3 eggs to clear. Reference: Kan-Ichiro Morishima (1921
(11) Filter thru cotton and paper. p. 277).
5. NaCl 1.0 g.
745. Heller's Urine Peptone Solution
6. K2HPO4 10 g.
Wroblewski's Suprarenal Capsule In- Wilcox's Glucose Veal Infusion Broth. 823
fusion Broth 795 Park, Williams and Krumwiede's
Harvey's Organ Infusion Broth 796 Glycerol Veal Infusion Broth 824
Kligler's Blood Clot Infusion Broth. 797 E2. Additional constituents of unknown
Boyer's Bone Infusion Broth 801 Thjotta & Gunderson's Blood Veal
Infusion Solution 828
Ball's Animal Infusion Broth 802
Robinson and Meader's Liver Veal
D2. Extracts specified.
Infusion Broth 829
El. Containing nitrates.
Starin and Dack's Casein Digest Veal
Stutzer's Nitrate Extract Broth.... 803
Infusion Broth 830
Davis and Ferry's Nitrate Bouillon
Bacto Veal Infusion Medium (De-
Extract Broth 804
hydrated) 831
Ej. Not containing nitrates.
Bruschettinis' Blood Egg Broth
Fi. Sodium chloride added. (Klimmer) 832
Debrand's Extract Broth 805
D2.t Liver infusion employed.
Barman and Rettger's Extract Pro- Jackson and Muer's Glucose Liver
teose Solution 806
Infusion 833
Guth's Selenium Extract Broth 807
834
Haslam's Brain Liver Infusion Broth.
Herman and Rettger's Salt Extract D3. Heart infusion employed.
Broth 808
Mueller's Heart Infusion Aminoid
Herman and Rettger's Trypsinized Solution 835
Extract Broth 809
Mueller's Heart Infusion Peptone
Olszewski and Kohler's Trypsinized Solution 836
Extract Broth 810 Huntoon's Hormone Heart Infusion
Bacto Cooledge Broth (Dehydrated) . 811 Broth 837
Fj. Sodium chloride not added. MacNoughton's Blood Infusion Broth. 838
Heinemann's Bouillon 812 D4. Beef (meat) infusions employed.
Wyant's Bouillon Cube Broth 813 El. All additional constituents of known
D3. Whether infusions or extracts employed composition.
not specified.
El. Nitrates added. * See next page for C2
Heinemann's Nitrate Broth 814 t See D3 to Ds.
212 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Ficker and Hoffmann's Caffeine In- Wade and Manalang's Blood Infusion
fusion Broth 839 Broth 866
Ayers and Rupp's Hippurate Infusion Harvey's Ascitic Fluid Infusion Broth. 867
Broth 840 Stryker's Serum Infusion Broth 868
MacConkey's Bile Salt Infusion Foster's Serum Infusion Broth 869
Broth (Heinemann) 841 Beach and Hasting's Serum Infusion
Fz. Not containing additional organic and Extract Broth 870
nitrogen. Harvey's Blood Infusion Broth 871
F4. Other materials of unknown chemical
Gi. Carbohydrates added.
Smith's Glucose Infusion Broth 841a composition used.
Mueller's Meat Infusion Broth 842 Park, Williams and Krumwiede's
Robinson and Rettger's Glucose In- Potato Infusion Broth 872
Harvey's Starch Infusion Broth.... 850 Menten & Manning's Lactose Bile In-
fusion Solution 880
Loeffler's Malachite Green Infusion
Broth : 851 Hitchen's Glucose Agar Infusion
Duval and Lewis' Inulin Bouillon. 852
. . .
Solution (Mulsow) 881
Kreidler's Trypsinized Broth 882
Gi. Carbohydrates not added.
Bulir's Mannitol Infusion Broth 853 Ds. Infusions other than above employed.
Richardson's Ctocinoma Infusion
Harvey's Ferric Tartrate Infusion
Broth 854 Broth 883
Harvey's Lead Acetate Infusion Siebert's Horse Meat Infusion Broth . 884
Broth
Jordan's Phenol Infusion Broth.... 857
Khouvine's Fecal Infusion Broth... 887
Cutler's Blood Clot Infusion Broth
E2. Chemical composition of at least one
(Stitt) 888
of the additional constituents not known.
Cj.* Extracts specified.
Fi. Egg used.
Di.* All additional constituents of known
Harvey's Egg Infusion Broth 858
chemical composition.
Olitsky and Kligler's Egg White In-
El. Containing additional organic nitrogen.
fusion Broth 859
Percival's Urea Extract Broth 889
Weiss & Wilkes-Weiss' Egg "Hor-
Stutzer's Dinitro Benzol Extract
mone" Broth 860
Broth 890
F2. Animal tissue used.
Harrison and Vanderleck's Aesculin
Kohman's Brain Infusion Medium . . 861
Extract Broth 891
Kreidler's Glucose Brain Broth 862
Sawin's Glycocholate Extract Broth. 892
Kligler's Heart Infusion Solutions. . 863
Olszewski and Kohler's Trypsinized
Havens & Taylor's Kidney & Blood Bile Salt Extract Broth 893
Infusion Broth 864 Frieber's Tryptophane Extract Broth. 894
Robertson's Cooked Meat Medium
(Torrey) 865 * See next page for C3 and Dj.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 213
Hegner and Becker's Blood Agar Thjotta and Avery's Yeast Ascitic
Solution 945 Fluid Bouillon 972
E2.* Unknown constituents of animal origin Thjotta and Avery's Yeast Serum
only. Bouillon 973
Fi. Animal tissue or cells employed. Thjotta and Avery's Yeast Blood
Gi. Blood cells added. Bouillon 974
Thjotta and Avery's Blood Cell Thjotta and Avery's Yeast Blood Cell
Bouillon 946 Bouillon 975
Gj. Heart, kidney or other tissue added. Thjotta and Avery's Yeast Hemo-
Smith, Brown and Walker's Tissue globin Bouillon 976
Bouillon 947 Savini and Savini-Castano's Bacteria
Orr's Glucose Heart Bouillon 948 Blood Bouillon 977
Park, Williams and Krumwiede's
Meat Medium 949 748. Committee A. P. H. A. (1899) Basal
Kligler's Tissue Infusion Bouillon.. 950 Infusion Broth
G3. Eggs or derivatives added.
Capaldi's Egg Yolk Bouillon 951 Constituents:
Oberstadt's Egg Albumin Bouillon. . 952 1. Distilled water 1000.0 cc.
Egg Albumin Bouillon. 953
Lipschiitz's . 2. Meat 500.0 g.
Kahn's Casein Digest Egg Albumin 3. Peptone (1.0%) 10.0 g.
(8) Heat (7) in small containers in the Sterilization: Method of sterilization not
Arnold sterilizer for 30 minutes on given. Sterilize in large Erlenmeyer
3 successive days. (Sterilize the flasks before filtering.
inulin solution in the autoclave.) Use: To study the fermentation of sugars,
(2) Add a culture of B. coli and incubate (3) Add 1.0% peptone and 0.5% NaCl.
the mixture at 37 for 24 hours. (4) Sterilize at 15 pounds for 15
(5) Press out meat, and the remaining solution prepared by dissolving
meat mixed again with water and sugar in sterile water and heating
pressed a second time. in autoclave for 10 minutes at
(6) Mix both fluids and bring to 10 pounds.
1000.0 cc. volume. (6) Add 5.0 cc. sterile litmus or 5.0 cc.
(7) Dissolve 3 and 4 in boiling (6) of 2.0% phenol red and 1.2 cc. of
(8) Add 5 and boil until the reaction is decolorized 1.0% aqueous solution
about 0.3 acid to phenolphthalein. of china blue per 100.0 cc. of broth.
(Requires from 30 to 60 minutes.) Adjust to pH = 7.0-7.1 by means
(7)
(2) Strain out the meat juice and 750. Hopkins and Lang's Basal Veal
make up to 1000.0 cc. with dis- Infusion Broth
tilled water.
Constituents:
(3) Add a 24 hour broth culture of 1. Veal Infusion 1000.0 cc.
B. coli to (2).
2. Peptone (Witte) 10.0 g.
(4) Incubate at 37C. for 12 to 16 XaCl
3. 5.0 g.
hours no longer. Preparation
(5) Mix 1.0% (10.0 g.) peptone into a
(1) Preparation of veal infusion not
thin paste with a little water and
specified.
add to (4). Dissolve 2 and 3 in (1),
(2)
(6) Heat in the autoclave for 20
Adjust the reaction to -f-0.3.
(3)
minutes or in the steamer for
(4) Dissolve 1.0% of one of the added
one hour.
nutrients in (3).
(7) Neutralize to phenolphthalein.
Sterilization: Method not given.
(8) Boil over a free flame for 3 to
Use: To study fermentation.
5 minutes.
Variants: The author used 2.0% Witte's
(9) Add 1.0% glucose (or any other
peptone instead of 1.0%.
desired sugar) and 10.0 cc. of a
Added nutrients: The author added 1.0%
0.5% solution of neutral red and of one of the following:
stir until the sugar is dissolved.
glucose mannitol
(10) Filter until clear.
lactose inulin
(11) Distribute into fermentation
sucrose soluble starch
tubes.
salicin arabinose
(12) Sterilize in the autoclave or in
raffinose
flowing steam.
Reference: Hopkins and Lang (1914 p. 72).
(e) Baker prepared a Brom thymol blue
medium as follows: 751. Harvey's Basal Carbonate Infusion
(1) Digest meat and water for two Broth
hours. Cook. Filter thru ab-
Constituents:
sorbent cotton and sterilize in
1. Water 1000.0 cc.
autoclave at 18 pounds for 20
2. Beef 500.0 g.
minutes.
3. Peptone 10.0 g.
(2) Inoculate cold broth with Bad.
4. NaCl 5.0 g.
saccharolyte (Rivas), incubate at
5. CaCOa 5.0 to 20.0 g.
37C. for 48 hours.
Preparation
(3) Sterilize in Arnold for 20 minutes.
(1) See Dunham's Meat Infusion Pep-
(4) Add peptone and NaCl. tone Solution variant (bb) 750 for
(5) Adjust to pH = 7.0 with brom- preparation of broth from water,
thymol blue. peptone and beef.
(6) Steam for 20 minutes and readjust (2) Dissolve 1.0% of one of the added
reaction. nutrients in (1).
(7) Filter medium. (3) Add from 5.0 g. to 20.0 g. CaCOj to
(8) Add 12.0 cc. per liter medium of (2).
0.2% alcoholic solution of brom- Sterilization: Method not given.
thymol blue. Tube. Use: To study fermentation.
(9) Sterilize at 18 pounds for 20 Added nutrients: The author added 10%.
minutes. of any desired carbohydrate, alcohol,
(10) Add sterile sugar solution under etc.
aseptic conditions. Reference: Harvey (1921-22 p. 107).
(2) Dissolve 3.0% of one of the added justed to a faint pink using phenol
nutrients in (1). red as an indicator or neutralize to
(3) AddtheCaCOs to (2). phenolphthalein if the reaction does
Sterilization: Method not given. not come between neutral and +1.0.
Use: To study fermentation. Author used The committee used 0.5% carbo-
Friedlander's pneumobacillus. hydrate instead of 1.0% as in 1917.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 219
(b) Rogers, Clark and Davis used 5.0 g. (5) Prepare a fresh 10.0% watery sodium
K2HPO4 instead of 5.0 g. Na2HP04. sulfite solution.
They added 1.0% of one of the (6) Prepare a 0.025% watery chrysoidine
following materials: solution.
glucose rafhnose (7) To each 100.0 cc. of sterile (3) add
levulose starch 5 to 6 drops alcoholic fuchsin, 2.0 cc.
galactose inulin sodium sulfite solution, 0.5 to 1.0 cc.
adonitol mannitol chrysoidine solution and one of the
sucrose glycerol added nutrients.
lactose salicin (8) Distribute into tubes.
dulcitol Sterilization: Sterilize (3); method not
(c) Evans used 5.0 g. K2HPO4 instead given. Sterilize the tubed medium;
of 5.0 g. Na2HP04 and added 1.0% method not given.
of any desired carbohydrate, alco- Use: Differentiation of the colon-typhoid
hol, etc. group.
(d) Sherman and Albus used 3.0 g. beef Added nutrients: The author added 1.0%
extract instead of 4.0 g. and 5.0 g. of one of the following:
K2HPO4 instead of 5.0 g. Na2HP04. glucose xylose
They added 1.0% of one of the levulose arabinose
following test materials: galactose mannitol
glucose inulin maltose glycerol
galactose starch sucrose
levulose glycerol Reference: Stern (1916 p. 483).
maltose mannitol
lactose salicin
758. Winslow, Rothberg and Parsons Basal
sucrose dextrin Extract Broth
raffinose Constituents
They omitted the K2HPO4 when glu- 1. Water 1000.0 cc.
cose, galactose, levulose and maltose 2. Beef extract (Bacto) 3.0 g..
were added. 3. Peptone (Bacto) 5.0 g.
References: Rogers, Clark and Evans 4. Brom cresol purple (0.04%
(1914 p. Ill), Rogers, Clark and Davis alcoholic solution 27.0 cc.
(1914 p. 421), Evans (1916 p. 445), Sher- 5. Carbohydrate 5-0 g.
man and Albus (1918 p. 162). Preparation
(1) Dissolve 2 and 3 in 1.
757. Stern's Basal Fuchsin Sulphite Broth
(2) Add 27.0 cc. of a 0.04% alcoholic solu-
Constituents tion of brom cresol purple.
1. Water 1000.0 cc. (3) pH = 6.7 to 6.8.
2. Peptone 20.0 g. (4) Add 0.5% of one of the nutrients to
3. Meat extract 10.0 g. sterile (3).
4. NaCl 5.0 g. Sterilization: Sterilize at 15 pounds for 15
5. Fuchsin (10.0% alcoholic solution) minutes.
6. Na2S04 (10.0% water solution) Use: To study fermentation of carbohy-
7. Chrysoidine (0.25% solution) drates, alcohols, etc., by bacteria.
Preparation Author used streptococci.
(1) Dissolve 2, 3 and 4 in 1. Added nutrients: The author added 0.5%
(2) Adjust the reaction so that it is basic ofany desired carbohydrate, alcohol, etc.
to phenolphthalein. Reference: Winslow, Rothberg and Par-
(3) Boil and filter. sons (1923 p. 151).
(4) Prepare a saturated alcoholic fuchsin
solution. Add about 10.0 g. fuchsin
759. Elser and Huntoon's Basal NShrstofE
to 100.0 g. alcohol and allow to stand Heyden Broth
in the thermostat for 24 hours. Filter Constituents
and place in dropping glass. 1. Distilled water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 221
(4) Mix sterile (2) and sterile (3). Use: General culture medium and with the
(5) Tube. addition of carbohydrates to study fer-
(6) Incubate for 3 days to detect acci- mentation. This medium conforms to
dental contamination. the "Standard Methods 1920" formula.
Sterilization: Sterilize (2) in the usual Added nutrients:
manner (method not given). Sterilize (a) Digestive Ferments Co. prepare de-
(3) at 100C. for 10 minutes. hydrated glucose and lactose broth.
Use: To study fermentation of carbo- (b) Digestive Ferments Co. prepare au
hydrates, alcohols, etc. by bacteria. The Andrade Basic Medium to which they
author used meningococci. add 0.5% sucrose, lactose, mannitol,
Added nutrients: The authors added 1.0% maltose and glucose. This dehy-
of one of the following: drated medium is the same as the one
glucose maltose given above except that 0.025 g.
sucrose mannitol (0.0025%) of Difco Andrade Indicator
galactose dulcitol is added.
(16) Heat in the Arnold for 15 minutes (7) Prepare 12.0% solutions of one of the
and filter. added nutrients.
(17) Tube in 9.0 cc. quantities. (8) Add 0.5 cc. of sterile (7) to each tube
the added nutrients in the sugar free (9) Incubate 3 days at 37C. to determine
broth (16). sterility.
(19) Add 1.0 cc. of sterile (18) to each Sterilization: Sterilize (5) in the autoclave.
tube of (17).
sterile Sterilize (8) by heating in flowing steam
(20) Cap the tubes with a vaseline cap. at 100C. for 12 minutes.
(21) Heat in the Arnold for 10 minutes. Use: To study fermentation of carbohy-
Sterilization: Sterilize (17) in the auto- drates and alcohols by bacteria. Author
clave. Sterilize (18) by heating in the used gonococci.
Arnold at 100C. for 10 minutes. Added nutrients The authors added 12.0
: g.
sterile ascitic fluid. The fluid stored growth of the causative agent of
in the ice box for a long period to poliomyelitis, pleomorphic strepto-
hydrolyze the fermentable materials cocci, using a medium prepared as
that it might contain. follows:
224 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
successive days for 10 minutes, taking Use: Cultivation of highly parasitic and
care not to overheat. pathogenic bacteria.
Use: Enrichment of lactose fermenters. Added nutrients and variants:
The author reported that the addition of (a) The author mixed glycerol with equal
KI may tend to delay the anaerobic parts of urine, pus, milk, egg white or
growth while not interfere with aerobic other albuminous fluids.
multiplication. (b) The author used a mixture of equal
Added nutrients: The author added 0.5 g. parts of ascitic fluid and glycerol
glucose, 1.0 g. sucrose or 0.5 g. lactose. instead of ascitic fluid in step (5)
Reference: MacConkey (1905 p. 338). above.
Reference: Cantani (1910 p. 472).
770. Klimenko's Basal Glycerol Bouillon
772. Buerger's Basal Ascitic Fluid Bouillon
Constituents:
1. Peptone bouillon 1000.0 cc. Constituents:
2. Glycerol (1 .0%) 10.0 g. 1. Bouillon sugar free 1000.0 cc.
Preparation 2. Ascitic fluid 333.0 cc.
(1) Preparation of peptone bouillon not 3. Litmus
given. Preparation:
(2) Add (1.0%) glycerol to (1). (1) Preparation of sugar free bouillon
(3) Mix equal parts of (2) and one of the not given.
added nutrients. (2) Add litmus solution to (1) until a
Sterilization: Not specified. desirable color is obtained.
Use: Cultivation of whooping cough bacil- (3) Add I volume of sterile ascitic fluid to
lus. Author reported that the organism sterile (2).
grows first on the surface and then on the (4) Add sterilewatery concentrated solu-
bottom of the tube. tions of one of the added nutrients to
Added nutrients: The author mixed equal (3) until a 1.0% strength is obtained.
parts of one of the following and the (5) Tube into sterile tubes.
basic solution: (6) Incubate in incubator to test sterility.
ascitic fluid Sterilization: Method of sterilization of (2)
blood serum not given. Sterilize the solution of
defibrinated blood added nutrients by heating on each of 3
Reference: Klimenko (1909 p. 312). successive days.
Use: To determine the fermentation of
771. Cantani's Basal Ascitic Fluid Bouillon
carbohydrates, alcohols, etc., by bac-
Constituents: teria. Buerger used streptococci.
1. Bouillon Added nutrients and variants:
2. Ascitic fiuid (a) The author added 1.0% of one of the
3. Glycerol following materials:
Preparation arabinose dulcitol
(1) Mix equal parts of glycerol and one of rhamnose sucrose
the added nutrients. Storing the glucose lactose
material with the glycerol tends to levulose maltos
sterilize the material. galactose dextrin
(2) After some time test the sterility mannitol inulin
of (1). (b) Elser and Huntoon prepared a sim-
(3) Exact composition of bouillon not ilar medium using one part ascitic
given. fluid with two parts bouillon. The
(4) Tube (3). procedure was as follows:
(5) Add 6 parts of sterile ascitic fluid to (1) Mix two parts sterile broth (exact
1 part (2). method of preparation or composi-
(6) Add 0.5 to 0.75 cc. of (5) to each tion not given) with one part sterile
sterile tube of (4). ascitic fluid. Method of steriliza-
Sterilization: Method not specified. tion not specified.
226 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Add 3.0 to 4.5 cc. of a watery solu- 774. Hiss' Basal Serum Bouillon
tion of Merck's highly sensitized Constituents:
litmus.
1. Bouillon (sugar free) 1000.0 cc.
(3) Prepare a 10.0% solution of one of 2. Serum (beef) 500.0 cc.
the following in distilled water: Preparation
glucose, galactose, levulose, lac-
(1) Method of preparation of sugar free
tose, maltose, sucrose, mannitol,
broth not given.
dulcitol, inulin and dextrin. Adjust (1) to 1.0% acid to phenol-
(2)
(4) Sterilize (3) at 100C. for 10
phthalein (neutral to litmus).
minutes. Mix 1 part sterile beef serum with 2
(3)
(5) Mix (2) and (4). parts sterile (2).
(6) Tube in sterile tubes. Dissolve one of the added nutrients
(4)
Incubate for 3 days to detect acci-
(7) in (3).
dental contamination. Sterilization: Sterilize at 65-68C. for one
Fermentation by the meningococci hour on 6 consecutive days.
was studied in these media. Use: Differentiation of streptococci and
References: Buerger (1907 p. 430), Elser pneumococci. Pneumococci fermented
and Huntoon (1909 p. 404).
starch. Streptococci did not. Strepto-
cocci and pneumococci fermented lac-
773. Akatsu's Basal Ascitic Fluid Bouillon saccharose and dextrin, produced
tose,
Constituents acid and formed a yellowish white
1. Bouillon 1000.0 cc. coagulum.
2. Ascitic fluid 1000.0 cc. Added nutrients and variants:
3. Tissue (a) The author added 1.0% sucrose, 1.0%
Preparation lactose, 1.0% glucose, 1.0% dextrin
(1) Composition or method of prepara- or 0.66% starch.
tion of bouillon not given. (b) Koch and Pokschischewsky prepared
(2) Mix equal parts of bouillon and similar media for the differentiation
ascitic fluid and tube in 10.0 cc. lots. of Streptococcus longus seu. erysipe-
(3) Add to each tube of (2) a piece of latos and Streptococcus equi. They
fresh tissue. reported that Streptococcus equi gave
(4) Add 1.0% of one of the added nu- a flocculent flaky sediment leaving a
trients to each tube. clear bouillon. Streptococcus longus
Sterilization: Not specified. seu erysipelatos gave a uniform tur-
Use : To study the fermentation of carbohy- bidity to the medium. (Medium
drates, alcohols, etc., by bacteria. containing no sugars). The media
Akatsu used spirochetes, Treponema pal- were prepared as follows:
lidum, Treponema calligyrum, Trepo- (1) Mix one part sterile horse serum
nema microdentium, Treponema mucosum and two parts ordinary bouillon.
and Spirochaeta refringens. Medium was (2) Sterilize on 3 successive days at
covered with a layer of sterile paraffin 60C. for one hour.
after inoculation and incubated at 36C. (3) Prepare a 10.0% solution of one of
Added nutrients: The author added 1.0% the following:
of one of the following materials: sorbitol maltose
amygdalin galactose dulcitol lactose
arabinose glycogen mannitol glucose
beerwort glucose levulose mannose
dextrin inulin galactose raffinose
mannitol lactose sucrose
raffinose levulose (4) Sterilize on 3 successive days for
sucrose maltose 2 or 3 minutes in streaming steam.
starch (5) Add 1.0 cc. of (4) to 9.0 cc. of (2),
Reference: Akatsu (1917 p. 376). under aseptic conditions.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 227
The medium was also employed with- Use: This solution is equivalent to a 1.0%
out the addition of carbohydrates, peptone broth.
alcohols, etc. Variants: Lohnis prepared the medium as
(c) Klimmer added 0.5 to 1.0% of any follows
desired carbohydrate, alcohol, etc., (1) Heat 22.0 g. of Ragit bouillon in a
and mixed one part
to bouillon, of liter of tap water in the steam steri-
serum with one to twenty parts of an hour.
lizer for
the sugar bouillon. (2) Filter thru filter paper.
References: Hiss (1901-05 p. 323), Koch (3) Add concentrated soda solution until
and Pokschischewsky (1913 p. 10), Klim- red litmus paper is turned slightly
mer (1923 p. 200). blue.
(4) Heat for 30 to 45 minutes in the steam
775. Holman's Basal Serum Bouillon sterilizer.
(5) Filter.
Constituents:
(6) Sterilization not specified.
1. Water 400.0 cc.
References: Marx (1910 p. 361), Lohnis
2. Bouillon, double strength. . 400.0 cc.
(1913 p. 14).
3. Andrade's Indicator 80.0 cc.
4. Beef serum 200.0 cc. 777. Bacto Dehydrated Broth
Preparation
Constituents:
(1) Preparation of double strength bouil-
1. Distilled water 1000.0 cc.
lon not given.
2. Bacto bouillon (0.8%) 8.0 g.
(2) Adjust (1) to +1.2.
Preparation
(3) Add 200.0 cc. of water to 400.0 cc. of
Dissolve (Prepared
(1) 2 in 1. accord-
(1).
ing to directions given on the bottle).
(4) Dissolve 8.0 g. of one of the added
(2) Initial pH = 7.1. (If sterilized at
nutrients in 80.0 cc. of Andrade's
15 pounds pressure for 20 minutes
indicator and add to (3).
pH = 6.5+.)
(5) Add 200.0 cc. of water to 200.0 cc. of
manner.
Sterilization: Sterilize in the usual
beef serum.
Use: General culture medium.
(6) Mix 400.0 cc. of sterile (5) and 600.0 cc.
References: Brown (1921 p. 562), Digestive
sterile (3).
Ferments Co. (1925 p. 10).
(7) Tube into sterile test tubes.
(8) Incubate 2 days to determine sterility. 778. Harvey and Iyengar's Dehydrated
Sterilization: Sterilize (4) by heating in Broth
flowing steam on each of 3 successive days.
Constituents: 1. Trypsinized mutton in-
Sterilize (5) by filtering thru a Berke-
fusion broth.
feld filter.
Preparation
Use: To study fermentation of carbo-
(1) Preparation of tryptic digest of
hydrates, alcohols, by bacteria.
etc.,
mutton infusion broth not given.
The author used streptococci. The pro- Adjust topH = 8.0.
(2) (1)
duction of acid coagulates the serum.
(3) Cut up agar fibre into small pieces.
Added nutrients and variants: The author
(4) Add (3) to (2) (6.0% by weight).
added 8.0 g. of any desired carbohydrate, Autoclave at 120 for an hour to
(5)
alcohol, etc. to the basic solution.
melt the agar.
References: Holman (1916 p. 385), Stitt
(6) Filter thru cotton, wool and muslin
(1923 p. 36). into a tin receptacle.
(7) Cut the agar out of the receptacle
776. Ragit's Dehydrated Broth
and into slices.
Constituents: (8) Pass slices thru meat mincing ma-
1. Water 1000.0 cc. chine with a finely perforated outlet
2. Ragit Bouillon (Marck) .... 22.0 g. disc.
Preparation: (1) Dissolve 2 in 1. (9) Spread minced nutrient agar on
Sterilization: Not specified. metal or other type of trays.
228 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(10) Dry in hot air oven or any other 5.0 g. NaCl and the whites of two
convenient way. eggs, beaten up in 2 or 3 volumes
(11) Store powder in sterile glass stop- of water.
pered bottle. (6) Boil under a gas flame for 15
(12) Add 6.0% by weight (11) to cold minutes.
water. (7) Adjust to faint alkalinity, using
(13) Extract (12) at room temperature phenolphthalein as an indicator,
for 2 hours. and the end point being a faint
(14) Filter. red color.
Sterilization: Method of sterilization of the (8) Pour into an iron kettle, add
not given.
filtrate 100.0 cc. distilled water, boil
Use: General culture medium. strongly for 5 minutes and filter.
possible.
two liter flask.
Pour the filtrate into a kettle, (6) Sterilize for 15 minutes at 115C.
(5)
and add 10.0 g. peptone (siccum), (d) Stutzer and Hartieb (1897).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 229
(2) Strain thru a piece of cheese (1) Remove all the fat and con-
(3) Heat between 50 and 60C. and (2) Add 1 liter of distilled water.
add 10.0 g. peptone and 5.0 g. (3) Place in a vessel for cooking and
NaCl. Stir constantly until solu- cook for 30 minutes at about
tion complete.
is 70C.
(4) Boil with constant stirring. (4) Filter thru paper and make up
(5) Filter thru paper. to one liter.
Reboil and filter again if the (5) Add 1.0% peptone (Witte) and
(6)
filtrate is turbid. 0.5% NaCl to (3).
(7) Cool to room temperature and The remainder of the preparation
make the volume to one liter. is identical with variant (j)
(8) Adjust the reaction. above, from step (5). These two
(9) Boil and filter. bouillons may be inoculated with
(10) Tube or flask. B. coll and incubated at 38 C.
(11 Sterilize by the fractional method for several hours to render them
in steam at 100C. or in the auto- sugar free.
clave at 120C. (I) Committee A. P. H. A. (1905).
(10) Test the reaction and readjust if free from fat and tissue as pos-
(4) Weigh the saucepan and cook for (5) Heat for 60 minutes in the steam
about 30 minutes. sterilizer.
(5) Strain thru cheese cloth, and (6) Filter thru filter paper.
press all the liquid out in a meat (7) Add concentrated soda solution
press. until red litmus paper is turned
(6) Replace the water lost by evap- slightly blue.
oration. (8) Heat for 30 to 45 minutes in the
(7) Dissolve 10.0 g. of Witte's pep- steam sterilizer.
tone in (6). (9) Filter.
(1) Mince 1.0 pound of fat free meat (1) Chop one pound of lean, fat free
and macerate in 1000.0 cc. cold beef with a knife or mincing
water over night. Or the mix- machine.
ture may be macerated at 40C. (2) Place (1) in a porcelain dish or
for 30 minutes instead. glass beaker and add 1 liter of
(8) Add 5 drops of a 0.5% alcoholic (10) Neutralize again and adjust the
solution of thymolphthalein to reaction to +10.
one dish. (11) Filter into sterile flasks or test
Add 10.0 cc. of N/1 HCl per liter (2) Mix thoroly and allow to stand
(12)
in a cool place (refrigerator) for
of filtrate. This gives a pH
= not more than 16 to 24 hours.
7.6.
(3) Strain the infusion thru a cheese
(13) Tube or flask and sterilize in the
cloth, thoroly pressing out all
autoclave for 20 minutes at 115C.
the juice.
(t) BezauQon (1920).
(4) Make up to 500.0 cc. by the addi-
(1) Soak 500.0 g. of finely chopped
tion of tap water if necessary.
lean beef (or other meat) in
(5) Place (4) in a sterile liter Erlen-
1000.0 cc. of water for 12 hours.
meyer flask.
(2) Collect the liquid from the meat
(6) Heat in the autoclave at 120C.
and add 10.0 g. peptone and 5.0 g.
for 30 minutes.
NaCl.
(7) Pour the contents of the flask into
(3) Autoclave for 10 minutes at an agate pail and add 500.0 oc.
115C. of tap water.
(4) Filter thru a wetted filter.
Add 1.0% Witte's peptone and
(8)
(5) Make slightly alkaline to litmus 0.5% NaCl.
by the addition of 10.0% soda (9) Thoroly mix 10.0 g. of egg albu-
tiolution. min with 100.0 cc. of tap water
(6) Autoclave for 15 minutes at and add to (8) or one well beaten
120C. egg white may be used.
(7) Filter. (10) Heat in the autoclave at 120C.
(8) Distribute as desired for one hour.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 233
(11) Adjust the Reaction to +1.5% by (1) Prepare beef infusion broth from
the addition of NaOH or HCI. 1000.0 cc. water, 300.0 g. lean
(12) Filter thru the same paper until beef and Parke Davis
10.0 g.
the medium is bright and clear. & Co. peptone (Procedure not
(13) Distribute as desired. given).
(14) Sterilize in autoclave
the at (2) Initial pH = 5.0 to 9.0, changing
120C. for 30 minutes. every 0.4 interval N/10
using
(w) Dopter and Sacquepee (1921). or N/1 NaOH and N/10 or N/1
(1) Add 1000.0 cc. of water to 500.0 g. HCI.
of finely chopped fat and tendon (3) Sterilize at 15 pounds for 15
free beef. minutes.
(2) Allow to stand in the ice box for (z) Randall and Hall (1921).
12 hours, or heat at 50 to 55 for (1) Soak ground lean beef in water
30 minutes. over night and then boil 10
(7) Filter thru a wetted filter paper. (aa) Ayers, Rupp and Mudge (1921).
(5) Boil for30 minutes. (5) Dissolve 2.0% Park Davis bac-
(6) Press the liquid from the meat teriological peptone and 0.5%
in a meat press. NaCl in (4).
(7) Mix the meat with more water, (6) Adjust to pH = 8.0.
and press out again. (7) Distribute in desired containers.
(8) Bring the volume of the com- (8) Pass steam thru the autoclave
bined liquids to 1 liter. for one hour after which slowly
(9) Dissolve 20.0 g. peptone and 5.0 g. raise the pressure to 10 pounds
NaCl in (8). (time about 30 minutes) and keep
(10) Adjust the reaction to 1.2% acid at this temperature for 30
with phenolphthalein as an indi- minutes.
cator. (hh) Stitt (1923).
(11) Filter until perfectly clear. (1) Cut up 500.0 g. fat-free meat in
(12) Sterilize in the autoclave. a sausage mill.
(ff) Pitfield (1922). (2) Pour 1000.0 cc. of water over (1).
(1) Cover 500.0 g. of finely cut fat (3) Keep in the ice chest over night.
free beef with 1000.0 cc. of water. (4) The following morning skim off
(2) Shake well and place on ice over the scum of fat by means of a
night. piece of absorbent cotton.
(3) Squeeze out the fluid by means of (5) Squeeze out the infusion thru a
a cloth and make up the volume strong muslin cloth, making the
to 1 liter. amount up to 1000.0 cc.
(4) Inoculate with a culture of the (6) Dissolve 1.0% Witte's peptone
colon bacillus. and 0.5% NaCl in (5). Mix the
(5) Allow to stand at room tempera- salt and peptone with a little of
ture over night. the infusion and prepare a paste
(6) Boil and add 10.0 g. Witte's pep- of themixture in a mortar before
tone and 5.0 g. NaCl. adding it to (5).
(7) Weigh the sauce pan and contents (7) Add sufficient normal NaOH to
and heat to 60C. make the reaction +1.0 to phenol-
(8) Make up the loss in weight by the phthalein.
addition of water. (8) Place in the inner chamber of a
(9) Neutralize to litmus. rice cooker and bring to boil.
(10) Boil 5 minutes. Boil for 20 minutes. It is neces-
(11) Make up the loss in weight by the sary to have NaCl or CaClj in
addition of water. the outer chamber. Do not stir
(12) Adjust the reaction as desired the medium.
(+0.5 to +1.5%). (9) Filter thru a wet filter paper.
(13) Filter. (10) Make up the volume to 1000.0 cc.
(14) Distribute into flasks or tubes. by the addition of distilled water.
(15) Sterilize(method not given). (11) Readjust or record the reaction.
(gg) Hartley (1922) used the following (12) Filter if the reaction is adjusted
medium for diphtheria toxin pro- in (11).
duction: (13) Sterilize in the autoclave at
(1) Soak 1 pound of lean finely 115C. for 15 minutes or in the
chopped horse meat in 1000.0 cc. Arnold on 3 successive days.
of tap water over night. (ii) Stitt (1923).
(2) Raise the temperature to 95C. Prepare the medium like the above
the following morning. variant (gg) (1) thru (12).
(3) Allow to cool slightly and filter (13) Inoculate with 5.0 cc. of a 24-hour
thru paper pulp. culture of the colon bacillus.
(4) Adjust the reaction of the filtrate (14) Incubate over night at 37C.
to pH = 8.0. (15) Boil in a sauce pan.
236 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Add 1.0% peptone and 0.5% (6) Strain thru cheese cloth.
(8)
NaCl. Squeeze by twisting the cloth or
Steam for an hour in a double use the meat press.
(9)
walled pot (one hour from the (7) This may be sterilized in flasks
time the water boils). and stored for future use. This
(10) Add normal NaOH solution until is meat infusion.
the liquid turns a piece of mois- (8) If used at once, add 1.0% peptone
qu6pee (1921 p. 117), Abbott (1921 p. 124), (3) Dissolve 3, 4, 5 and 6 in (1).
Foster and Randall (1921 p. 151), Randall (4) Add enough N/1 NaOH to give final
and Hall (1921 p. 347), Ayers, Rupp and reaction pH = 8.0 to 8.2.
Mudge (1921 p. 258), Harvey (1921-22 (5) Steam 15 minutes and check reaction.
pp. 67, 68), Heinemann (1922 p. 31), Pit- (6) Distribute as desired.
field (1922 p. 13), Hartley and Hartley Sterilization: Sterilize at 115 for 20
(1922 p. 460), Stitt (1923 p. 30), Klimmer minutes.
(1923 p. 192), Frost (1923 p. 64), Cunning- Use: Cultivation of Bad. diphtheriae and
ham (1924 p. 11), Park, Williams and toxin production. Authors reported good
Krumwiede (1924 p. 96), Kreidler (1926 growth.
p. 190). Reference: Davis and Ferry (1919 p. 236).
780. Jensen's Nitrate Infusion Broth 782. LoefHer's Infusion Broth (Roux and
Rochaix)
Constituents:
1. Water 1000.0 cc. Constituents
2. Meat 500.0 g. 1. Distilled water 1000.0 cc.
3. NaCl 5.0 g. 2. Beef 500.0 g.
4. Peptone 10.0 g. 3. NaCl 5.0 g.
5. NaNOs 3.0 g. 4. Peptone 20.0 to 25.0 g.
238 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(d) China blue 0.025 g. + rosolic acid or gested various methods of preparing this
its sodium salt 0.05 g. medium:
(e) China green (0.2% solution) 15.0 cc. (a) Dernby and David.
(f) Malachite green 1.0 g. (1) Immerse 6.0 g. of fresh finely
(g) China blue 0.025 g. + phenol red chopped veal in 12 liters of tap
(phenol sulphone phthalein) 0.01 g. water and add 70.0 g. of ordinary
Reference: Muller (1899 p. 805), Harvey distillers yeast.
(1921-22 pp. 88-92), Omelianski (1903 (2) Incubate at 36C. for 16 hours.
p. 4). (3) Press thru cheese cloth.
(4) Add 1.5% Witte's peptone and
785. Robinson and Rettger's Infusion
0.4% NaCl.
Opsine Solution Adjust to pH = 7.2 or 7.3.
(5)
Constituents: (6) Boil several minutes.
1. Beef Infusion 1000.0 cc. Filter while warm thru paper.
(7)
2. Opsine 20.0 g. Distribute in liter flasks.
(8)
Preparation (9) Sterilize at 110 for 20 minutes.
(1) Method of preparation or composi- (b) Dernby and Blanc.
tion of beef infusion not given. (1) Chop veal finely and allow to
(2) Dissolve 2 in (1). autolyzeat37for24hours. Then
(3) Adjust reaction faintly alkaline to boil and filter.
litmus. (2) Dissolve 2.5 g. XaCl and 5.0 g.
(4) Distribute in 20.0 cc. lots in 150.0 cc. peptone in (1).
Erlenmeyer flasks. (3) Adjust to desired pH with XaOH
Sterilization: Sterilize at 12 to 14 pounds and HCl.
pressure for 15 minutes. (4) Sterilize at 110 for 20 minutes.
Use: Production of diphtheria toxin by (c) Harvey.
B. diphtheriae. The author reported (1) Mince finely fat-free veal.
that the addition of 0.1% glucose had (2) Add 500.0 g. to 1000.0 cc. tap water.
little effect. M.L.D. = 0.035 cc. (3) Add an 18 hour culture of B. coli.
Variants (4) Incubate 20 hours at 22C.
(a) The author added 0.1% glucose with (5) Heat the mixture 2 hours at a
or without 0.5 g. XaCl. temperature not exceeding 50C.
(b) The author added 0.5 g. XaCl. (6) Skim off fat on the surface.
Reference: Robinson and Rettger (1917 (7) Raise to boiling point.
p. 367). (8) Boil 10 minutes.
(9) Pour the mixture onto a wet, thick,
786. Warden, Connell and Holly's Veal
clean cloth.
Infusion Broth
(10) Collect the fluid which drains thru
Constituents the cloth together with that ob-
1. Veal Infusion 1000.0 cc. tained by squeezing the meat in
2. Peptone (Difco proteose)... 20.0 g. the cloth.
3. NaCl 5.0 g. (11) Filter the fluid collected thru well
Preparation wetted, thick filter paper.
(1) Method of preparation of veal infu- (12) Add to the filtrate: peptone
sion not given. 20.0 g., sodium chloride 5.0 g.
240 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(13) Bring the volume up to 1000.0 cc. Variants Omit the sodium and potassium
:
(4) Add peptone (1.0%) and NaCl feld candle (length of extraction not
(0.5%). given).
(5) Titrate to pH = 8.2. (5) Decant the ether extract, evaporate
(6) Autoclave at 15 pounds pressure nearly to dryness under suction, and
for 20 minutes. take up the residue in an amount of
(e) Stitt used the medium as a bile sub- saline equal to the original volume.
stitute. He used 500.0 g. of ground (6) Add 1.0 cc. of (4) or (5) or a mixture
beef liver, 1 liter of water and 1.0% of the two to 5.0 cc. of (1).
peptone. The procedure was the Sterilization: Method not given.
same as for Stitt's variant of Dun- Use : To show growth accessory substances
ham's Infusion Broth (see variant for pathogenic bacteria. Author reported
791. Owen, Martin and Pitts' Trypsin 793. Harvey's Heart Infusion Broth
Beef Tea
Constituents: Ox
heart Bouillon.
1.
Constituents Preparation
1. Bouillon (Beef tea) (1) Prepare bouillon 1% acid to phenol-
2. Trypsin phthalein from ox heart which has
Preparation been "hung" 2 days.
(1) Add 10.0 cc. of trypsin (Liquor pan- (2) Sow with B. lactis aerogenes.
creatica. Digestive Ferments Co.) to (3) Incubate 48 hours.
90.0 cc. of sterile bouillon (Beef tea). (4) Steam 20 minutes.
(2) Distribute in 5.0 to 10.0 cc. lots in (5) Make reaction 1% acid to phenol-
sterile test tubes. phthalein.
Sterilization: Method of sterilization of (6) Sow with B. coll.
wetted, thick filter paper. (1) Grind beef bones to small pieces.
(9) Add peptone, 5.0 g. (2) Mix 500.0 g.of (1) with 900.0 g. water
(10) Steam 45 minutes. and 100.0 cc. normal HCl.
(11) Bring the volume up to 1000.0 cc. (3) Allow to stand for 12 to 24 hours.
by the addition of water. (4) Heat in the autoclave at 125 to
(12) Steam 30 minutes. 130C. for 30 minutes.
(13) Filter while hot, thru well-wetted, (5) Cool and then filter thru linen.
thick filter paper. (6) Add 15 parts of peptone to 1000
(14) Distribute into flasks or test tubes. parts of the liquid.
Sterilization: Sterilize in streaming steam. (7) Neutralize by the addition of a
Use: General culture medium. dilute soda solution. (Indicator not
Reference: Harvey (1921-22 p. 69). specified.)
(8) Heat in the autoclave at 125-130
800. Richardson's Mucosa Infusion Broth for 30 minutes.
Constituents : (9) Filter while hot.
1. Distilled water 500.0 cc. (10) Allow to stand for one day in the
2. Mucosa, hog intestine 250.0 g. cold.
3. Peptone 5.0 g. (11) Filter again.
4. NaCl 2.5 g. Sterilization: Sterilize at 120 (time not
Preparation : specified).
(1) Take the small hog in
intestine of a Use: General culture medium. Author
fresh without cutting,
condition, cultivated streptococci, Streptococcus
wash thoroly in running water, until pyogenes and other organisms.
the water runs perfectly clear. Reference: Boyer (1918 p. 230).
(2) Lay the intestine open and scrape
802. Ball's Animal Infusion Broth
off the mucosa with a glass slide
(100.0 g. of mucosa is easily obtained Use the flesh of guinea pigs as well as
from one hog). other experimental animals instead of beef
(3) To 250.0 cc. of the mucosa add in the preparation of Dunham's Infusion
500.0 cc. of distilled water. Broth (779).
(4) Boil for 30 minutes and cool. Reference: Ball (1919 p. 84).
(5) Boil again, neutralize and boil once
803. Stutzer's Nitrate Extract Broth
more.
(6) Filter. Constituents:
(7) Boil and 5.0 g. peptone and 2.5 g. 1. Water.
NaCl. 2. Peptone.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 245
growth. necessary,
Reference: Davis and Ferry (1919 p. 236). (11) Add 0.5% to 1.5% of normal HCl.
246 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(12) Heat until the precipitate appears Witte's peptone. The medium was
flaky. prepared as follows
(13) Filter thru moistened filter paper. (1) Make a paste of the peptone.
(14) Tube. (2) Dissolve (1) in 1.
(15) Sterilize for 20 minutes in a steam (3) Add 2 and 3 to (2).
sterilizer on 3 consecutive days or (4) Boil and stir for 30 minutes.
in the autoclave at 120 for 20 (5) Make up the loss due to evapora-
minutes. tion by the addition of water.
(c) Roux and Rochaix (1911) did not (6) Bring to the boiling point.
specify the type of peptone used. (7) Adjust the reaction as desired.
(d) Day and Baker (1912-13) specified (8) Filter when hot and again when
Lemco meat extract and Witte's pep- cool.
tone. They adjusted the reaction to (9) Sterilize in the autoclave.
+1 and cultivated an organism caus- (j) Brussoff (1918) cultivated sludge
ing ropiness in beer. forms on a medium containing 5.0 g.
(e) Lohnis (1913) used Witte's peptone NaCl, 10.0 g. Witte's peptone, and
and adjusted the reaction alkaline 10.0 g. Liebig's meat extract in a
to litmus. liter of tap water rich in potassium.
(f) Rideal and Walker (1913) used 20.0 g. (k) Foster and Randall (1921) studied the
Liebig'smeat extract, 20.0 g. Witte's changes in pH values of a medium
peptone and 10.0 g. NaCl. The me- due to autoclaving and standing.
dium was used for the growth of B. They used 5.0 g. NaCl, 10.0 g. Parke-
typhosus in the Rideal-Walker test Davis & Co. peptone, 3.0 g. of Liebig's
of disinfectants. beef extract, or 50.0 g. of Bacto beef
(1) Dissolve the constituents in water in a liter of distilled water.
by boiling for 30 minutes. (1) Harvey used 3.0 to 5.0 g. of Lemco
(2) Filter. meat extract and prepared the me-
(3) Neutralize to phenolphthalein. dium as follows:
(4) Add 15.0 cc. of N/1 HCl. This (1) Add the whites of 2 eggs to
gives a +1.5 reaction. 1000.0 cc. water.
(5) Make up to 1 liter. (2) Add to the mixture by degrees to
(6) Filter. make a suspension; Lemco 3 to
(7) Sterilize (method not given) in 5.0 g., peptone 10.0 g., sodium
500.0 cc. lots. chloride 5.0 g.
(8) Sterilize again on the next day. (3) Steam or boil 45 minutes.
(9) Tube in 5.0 cc. quantities into (4) Filter while hot thru well-wetted,
sterile test tubes. thick filter paper or thru 2 layers
(10) Plug with sterile cotton. of absorbent cotton wool.
(11) Place in the steam sterilizer for (5) Bring the volume up to 1000.0 cc.
about 30 minutes. by the addition of water.
(g) Brussoff (1916) used 5.0 g. of Merck's (6) Estimate and adjust to a definite
iron peptone and 2.5 g. NaCl. The pH value or faintly alkaline to
medium was used for the cultivation litmus or 1.0% acid to phenol-
of Ferribacterium duplex. A slight phthalein.
yellow membrane was formed after (7) Sterilize in streaming steam or in
5 or 10 days incubation. the autoclave.
(h) Berman and Rettger (1916) studied (m) Pitfield (1922) used 3.0 g. Liebig's
erepsin production by members of beef extract, 10.0 g. Witte's peptone
the colon-typhoid group using 2.5 g. and 5.0 g. of NaCl per liter. The
Liebig's meat extract and did not medium was prepared as follows:
specify the type of peptone (10.0 g.) (1) Dissolve the constituents in
used, 5.0 g. NaCl was also used, 1000.0 cc. of water.
(i) Roddy used 2.0 g. Liebig's
(1917) (2) Weigh the saucepan and contents
beef extract, 5.0 g. NaCl, and 10.0 g. and heat to 60C.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 247
(3) Make up the loss in weight by the did not specify the type of peptone
addition of water. emploj'ed.
(4) Neutralize to litmus, (r) Cunningham (1924) prepared the
(5) Boil 5 minutes. medium as follows:
(6) Make up the loss in weight by the (1) Weigh out 10.0 g. peptone, 5.0 g.
addition of water. NaCl and 10.0 g. of Liebig's meat
(7) Adjust the reaction as desired extract. Weigh out the extract on
(+0.5 to +1.5%). a piece of paper.
thru paper.
(8) Filter (2) Place (1) into pot and add
(9) Distribute into flasks or tubes. 1000.0 cc. of tap water.
(10) Sterilize (method not given). (3) Steam for an hour in a double
(n) Dopter and Sacquepee (1921) did not walled pot (one hour from the time
specify the use of Liebig's meat ex- the water boils).
tract, nor they specify the
did (4) Add normal NaOH solution until
amount of NaCl or peptone used. the liquid turns a piece of mois-
(o) Stitt (1923) prepared the medium as tened tumeric paper dipped into
follows: it faintly but distinctly brown.
(1) Place 3.0 g. Liebig's meat extract, The reaction is acid to phenol-
10.0 g. peptone, and 5.0 g. NaCl phthalein, pH varies from 7.7
in a mortar. to 8.0.
(2) Dissolve the white of one or two (5) Heat in an autoclave.
eggs in 1000.0 cc. of water. (6) Filter thru gray coarse filter paper
(3) Add (2) little by little to (1) until until clear.
a brownish solution is obtained. (7) Make up the volume to 1 liter with
(4) Pour (3) into the inner compart- distilled water.
ment of a rice cooker. (8) Tube in 8.0 cc. lots.
(5) Apply heat to the outer compart- (9) Sterilize at 22.5 pounds pressure.
ment containing NaCl or CaCl2. References: Debrand (1900 p. 759), Smith
(6) Bring to a boil and boil for 15 to (1902 p. 82), Frost (1903 p. 6), Roux and
20 minutes. Do not stir. Rochaix (1911 p. 108), Day and Baker
(7) Place the inner compartment on (1912-13 p. 435), Lohnis (1913 p. 14),
a scales, and its counterpoise and Rideal and Walker (1913 p. 575), Brussoff
a one kilo weight on the other side. (1916 p. 552), Berman and Rettger (1916
Add water to the medium until p. 537), Roddy (1917 p. 41), Brussoff
the two arms balance. (1918 p. 205), Foster and Randall (1921
(8) Filter. pp. 152, 153), Pitfield (1922 p. 114), Dopter
(9) Sterilize in the autoclave at 115C. and Sacquepee (1921 p. 118), Stitt (1923
for 15 minutes or in the Arnold on p. 33), Manwaring, Boyd and Okami (1923
3 successive days. p. 307), Park, Williams and Krumwiede
(10) The reactionrarely exceeds +0.75 (1924 p. 97), Cunningham (1924 p. 11),
(from +0.6 to +0.9) and does not Besson (1920 p. 30), Harvey (1921-22
require adjusting. p. 68).
(p) Manwaring, Boyd and Okami (1923)
cultivated S. cholerae in a medium
806. Berman and Rettger's Extract Proteose
Solution
containing 10.0 g. peptone, 2.5 g.
beef extract, and 5.0 g. NaCl per Constituents
liter. The reaction was adjusted to 1. Water 1000.0 cc.
be identical to that of Locke's solu- 2. Proteose 2.5, 5.0, 8.0 g.
tion (0.015% NaHCOs). 3. Beef e.xtract 2.5 or 5.0 g.
(q) Park, Williams and Krumwiede (1924) 4. NaCl 5.0 g.
suggested the use of 2.0 to 5.0 g. Preparation
Armour or other commercial beef (1) Prepare proteose by salting out the
extracts in addition to Liebig's proteose from commercial peptone
product. They used 5.0 g. NaCl, but (details of the method not given).
248 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Dissolve 2.5, 5.0 or 8.0 g. of (1), 2.5 pany's amenoids instead of Witte's
or 5.0 g. of beef extract and 5.0 g. peptone.
NaCl ia a liter of water. Reference: Berman and Rettger (1918
Sterilization: Not specified. pp. 371-377).
Use: To study bacterial nutrition.
809. Berman and Rettger's Trypsinized
Reference: Berman and Rettger (1918
Extract Broth
pp. 377-380).
Constituents:
807. Guth's Selenium Extract Broth 1. Water 1000.0 cc.
2. Beef e.xtract 2.5 g.
Constituents 3. NaCl 5.0 g.
1. Water 1000.0 cc.
4. Peptone (Witte)
2. Meat extract (Liebig) . 10.0 g.
Preparation
3. Peptone (Witte) 10.0 g.
(1) Prepare peptone digest by digesting
4. NaCl 5.0 g.
at 45C. for several hours solutions
5. Sodium selenicate of Witte's peptone with commercial
(Merck) 1.0 to 2.5 g. Then sterilize and add ex-
trypsin.
Preparation :
tractand NaCl. The amount of pep-
(1) Dissolve 2, 3 and 4 in 1.
tone was not specified.
(2) Adjust to neutral using litmus as an Tube in 10.0 cc. lots.
(2)
indicator. Sterilization: Method not given.
Dissolve 5.0 g. Merck's sodium Au-
(3) Use: To study bacterial nutrition.
selenicate in 50.0 cc. distilled water. thors reported that more nitrogen was
(4) Add normal HCl to (3) until the
available in this medium than in similar
reaction is neutral to litmus (requires undigested media.
about 20.0 cc). Reference: Berman and Rettger (1918
(5) Make (4) up to 100.0 cc.
pp. 383-384).
(6) Heat in the autoclave for about 15
minutes. 810. Olszewski and Kohler's Trypsinized
(7) Tube (1) in 10.0 cc. quantities. Extract Broth
(8) Add 0.3 cc. of (6) to each tube of (7)
Constituents :
Sterilization: Not specified.
1. Water 1000.0 cc.
Use: Enrichment of typhoid bacilli. Colon Peptone 10.0 g.
2.
bacilli are inhibited. Liebig's meat extract 5.0 g.
3.
Reference: Guth (1915-16 p. 490). 4. NaCl 5.0 g.
5. Physiological salt solution. 3000.0 g.
808. Berman and Rettger's Salt Extract Preparation :
Broth
(1) Prepare ordinary nutrient bouillon
3. Liebig's beef extract 2.5 g. 7.0 cc. N/L soda solution after neu-
4. NaCl 5.0 g. tralizing to litmus.
ments, Park Davis, Eimer and Amend's solution with 3 parts physiological
peptone or Arlington Chemical Com- salt solution.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 249
Preparation autoclave.
Use: Substitute for beef extract or meat in
(1) Weigh a saucepan and measure
300.0 cc. tap water into it.
nutrient media. A very satisfactory sub-
(2) Dissolve 3.0 g. of peptone in (1)
stitute for meat. Can also be used in
when hot.
special media omitting the peptone.
Make up the loss due to evaporation Reference: Wyant (1920 p. 189).
(3)
by adding water.
(4) Dissolve 1.0 g. of beef extract in (3). 814. Heinemann's Nitrate Broth
(5) Adjust the reaction to alkaline to Constituents
litmus or neutral to phenolphthalein 1. Bouillon 1000.0 cc.
and add 0.5% normal HCl. 2. KNO3 5.0 g.
(6) Distribute into Erlenmeyer flasks. Preparation: (1) Dissolve 2 in 1.
(b) Lohnis added about 0.1% NaNOs to zone of about 1 cm. on the surface of
bouillon. the bouillon.
Percival added 3.0 g. NaNO, to (f) Signorelli added 0.5 cc. of a 1.0%,
(c)
bouillon. dahlia solution, 10.0 cc. of a 1.0%
Preparation: (1) Add 1.0% of a saturated was added after the culture had
solution of neutral red to nutrient developed the vibrio absorbed the
bouillon. dye. With erythrosin and safranin
Sterilization: Not specified. the bacteria were well colored, but
of Bacillus coli in water the medium was not decolorized.
Use: Detection
Bacillus coli gave a canary (g) Groenewege
added 10.0 drops of
analysis.
yellow color to the medium. Different methylene blue to 150.0 cc. of bouil-
lon. The medium was used to study
authors have added various indicators for
other special purposes. These will be the reduction of Phytobacter lyco-
indicated under variants.
persicum n. sp. the cause of tomato
rot. Partial reduction occurred.
Variants
Gordon reported that Streptococcus (h) Botez added 2.0 cc. of a 5.0%o methyl
(a)
brevis decolorized bouillon contain- violet solution to 100.0 cc. of bouillon.
ing 2.0 cc. of a 2.0%o watery solution He reported that B. typhosus grew
of neutral red per liter while Strepto-
well. Did not decolorize (reduce)
methyl violet. Para typhoid A
coccus longus did not decolorize the
medium. changed the color to a pale violet.
(b) Heinemann added 10.0% of a 1.0% Paratyphoid B completely decolorized
the medium after 48 hours. B. coli
litmus solution to nutrient bouillon.
completely decolorized the medium
(c) Calandra added 4 drops of a 1.0%
of Brilliant cresyl after 48 hours. B. fecal alcaligenes
aqueous solution
blue to 10.0 cc. bouillon. He re- did not reduce methyl violet. B.
ported that typhoid bacilli caused no Shiga did not grow. Cholera vibrio
change. B. coli after 24 hours gave grew slowly and did not change the
color after 10 days. B. pyocyaneus
a blue colored layer about i cm. high
on the upper surface. After 48 hours reduced methyl violet,
Tanner added 2.0 cc. of 1.0%o neutral
the blue color persisted in the upper (i)
layer. The remainder of the tube red solution to 1000.0 cc. of bouillon
was decolorized. containing 1.0% peptone and 0.5%
(d) Calandra added 3 drops of a 1.0% NaCl.
congo red solution to 10.0 cc. of (j) Heinemann added 2.0 to 3.0 cc. of a
bouillon. reported that typhoid
He 2.0%, solution of Hochst's 120" mala-
Preparation 435).
818. Muller and Malvoz's Iodine Bouillon 820. Stitt's Carbonate Bouillon
Constituents: Constituents:
1. Bouillon 1000.0 cc. 1. Bouillon.
821. Dalimier and Lancereau's Opsine (4) Strain broth thru cheese cloth.
Bouillon (5) Measure broth and add 3, 4 and 5.
(11) Distribute 800.0 cc. in 2 liter flasks. Reference: Park, Williams and Krum-
(12) Autoclave for 30 minutes at 15 wiede (1924 p. 133).
pounds pressure.
825. Reeser's Potato Veal Infusion Broth
(13) Add 8.0 cc. of a 10.0% glucose solu-
tion, sterilized in the Arnold on Constituents
each of 3 successive days, for 1. Distilled water 3000.0 cc.
30 minutes to each flask. 2. Potato
References: Wilcox (1916 p. 334), Walbum 3. Peptone 30.0 g.
(1922 p. 25) taken from (1922 p. 109), 4. NaCl 15.0 g.
Park, Williams and Krumwiede (1924 5. Glycerin 150.0 g.
pp. 132, 133). 6. Veal 1500.0 g.
Preparation
824. Park, Williams and Krumwiede's (1) Peel potatoes and carefully remove
Glycerol Veal Infusion Broth all eyes.
each flask.
bit blood to necessary.
(5) Heat the mixture over a water bath (7) Filter thru wire gauze and sediment
at 75C. until the blood coagulates allowed to settle.
and settles on standing (3 to 5 minutes (8) Distribute in 125.0 cc. lots in 300.0 cc.
after reaching the temperature). Florence flasks.
Sterilization: Method not given. (9) Kill a large guinea pig by a blow on
Use Cultivation of B. infliienzae and toxin
: the head and plunge the guinea pig
production. After incubation at 37C. into a 0.5% lysol solution for a few
for 24 hours, centrifuge to remove the minutes.
coarse particles and then filter thru a (10) Open the body cavity aseptically and
mandler candle filter. remove the liver.
Reference: Albert and Kelman (1919 p. (11) Introduce a piece of liver slightly
434). larger than a 25 cent piece into each
flask containing 125.0 cc. sterile
828. Thjotta and Gundersen's Blood Veal medium.
Infusion Solution by heating at 15
Sterilization: Sterilize (8)
Constituents: pounds pressure for 15 minutes.
1. Veal broth. Use: Cultivation of diphtheria bacilli, and
2. Sodium phosphate (secondary) production of toxin by the Park Williams
(NajHPOO. strain ^8. Contamination from liver
3. Blood. may be detected in smear preparations
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 255
and also
at the time of testing the toxin 2. Blood, defibrinated, rabbit
by the odor of the broth. or dog 100.0 cc.
Reference: Robinson and Meader (1920 3. Egg yolk.
p. 107). Preparation: (1) Add 50.0 cc. of egg yolk
and 100.0 cc. of dog or rabbit defibrinated
830. Starln and Dack's Casein Digest Veal
blood to 1000.0 cc. of veal infusion broth.
Infusion Broth
Sterilization: Method not given.
Constituents Use: Cultivation of tubercle bacilli.
1. Veal infusion 1000.0 cc. Reference: Klimmer (1923 p. 224).
2. Peptone 10.0 g.
3. NaCl 5.0 g. 833. Jackson and Muer's Glucose Liver
4. Casein digest (Wolf's) 50.0 g. Infusion
Preparation
Constituents:
(1) Method of preparation or composi-
1. Water 1000.0 cc.
tion of veal infusion not given.
2. Liver, beef 500.0 g.
(2) Dissolve 2, 3 and 4 in (1). The prepa-
3. Peptone (Witte) 10.0 g.
ration of Wolf's casein digest was not
4. Glucose 10.0 g.
given by the authors. It was pre-
pared according to Kahn's method.
5. K2HPO4 1.0 g.
Preparation
See 648.
(1) Chop 2 into small pieces and add 1.
(3) Filter to remove any precipitated
Weigh the infusion and container.
material.
(2) Boil slowly for 2 hours in a double
(4) Adjust reaction to pH = 7.8 to 8.0
boiler, starting cold and stirring it
Sterilization: Method not given.
occasionally.
Use: Cultivation of Clostridium botulinum.
(3) Make up the loss in weight by evapo-
Reference: Starin and Dack (1923 p. 173).
ration and strain thru a wire strainer.
831. Bacto Veal Infusion Medium (4) To the filtrate add 3, 4 and 5. Weigh
(Dehydrated) the infusion and container.
(a) Harvey prepared a similar medium p. 290), (1911 p. 727), Levine (1921 p. 110),
add (6) until the pieces of liver are (b) Long and Cornwell attempted to cul-
6. CaClz 0.2 g.
HgS04 filtrate frac-
[tion (14) 0.5 or 1.0 cc.
7. K2HPO4 2.0 g.
8. Glucose 2.0 g. Decolorized infu-
9. Phenol red (0.02% soln.) .... 80.0 cc. sion (2) 25.0 cc.
Preparation Glucose salt solu-
(1) Prepare a beef heart infusion by tion (9) 25.0 cc.
(b)
heating 1 pound of chopped beef HgS04 filtrate frac-
heart with 500.0 cc. of water to the tion (14) 1.0 cc.
an enzyme digest of milk to the amino 837. Huntoon's Hormone Heart Infusion
acid stage. Author reported no growth Broth
in medium (15b).
Constituents
Reference: Mueller (1922 p. 331).
Water
1. 1000.0 cc.
2. Heart, beef 500.0 g.
836. Mueller's Heart Infusion Peptone 3. Peptone (Bacto) 10.0 g.
Solutions 4. Gelatin 10.0 g.
5. Salt 5.0 g.
Constituents
6. Whole egg 1
Water (tap) 500.0 cc
7. Glucose 1.5 g.
Heart, beef 1.0 lb
8. Laked blood
Peptone (Difco) 10.0
Preparation :
NaCl 10.0
(1) Mix Bacto peptone, 10.0 g.
10.0 g.
MgS04 0.4
gelatin, 5.0 g. NaCl, one whole egg
6. CaCl, 0.2
and 500.0 g. of finely chopped beef
7. K0HPO4 2.0 g.
heart in a liter of water. Place in an
8. Glucose 2.0 g.
enamel ware vessel or a large coffee
9. Phenol red (0.02% soln.) .... 80.0 cc.
pot.
Preparation
(2) Heat over a free flame with constant
(1) Prepare beef heart infusion by heat-
stirring until the red color of the
ing 1 pound of chopped beef heart
meat infusion changes to brown at a
with 500.0 cc. of water to the boiling
temperature of about 68C. Do not
point. Strain and filter.
go beyond this temperature.
(2) Decolorize by boiling for 25 minutes
(3) Adjust to slightly alkaline to litmus
with 10.0% "Norit" a commercial
with N/1 NaOH and then add 1.0 cc.
grade of wood charcoal.
per liter of medium.
(3) Filter thru paper.
(4) Cover the vessel and place in an
(4) Make up one of the following media:
Arnold sterilizer or in a water bath
Decolorized infu-
at 100 for one hour.
sion 25.0 cc.
Glucose salt solu-
(5) Remove the vessel from the sterilizer
(a)
and separate with a glass rod the firm
tion 25.0 cc.
clot which has formed from the side
Peptone (Difco) .... 0.0 or 0.5 g.
of the vessel.
Infusion (not de-
(6) Return to the Arnold sterilizer at
colorized) 25.0 cc.
(b) Glucose salt solu-
100 for U hours.
(7) Remove the vessel and allow to stand
tion 25.0 cc.
at room temperature for about 10
Peptone 0.0 or 0.5 g.
minutes in a slightly inclined posi-
'Water 25.0 cc. tion.
Glucose salt solu-
(8) Pipette off the fluid portion or
(c)
jtion 25.0 cc. decant. If it is poured thru a fine
[Peptone 0.5 g. wire sieve, many of the fine pieces
(5) Bring each lot to pH = 7.4 to 7.8. of meat clot may be caught. (Avoid
(6) Filter if necessary. filtering thru cheese cloth, cotton
(7) Tube. or other adsorptive materials.)
Sterilization: Sterilize at 10 pounds pres- (9) Allow (8) to stand in tall cylinders
sure for ten minutes. for 15 to 20 minutes until the fat
Use: To study food requirements of strep- present has risen to the surface and
tococci and pneumococci. Author re- removed.
ported that no growth in (3) (a) occurred (10) The medium may be further cleared
without peptone, no growth in (3) (c). by filtering thru glass wool, asbestos
All others gave growth. wool, sedimentation or centrifu-
Reference: Mueller (1922 p. 325). gation.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 259
(11) Add 0.15% dextrose and enough (5) Allow to stand at room temperature
laked blood to give a slight pink over night.
tint. Sterilization: Not specified.
(12) Tube in 10.0 cc. lots. Use: Isolation of gonococci.
by the intermittent
Sterilization: Sterilize Reference: Macnoughton (1923 p. 297).
method.
839. Ficker and Hoffmann's Caffeine
Use: To cultivate highly pathogenic organ-
Infusion Broth
isms. Author reported that if spinal
fluid be taken from a meningitis case in Constituents:
which no organism can be found in 1. Distilled water 4000.0 cc.
smears, a distinct growth appeared in 2. Beef 1000.0 g.
this medium after 9 hours rendering a 3. Peptone (Witte) (6.2%) .... 120.0 g.
positive diagnosis possible. Organisms 4. NaCl (0.5%) 10.0 g.
multiplied in the leukocytes transferred. 5. Caffein (0.6%) 24.0 g.
Variants 6. Crystal violet (0.1%)
(a) Author used fresh beef steak instead (solution) 28.0 cc.
of heart. Preparation
(b) Bailey prepared a similar medium as (1) Pour 2 liters of distilled water over
follows: 1000.0 g. finely chopped lean beef
(1) Dissolve 10.0 g. gelatin in 1000.0 cc. in an enamel kettle.
distilled water and heat to 50 to (2) Weigh the kettle and contents.
60C. (3) Heat at 50 to 55C. for 30 minutes.
(2) Add 500.0 g. of moderately fine (4) Stir with a glass rod and heat to
chopped beef to (1). boiling.
(3) Bring to a boil and cook slowly for (5) Weigh and add distilled water to
15 to 20 minutes. make up the loss in weight.
(4) Filter thru a 16 mesh (cullender (6) Filter thru filter gauze.
type) until clear. (7) Measure and add 6.0% (not given as
(5) Add 10.0 g. peptone and 5.0 g. 0.6%) Witte peptone and 0.5% NaCl.
NaCl. (8) Heat until the peptone is dissolved.
(6) Boil 5 minutes. (9) Filter.
(7) Adjust to the desired reaction (pH (10) Distribute in Erlenmeyer or beer
= 7.5). flasks.
(8) Allow to stand several minutes (11) Provide each flask with an absorbent
and decant the supernatant fluid. paper cap.
(9) Tube. (12) Prepare a 1.2% caffeine solution in
(10) Sterilize fractionally or at 5 sterile distilled water. Shake, but
pounds pressure for 5 minutes. heating is not necessary to obtain
Reference: Huntoon (1918 p. 172). Bailey complete solution. Weigh the caf-
(1925 p. 341). feine on a chemical balance.
(13) Measure 100.0 cc. of sterile (11) into
838. Macnoughton's Blood Infusion Broth
a sterile Erlenmeyer flask and adjust
Constituents to 2.7 to phenolphthalein.
1. Heart infusion broth. (14) Measure 105 cc. of (12) in a sterile
2. Blood. measuring flask and add to each
Preparation 100.0 cc. of cool (13) under aseptic
(1 Prepare infusion broth using ox heart, conditions. Do not heat after the
and adjust to pH between 7.4 and 7.5. addition of caffeine.
(2) Tube in 10.0 cc. lots. (15) Prepare a 0.1% crystal violet solu-
(3) The evening before the medium is re- tion (use chemical balance) in sterile
quired, add 1.0 cc. of sterile human distilled cold water.
blood to each 10.0 cc. of (2). (16) Add 1.4 cc. of (15) by means of a
(4) Thoroughly mix by rolling the tubes sterile pipette to each flask (14).
between the hands. Sterilization: Sterilize (1) in streaming
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
and 0.5% NaCl. Dissolve by gentle (b) Copeland and Boynton used a me-
heat. dium prepared as follows to study
(7) Add N/1 NaaCOa until 0.8% acidity the Voges-Proskauer reaction by the
is reached. colon group.
(8) Boil or steam 20 or 30 minutes. (1) Prepare an infusion from fresh
(9) Filter. beef steak.
(10) Distribute into Fernbach flasks in (2) Dissolve 10.0 g. Witte's peptone,
layers 2.5 ctm. deep. Each flask 5.0 g. NaCl and 10.0 g. of water-
should have two or three cotton- free glucose in (1).
plugged openings. (3) Adjust to 1.0% acid to phenol-
(11) Add 0.1% glucose to each sterile flask phthalein.
of (10) from a sterile 20.0% glucose (4) Sterilization not specified.
solution. (c) Avery and Cullen prepared a medium
Sterilization: Sterilize (10) by heating in as follows:
the autoclave at 110 to 115 for 30 (1) Prepare a beef infusion.
minutes. Method of sterilization of glu- (2) Dissolve 10.0 g. peptone, 10.0 g.
cose solution not specified. glucose and 5.0 g. NaCl in (1).
Use: Cultivation of B. diphtheriae and (3) Adjust to pH = 7.6 to 7.8.
given under variants, all media not con- Variants: Authors adjusted the reaction to
taining peptone gave poor growth. + 1.0 to phenolphthalein instead of +0.6,
(5) Adjust the reaction to +0.6 to phenol- (2) Add 2 and 3 to (1).
phthalein. (3) Adjust reaction to pH = 7.5.
(6) Filter and distribute into 150.0 cc. (4) Add castor oil soap (sodium ricino-
Erlenmeyer flasks in 20.0 cc. lots. late) or other surface reducing ma-
(7) After sterilization add enough sterile terial in sufficient amounts to lower
glucose to each flask to make 0.1% the surface tension to about 50 and
glucose. 43 dynes.
Sterilization: Sterilize at 12 to 14 pounds Sterilization: Method not given.
pressure for 15 minutes. Use: To study effect of surface tension on
Use: Cultivation of B. diphtheriae and growth of streptococci. Authors re-
toxin production. ported that in general when the surface
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 263
(4) Skim off fat floating on the surface, Use: Enrichment of the colon-typhoid
(5) Raise the temperature to boiling group.
point. Variants: Klimmer prepared the medium
(6) Boil 10 minutes. as follows:
(7) Pour the mixture on to a wet, thick, (1) Dissolve 2, 3, 4, 5, 6 and 7 in meat
clean cloth. infusion broth.
(8) Add sodium chloride 5.0 g. to the (2) Flask in 100.0 cc. quantities.
filtrate. (3) Sterilize on each of 3 successive days
(9) Steam 45 minutes. for 10 minutes in streaming steam.
(10) Bring the volume up to 1000.0 cc. (4) Add 3.0 cc. of a 2.0% solution of 120
by the addition of water. Hochst malachite green to each
. (11) Estimate and adjust the reaction, 100.0 cc. lot of sterile (2).
(12) Steam for 30 minutes. (5) Tube in 3.0 or 4.0 cc. lots in sterile
(13) Filter while hot thru well-wetted, tubes.
thick filter paper. References: Loeffler (1906 pp. 289, 295),
(14) Make a paste of 10.0 g. of corn flour Klimmer (1923 p. 213).
starch and 200.0 cc. of (13).
(15) Heat 20 minutes at 20 pounds pres-
852. Duval and Lewis' Inulin Bouillon
sure. Constituents
(16) Add 10.0 g. peptone, 5.0 g. NaCl 1. Meat infusion broth 1000.0 cc.
and 800.0 cc. of (13) to (15). 2. Inulin 10.0 g.
(17) Steam 45 minutes. Preparation
(18) Make the reaction 0.2% acid to (1) Prepare boiullon according to stand-
phenolphthalein. ard methods from beef. (Method or
(19) Steam 30 minutes. reference not given.)
(20) Filter while hot thru well-wetted, (2) Adjust from 0.2 to 0.4% normal to
thick filter paper. phenolphthalein. After sterilization
(21) Add
saturated litmus solution to reaction from 0.5 to 0.8%,
make a deep
blue color. (3) Distribute into clean tubes in 9.0 cc.
(22) Distribute into test tubes. lots.
Sterilization: Sterilize at 100C, for 20 Prepare a 10.0% inulin solution in
(4)
minutes on 3 days. distilled water.
Use: Cultivation of meningococci. (5) Add 1.0 cc. of sterile (4) to each 9.0 cc.
Reference: Harvey (1921-22 p. 112), of sterile (3).
Sterilization: Sterilize (3) method not
851. Loeffler's Malachite Green Infusion
given. Sterilize (4) in the autoclave at
Broth
15 pounds pressure for 15 minutes.
Constituents Use: Cultivation of pneumococci.
1. Beef infusion 1000.0 cc. Reference: Duval and Lewis (1905 p. 484).
2. Peptone (2.0%) 20.0 g,
Lactose (5.0%)
853. Bulir's Mannitol Infusion Broth
3. 50.0 g.
4. Dextrose (1.0%) 10.0 g. Constituents
5. Sodium sulfate (0.5%) 5.0 g. 1. Water 2000.0 cc.
6. KNO3 (2.0%) 20.0 g. 2. Beef 1000.0 g.
7. KNO2(1.0%) 10.0 g. 3. Peptone (Witte) 25.0 g.
8. Malachite green 2% solution 30.0 cc. 4. NaCl 15.0 g.
Preparation 5. Mannite 30.0 g.
(1) Prepare a beef infusion from one 6. Neutral red.
pound of beef. Preparation
(2) Dissolve 2, 3,t, 5, 6 and 7 in (1). (1) Macerate 1000.0 g. finely chopped
(3) Add 3.0% of 2.0% malachite green lean beef with 2000.0 cc. water for
solution to (2), 24 hours.
Sterilization: Sterilizeon each of 3 suc- (2) Filter thru linen, and press the meat
cessive days for 10 minutes in streaming free from water.
steam. (3) To the liter of meat infusion thus
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 265
obtained add 25.0 g. Witte's peptone, 855. Harvey's Lead Acetate Infusion
15.0 g. NaCl and 30.0 g. mannite. Broth
(4) Boil for 1.5 hours over a flame. Constituents
(5) Neutralize by the addition of soda. 1. Water 1000.0 cc.
(6) Filter. 2. Peptone 10.0 g.
(7) Dissolve 0.1 g. neutral red in 100.0 g. 3. Beef 500.0 g.
distilled water. 4. NaCl 5.0 g.
(8) When ready for use add 3.0 cc. of 5. Lead acetate 1.0 g.
sterile (7) to each 150.0 cc. of the Preparation
medium. (1) Prepare meat infusion broth accord-
(9) Distribute in 15 to 20.0 cc. lots in ing to Harvey's method, see variant
fermentation tubes. (bb) 665.
Sterilization: Sterilize streaming
(6) in (2) Add 1.0 g. of lead acetate to (1).
steam. Sterilize (7) method not given. Sterilization: Not specified.
Use: Detection of Bacterium coli in water. Use: General culture medium.
When desired for use, add 100.0 cc. of the Reference: Harvey (1921-22 p. 107).
water being investigated to 50.0 cc. of
sterile (6). Then add 3.0 cc. of neutral 856. Omeliansky's Formate Infusion Broth
red solution as indicated in step (8). Constituents
The author reported that after 12 to 24 1. Meat infusion broth 1000.0 cc.
hours incubation at 46C. that the pres- 2. Sodium formate 5.0 g.
ence of Bad. coli was indicated by a 3. Phenolphthalein drops
uniform turbidity in the fermentation Preparation
tube, the formation of gas, and a change (1) Prepare meat infusion broth from
in color from a red to a yellow-greenish meat and peptone.
fluorescence. (2) Add 0.5% sodium formate to (1).
Variants: Klimmer added 25.0 g. Witte's (3) Add several drops of phenolphthalein.
peptone, 15.0 g. NaCl and 30.0 g. man- Sterilization: Not specified.
nitol in a liter of meat infusion, boiled Use: Differentiation of colon typhoid
1.5 hours, neutralized, tubed and steril- group. Author reported that Bad. typhi
ized in streaming steam for 2 hours. The abdom. did not decompose the formate.
addition of neutral red or test water not Bad. coli commune decomposed the
specified. formate with the production of gas.
References: Bulir (1907 p. 11), Klimmer Bad. paratyphi A and B decomposed the
(1923 p. 216). formate with production gas. Bact.
dysenteriae and Bac. faecalis alcaligenes
854. Harvey's Ferric Tartrate Infusion did not decompose the formate. Some
Broth of the organisms not producing gas may
turn the phenolphthalein red due to the
Constituents: decomposition of some albuminous ma-
1. Water 1000.0 cc.
terial to amino acids or other compounds.
2. Beef 500.0 g.
Variants: The author omitted the phenol-
3. Peptone 10.0 g.
phthalein.
4. NaCl 5.0 g.
Reference: Omeliansky (1905 p. 674, 1906-07
5. Ferric tartrate 1.0 g.
p. 158).
Preparation
(1) Prepare infusion broth according to 857. Jordan's Phenol Infusion Broth
Harvey's method, see variant (bb) Constituents:
665. 1. Infusion broth 900.0 cc,
(2) Add 1.0 g. of ferric tartrate to a liter 2. Phenol (1.0% soln.) 100.0 cc.
of (1). Preparation
Sterilization: Not specified. (1) Prepare infusion broth according to
Use: General culture medium. standard methods (see committee
Reference: Harvey (1921-22 p. 107). A. P. H. A. 1899, 665).
266 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
12.0 cc.
graduate.
Preparation
(2) Shake constantly, adding little by
(1) Prepare meat infusion broth accord-
little three times their volume of dis-
ing to Harvey's method (see variant
tilled water.
(bb) 665).
Prepare an egg stock solution by (3) Add 2.0 cc. of 10.0% soda solution
(2)
(0.5 cc.per 100.0 cc). Mix well.
mixing the whites of 4 eggs, the yolks
(4) Heat in the autoclave at 115C.
of 2 eggs and 12.0 cc. normal NaOH.
(5) Prepare meat infusion peptone solu-
(3) Add 1000.0 cc. of water to (2).
tion (500.0 g. beef, 5.0 g. NaCl and
(4) Heat very slowly to 90 C.
20.0 g. peptone per liter).
(5) Distribute in flasks.
(6) Mix one part (4) with 5 parts (5)
(6) Mix equal parts of (5) and sterile (1)
(7) Distribute.
when ready for use.
(8) Sterilize for 15 minutes at 112C.
Sterilization: Sterilization of (1) in the
autoclave or steamer. Sterilize (5) in the
860. Weiss and Wilkes-Weiss Egg
autoclave.
"Hormone" Broth
Use: General culture medium.
Variant: Harvey used a similar egg medium Constituents
by preparing the egg stock solution as 1. Hormone broth 400.0 cc.
indicated above, or by mixing the whites 2. Whole egg 100.0 cc.
of 4 eggs and 8.0 cc. of normal NaOH with Preparation: (1) Mix whole egg and "hor-
660.0 cc. of water and then diluting to a mone" broth in the ratio of 1:4.
liter. When ready for use add 1 part of Sterilization: Heat for 30 minutes in the
the egg stock solution, prepared as indi- Arnold sterilizer.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 267
if a small quantity of agar be added (1) Chop beef heart into small pieces.
(2) Divide (1) into six equal portions.
(0.2%).
(3) Take up each portion of (2)
with
Reference: Weiss and Wilkes-Weiss (1924
9 volumes of saline solution.
p. 226).
(4) Treat each one of the suspensions
in
861. Kohman's Brain Infusion Medium one of the following ways:
(a) Keep in the ice box over night and
Constituents
1. Meat infusion broth steam in the Arnold sterilizer for
2. Brain. an hour.
Grind beef brain and add (b) Keep in the ice box over night and
Preparation: (1)
meat infusion broth to cover. filterthru a Berkefeld candle.
sufficient
Sterilization: Sterilize in the autoclave. (c) Keep at 55C. over night and heat
in the Arnold sterilizer for an hour.
Use: To cultivate meningococci and to
(d) Keep at 55C. over night and filter
determine the effect of oxygen tension
thru a Berkefeld candle.
on growth.
Reference: Kohman (1919 p. 577). (e) Extract by boiling one hour and
then steam for an hour in the
862. Kreidler's Glucose Brain Broth Arnold.
(f) Extract by boiling for one hour.
Constituents
(5) Take one of (4) and add
1.0 cc. of to
1. Water 1000.0 cc.
meat infusion broth.
sterile
2. Beef 500.0 g.
Sterilization: Method not given.
3. Peptone (Difco) 10.0 g.
Use: To study the effect of growth stimu-
4. NaCl 5.0 g.
lating materials on the growth of patho-
5. Glucose (1.0%) 10.0 g.
genic bacteria. Author reported that
6. Brain.
heat destroyed the growth stimulating
Preparation
materials.
(1) Infuse 500.0 g. of finely chopped lean
Reference: Kligler (1919 p. 43).
beef in 1000.0 cc. water in an ice box
over night.
(2) Boil 30 minutes over a free flame. 864. Havens and Taylor's Kidney and Blood
Filter through gauze and then Infusion Broth
(3)
through paper to remove the fat.
Constituents
(4) Make up to 1000.0 cc. volume. 1000.0 cc.
and 1. Meat infusion
(5) Add 10.0 g. peptone (Difco)
10.0 g.
2. Peptone (1.0%)
5.0 g. NaCl.
3. Na2HP04 (1.0%) 10.0 g.
(6) Boil to dissolve.
4. Glucose (0.5%) 5.0 g.
(7) Adjust to pH = 7.8.
5. Kidney (rabbit).
(8) Filter through paper.
6. Blood, defibrinated, (sheep or rabbit).
(9) Add 10.0 g. glucose.
Preparation
(10) Fill long tubes and add several pieces
(1) 3 and 4 to 1000.0 cc. of ordinary
Add 2,
of calf brain, one cubic centimeter in
meat infusion made with distilled
size, washed in running water.
water.
Sterilization: Autoclave at 12 pounds pres-
(2) Adjust to pH 8.0 to 8.2.
sure for 20 minutes.
Tube in 10.0 cc. quantities.
Use: To study the bacteriology of endo- (3)
Use: Cultivation of hemolytic strepto- (6) Reduce the filtrate to 1.0% acid.
cocci. (7) Mix equal parts of sterile (6) and
Reference: Havens and Taylor (1921 sterile (1).
p. 313). Sterilization: Method of sterilization of (1)
not given. Sterilize (6) by repeated
865. Robertson's Cooked Meat Medium
heating at 65C. or by filtering.
(Torrey)
Use: Cultivation of Bacillus influenzae,
Constituents The author reported that better growth
1. Water 1000.0 cc. was obtained using double strength
2. Beef heart 500.0 g. broth.
3. Peptone 10.0 g. Variants: Author mixed (6) with equal
Preparation parts of double strength broth.
(1) Mix 500.0 g. finely minced fresh beef Reference: Wade and Manalang (1920
heart with 10.0 g. peptone and a liter p. 98).
of water.
867. Harvey's Ascitic Fluid Infusion Broth
(2) Cook in a double boiler with just
enough heat to cause a slight simmer- Constituents:
ing for about 10 minutes. 1. Water 1000.0 cc.
(3) Adjust to pH = 7.2. 2. Peptone 10.0 g.
(4) Continue to cook for 1| hours. 3. Beef 500.0 g.
(5) Readjust the reaction if necessary. 4. NaCl 5.0 g.
(6) Decant the broth. 5. Ascitic fluid 500.0 cc.
(7) Place the meat in test tubes to a Preparation
height of 5 cm. (1) Prepare meat infusion broth accord-
(8) Add 5.0 cc. of sterile broth to each ing to Harvey's method, (see variant
sterile tube of (7). (bb) 665).
Sterilization: Sterilize flasks of (6) and (2) Mix 2 parts (1) with one part ascitic
tubes of (7) separately in the autoclave fluid.
at 15 pounds pressure for 15 minutes. (3) Test sterility before use by incuba-
Following mixing, place the tubes in the tion 48 hours.
Arnold sterilizer on each of two successive Sterilization: Sterilize in the water bath
days for 20 minutes. 30 minutes at 56C., on each of five suc-
Use: Analysis of fecal flora. cessive days.
Reference: Torrey (1926 p. 355). Use: Cultivation of parasitic and saphro-
phytic bacteria.
866. Wade and Manalang's Blood Infusion
Reference: Harvey (1921-22 p. 83).
Broth
868. Stryker's Serum Infusion Broth
Constituents
1. Distilled water. Constituents
2. Meat infusion broth. 1. Meat infusion broth 900.0 cc.
3. Blood. 2. Serum 100.0 cc.
Preparation Preparation
(1) The beef infusion broth contains (1) Prepare meat infusion broth.
Witte's peptone and NaCl in various (2) Mix 1 part highly potent anti-pneu-
concentrations. Exact amounts not mococcus horse serum with 9 parts
given. normal horse serum.
(2) Thoroly lake sheep or horse blood by (3) Mix 100.0 cc. of (2) with 900.0 cc.
adding 20.0 cc. blood to 100.0 cc. dis- of (1).
tilled water. Sterilization: Method not specified.
(3) Heat to 80 to 85C. Use: To study variation in pneumococcus.
(4) Precipitate the proteins while hot by Other investigators used similar media
adding strong hydrochloric acid. for different purposes.
(5) Filter first thru gauze, then thru Variants
paper. (a) The author used plain serum instead
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 269
(b) Lucke and Rea cultivated strepto- (2) Adjust (1) to pH = 6.3.
cocci on a similar medium. They (3) Prepare (2) 10.0% glucose solution.
reported that typical streptococci (4) Mix (1) and (2).
gave a heavy white sediment consist- (5) Add sterile 3 to sterile (4) in suflB-
ing of albumin. Their medium was cient quantity to give a 1.0% con-
prepared as follows: centration of glucose.
(1) Prepare a meat infusion broth (2.0% Sterilization: Sterilize (3) method not
peptone). given. Sterilize (4) at 15 pounds pressure
(2) Boil 1000.0 CO. of (1) to 80.0% of its for 15 minutes.
original volume and adjust to 0.4+. Use: To study protein and carbohydrate
(3) Tube and sterilize for 3 days in metabolism of Streptococcus hemolyticus
steam. General culture medium.
(4) Obtain horse serum aseptically and Variants: Harvey cultivated meningococci
heat to 60C. for 1 hour in a water on a medium containing 1 part unheated
bath to destroy antihemolysins. clear sterile horse serum to 20 parts in-
(5) Add 2.0 cc. of (4) to each 8.0 cc. of fusion broth (see variant (bb) 770) to
(3). which was added 1.0% glucose.
(c) Stevens, Brady and West studied the References: Foster (1921 p. 222), Harvey
and hemol-
relation between virulence (1921-22 p. 80).
ysin. They reported that a strain
of streptococcus whose virulence had 870. Beach and Easting's Serum Infusion
been did not produce
increased and Extract Broth
greater concentrations of hemolysin.
Constituents:
Their medium was prepared as
1. Water 1000.0 cc.
follows Lean beef 450.0 g.
2.
(1) Prepare a meat infusion broth with 3. Peptone (Difco) 15.0 g.
2.0% peptone. 4. K2HPO4 7.5 g.
(2) Titrate (1) so that it is pH = 7.6
5. Liebig's beef extract 4.0 g.
after sterilization (method not
Glycerol 55.0 cc.
6.
mentioned). Aminoid peptone from beef
7.
(3) Distribute into 250.0 cc. Pyrex (Arlco) 5.0 g.
flasks in 80.0 cc. lots.
8. Serum (sterile blood) 100.0 cc.
(4) Add 20.0 cc. of fresh horse serum to
Preparation
each flask. with
lean beef
(1) Extract 450.0 g.
(5) Inactivate the contents at 56C. on 1000.0 cc. water for 3 hours. Bring
3 successive days, and store on ice
the water to 45C. After an hour
until ready for use.
raise the temperature to 50C. and
(d) Harvey mixed 2 parts serum with
during the third hour to 55C. Stir
1 part infusion broth, (see variant frequently.
(bb) 665). The mixture was steri-
Remove the major portion of the
(2)
lized for 60 minutes at 57C. on each liquid from the meat.
of 2 successive days.
(3) Heat meat and remaining liquid to
References: Stryker (1916 p. 50), Lucke
100C. This causes the meat par-
and Rea (1919 p. 535), Stevens (1921
ticles to shrink giving the maximum
p. 224), Harvey (1921-22 p. 82). amount of liquid.
(4) To the liquid (2) plus that of (3)
869. Foster's Serum Infusion Broth add 10.0 g. peptone, 5.0 g. K2HPO4,
Constituents: 2.0 g. Liebig's beef extract and
1. Beef infusion broth 1000.0 cc. 50.0 cc. of glycerol.
2. Glucose 10.0 g. (5) Adjust the reaction to pH of 7.5
3. Horse serum broth 5.0 g. to 7.8.
270 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(6) Distribute in 100.0 cc. lots in 872. Park, Williams and Krumwiede's
500.0 cc. Erlenmeyer flasks. Potato Infusion Broth
(7) Seed half the sterilized flasks with Constituents:
the grass bacillus of Karlensky and 1. Water 1000.0 cc.
the remainder with the glass bacillus 2. Meat infusion 1000.0 cc.
of Moeller. 3. Potato 1.0 lb.
(8) Incubate at 37.5C. for two weeks. 4. Peptone 20.0 g.
(9) Then heat to 100C.
5. NaCl 10.0 g.
(10) Cultures are then filtered and fil-
Preparation
trate made up to 1000.0 cc.
(1) Grate 1.0 pound of white potatoes
(11) Add 5.0 g. peptone, 2.5 g. K2HPO4, or run thru a chopping machine.
2.0 g. Liebig's beef extract, 50.0 cc. Soak in a liter of water over night.
(2)
glycerol and 5.0 g. of aminoid pep- Heat to boiling.
(3)
tone from beef (Arlco). Press thru cheese cloth.
(4)
(12) The reaction is left unchanged. (5) Add one egg per liter.
(13) Filter and distribute 50.0 cc. lots
(6) Autoclave one hour to clarify.
in 500.0 cc. Erlenmeyer flasks. (7) Filter thru cotton (very tedious).
(14) Add 5.0 cc. of sterile horse blood (8) Flask.
serum (serum must be raw) just (9) Soak 1 pound of finely chopped lean
before inoculation. beef in a liter of tap water over night
Sterilization: Sterilize (6) and (13) sepa- in the ice box or at room tem-
rately in the autoclave, time not given. perature.
Use: Preparation of johnin. Johnin is a (10) Weigh (9) and heat at 45C. for 1
material used for detection of Johnin's hour and then boil for 30 minutes.
disease, much as tuberculin is used for (11) Make up the loss in weight by the
tuberculosis. The medium was inocu- addition of hot water.
lated heavily with the organism causing (12) Strain thru cheese cloth and squeeze
Johnin's disease. Cover the flasks with by twisting the cloth or use a meat
tin foil to prevent evaporation and incu- press.
bate 10 to 16 weeks. (13) Mix equal parts of (8) and the juice
Reference: Beach and Hastings (1922 from (12).
(5) Incubate until a faint haze is formed (4) Heat to 80C. to stop digestion.
(usually 1 to 2 hours). (5) Pass thru a -layer of absorbent
(6) Cover with a layer of sterile paraffin. cotton.
Sterilization: Steam (6) at 100 for 15 to (6) The digest may be stored without
30 minutes. sterilization.
Use: Cultivation of Bacleriun pneumo- (7) Heat the filtrate from (5) to 70 C.
sintes. The medium was inoculated (8) Neutralize to litmus at 70C.
while the layer of paraffin was still (9) Mi.x an equal volume of (5) and infu-
liquid. sion broth, prepared according to
Reference: Olitsky and Gates (1922 p. 818), Park, Williams and Krumwiede
(see variant (ii) medium 779).
876. Ogata's Porphyra Infusion Broth
(10) Adjust to the desired reaction at
Constituents room temperature.
1. Meat infusion broth 1000.0 cc. (11) Autoclave for 15 minutes to clear.
2. Glucose 25.0 g. (12) Readjust the reaction as necessary.
3. Porphyra vulgaria 50.0 g. (13) Filter thru paper and cotton to
Preparation clear.
(1) Prepare meat infusion, using 500.0 g. Sterilization: Sterilize in the autoclave at
of meat per liter. 15 pounds pressure for 30 minutes.
(2) Dissolve 2 and 3 in (1). Use: General culture medium.
(3) Boil. Reference Park, Williams and Krumwiede
:
Constituents Preparation
1. Water 2000.0 cc. (1) Dissolve 200.0 cc. of strained horse
2. Meat 1.0 lb. blood clot, 10.0 g. peptone, 5.0 g.
3. Peptone (1.0%) 10.0 g. NaCl and 0.7 g. agar in sufficient
4. NaCl (0.5%) 5.0 g. beef infusion to make a liter.
5. Stomach (hog) 200.0 g. (2) Adjust the reaction to pH 5.4.
Preparation (3) Autoclave and filter while hot.
(1) Clean 5 pigs' stomachs, remove the (4) Distribute in 500.0 cc. lots in liter
fatand mince finely. (A number of flasks.
stomachs should be used to equalize (5) After sterilization, add sufficient
the peptone content. In this way sterile solutions of glucose and mal-
an almost average composition in tose to make a 3.0% solution of each
peptone is obtained.) of the sugars.
(2) Mix 200.0 g. of (1), 10.0 g. HCl, pure, (6) Inoculate with yeast and keep at
and 1000.0 cc. of water at 50C. room temperature until ready for
(3) Incubate at 50C. for 20 to 24 hours use. (One week is sufficient.)
in a glass or porcelain vessel, not (7) Place the yeast cultures at 55C. over
enamel. It is most important not night and then bring rapidly to the
to allow the digest to come in con- boiling point over a free flame.
tact with any metal until neutral- (8) Heat at 95C. for 15 minutes.
ized. (9) The autolysate may be filtered thru
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 273
1. Infusion broth 100.0 cc. beef in 1000.0 cc. water in an ice box
2. Bile, fresh ox 900.0 cc. over night.
3. Peptone 10.0 g. (2) Boil 30 minutes over a free flame.
4. Lactose 10.0 g. (3) Filter thru gauze and then thru paper
(Exact amounts not given.) (8) Mix 500.0 cc. of (6) and (7).
and while hot add 2.0% glucose and be alkaline after sterilization.
Sterilization Method not given.
:
1.0% tartaric acid.
Use: Cultivation of plant actinomyces.
(6)
Cool in a flask boil again and filter
Variants: The author gave the following
and tube hot.
variants:
Sterilization : Sterilize in the Arnold steril-
izer 3 times, one-half hour each time.
(a) Omitted the K2HPO4 and glycerol.
When a tube
(b) Omitted the glycerol, used 5.0 g.
is to be inoculated, sterilize
for I hour just before inoculation.
K2HPO4 instead of 2.5 g. and added
1.0 g. K2CO3, and 1.5 g. MgS04.
Reference: Richardson (1900-01 p. 72).
The medium was filtered and steril-
ized.
884. Siebert's Horse Meat Infusion Broth
Reference: Peklo (1910 p. 551).
Constituents:
1. Water 1000.0 cc.
886. Hida's Horse Meat Infusion Broth
2. Horse meat 250.0 g. Constituents
3. Peptone (Witte's) 10.0 g. 1. Water 1000.0 cc.
4. NaCl 5.0 g. 2. Meat, horse 500.0 g.
5. Glycerol 37.0 g. 3. Burdock root 40.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 275
(2) Filter.
1. Water 1000.0 cc.
Add 20.0 g. Witte's peptone and 5.0 g. 2. Blood clot (human) 500.0 cc.
(3)
NaCl to the bluish filtrate. 3. NaCl (0.5%) 5.0 g.
toxin production. Author reported that Sterilization: Sterilize (4) for 20 minutes
Constituents :
Use: Indol production. Dilute one part of
1. Water 1000.0 cc. (6) with 5 parts physiological salt solution
7. Pancreatin 1.0 g.
894. Frieber's Tryptophane Extract Broth
Preparation
(1) Heat 2, 3, 4 and 5 in 1 over boiling Constituents :
Use: Presumptive test for B. coli. En- (2) Neutralize and add 6.0 cc. of normal
richment medium in water analysis. soda solution per liter.
Author reported that best results were ob- (3) Dissolve 0.03% tryptophane in (2).
tained when pancreatin was present. Sterilization: Not specified.
B. coli produced gas. Use: Indol production. Author reported
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 277
(2) Adjust the reaction to a slightly alka- (2) Adjust reaction to pH = 8.0.
line reaction by the addition of (3) Tube in 10-12 cc. lots.
Na^COs. Sterilization: Sterilize at 15 pounds pres-
Sterilization: Not specified. sure for 15 minutes.
Use: Cultivation of obligate anaerobes. Use To study change in reaction by actino-
:
(7) Make up (6) to 1000.0 cc. isms found in ice cream to give the
278 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
mosus, Mucor rhizopodiformis, Mucor 909. Albus and Holm's Beef Extract
stolonijer. Medium (Medium X)
References Banning (1902 pp. 395 and 425),
:
Constituents
La Garde (1911-12 p. 248).
:
Preparation and 6 in 1.
(1) Dissolve
:
2, 3, 4, 5
(1) Prepare a sugar free bouillon con- (2) Divide in four and keep one as
lots,
taining 1% Liebig's meat extract, 1% a control. To the other three add
peptone and 0.5% NaCl. varying quantities of sodium recino-
(2) Add about 20.0 cc. N/1 NaOH to late to lower the surface tension.
neutralize. Surface tension determined by weight
(3) Boil for about one hour. drop method following sterilization.
(4) Filter. Sterilization: Method not given.
(5) Add 1% mannitol and 5.0% litmus Use: To determine the eflfect of surface
solution. tension on growth of Lactobacillus bul-
(6) After sterilization, distribute in ster- garicus and Lactobacillus acidophilus.
ile tubes. The author reported that L. bulgaricus
steam bath.
Sterilization: Sterilize in a
was inhibited at a surface tension lower
Use: To determine fermentation of man- than 40 dynes while L. acidophilus ex-
nitol by the typhoid bacilli. hibited good growth in the same media
Reference: Jacobson (1910 p. 211). depressed to 36 dynes.
907. Harrison and van Der Leek's Citrate Variants: The authors used sodium tauro-
Aesculin Extract Broth cholate and sodium glycocholate instead
of sodium recinolate to lower the sur-
Preparation and composition the same face tension.
as for medium 891 except no bile salt Holm
Reference: Albus and (1926 p. 14).
is added.
908. De Gaetano's Potato Extract Broth 910. Flint's Serum Extract Broth
(Kamen) Constituents :
Ascitic fluid (5.0%) 50.0 cc. (6) Dissolve 75.0 and 25.0 g.
g. lactose
6.
Preparation NaCl Kubel and Tie-
in 600.0 cc. of
(3) Adjust to pH = 6.4, 6.8, 7.2, 7.6 or 8.0. method not given. Sterilize (7), method
(4) Heat for 15 minutes at 15 pounds not given.
pressure. Use: Indol production in water analysis.
(5) Filter. Before use mix 5 parts of sterile (5) with
Tube and sterilize. 6 parts of sterile (7) and distribute in
(6)
Sterilization: Sterilize (1) in the autoclave 10.0, 5.0, 2.5, and 1.0 cc. lots in such sized
by heating at 20 pounds pressure for 15 containers so that 100.0, 25.0 and 10.0 cc.
minutes. Sterilize the ascitic fluid by of water under investigation may be
autoclave. p. 312).
Use: Isolation of bacteria from infected Use: Enrichment of typhoid from the
teeth. blood. Saponin is added to lake the
Reference: Haden (1923 p. 831). erythrocytes.
Reference Otaki and Akimoto (1922
: p. 101)
914. Hoffmann and Fischer's Nutrose taken from (1923 p. 331).
Extract Broth (Heinemann)
917. Dimitroff's Egg Sea Water Medium
Constituents:
1. Water, tap 1000.0 cc. Constituents
2. Peptone 10.0 g. 1. Sea water (75.0%) 750.0 cc.
3. Extract, meat 3.3 g. 2. Extract broth (25.0%) 250.0 cc.
4. Nutrose (1.0%) 10.0 g. 3. Egg white.
5. Caffein (0.5%) 5.0 g. Preparation:
6. Crystal violet 1.0% solution. (1) Place small cubes of hard boiled egg
Preparation white in 10.0 cc. of beef extract broth.
(1) Prepare e.xtract broth according to (2) Mix 25.0% of sterile (1) with 75.0%
Heinemann's method using 1, 2 and sterile sea water.
923. Rivas' Glucose Bouillon (15) Distribute in tubes, and cover with
Constituents: a layer of sterile olive oil. Plug
1. Bouillon 958.0 cc. with cotton and seal with gum.
2. Glucose 10.0 g. This process should be done quickly.
3. Peptone 15.0 g. (16) Incubate at 37C. for 48 hours dis-
4. Ammono-sulpho-hydrate carding any tubes showing turbidity
water 40.0 cc. or any sign of color.
5. Sodium indigo sulphuric Sterilization: Sterilize (6) at 120C. for
acid (10.0% soln.) 2.0 cc. 15 minutes.
Preparation Use: Cultivation of anaerobes.
(1) Prepare bouillon. Variants The author omitted the ammono-
:
(2) Add 1.0% dextrose and 1.5% pep- sulpho-hydrate (steps (5) thru (14)), but
tone. prepared a 1.0% NaiSOs solution and
(3) Prepare a 10.0% sodium indigo sul- added 50.0 cc. to 948.0 cc. of glucose
phuric acid solution in distilled bouillon containing 2.0 cc. of a 1.0%
water. solution of sodium indigo sulphuric.
(4) Heat (3) at 100C. for one hour. The medium was covered with olive oil
(5) Prepare a 1.0% ammonia solution in as specified above. This medium is in-
sterile distilled water. one described above.
ferior to the
(9) To a series of tubes add 1 drop, minutes on each of two successive days,
2 drops, 3 drops, etc., of (5) to each taking care not to over heat.
tube. Shake thoroughly. Use: Enrichment of colon-typhoid group.
(10) To each tube of (9) add 3 drops of a Variants: Author added 10.0 or 20.0 g. KI
10.0% methylene blue solution in instead of 5.0 g.
Use: Presumptive test for B. coli. Author Reference: Holzel (1913 p. 149).
reported that dye inhibited other organ-
isms. 935. Heinemann's Glycerol Bouillon
References Hall and Ellefson (1918
: p. 337),
Constituents
Levine (1921 p. 110).
1. Bouillon 1000.0 cc.
932. Jouan's Citrate Lactose Bouillon 2. Glycerol (6.0%) 60.0 g.
(Besson) Preparation: (1) Add 6.0% glycerol to
bouillon.
Constituents:
Sterilization: Not specified.
1. Water 850.0 cc.
Use: General culture medium.
2. Bouillon 150.0 cc.
Variants
3. Lactose 10.0 g.
(a) Kendall, Day and Walker specified
4. Magnesium citrate 1-0 g.
the use of sugar free bouillon and
5. Litmus.
used 30.0 g. of glycerol instead of
Preparation
60.0 g. per liter.
(1) Dissolve 150.0 cc. of bouillon, 10.0 g.
(b) Owen also used a 3.0% glycerol bouil-
lactose and 1.0 g. of magnesium ci-
lon to determine the amount of
trate in 850.0 cc. of water.
alcohol formed in the fermentation
(2) Add sufficient litmus solution to give
of glycerol by Bacillus saccharalis.
a violet color.
References: Heinemann (1905 p. 127),
(3) Filter.
Kendall, Day and Walker (1914 p. 419),
(4) Tube.
Owen (1916 p. 241), Dopter and Sac-
Sterilization: Sterilize at 110 to 112C.
quepee (1921 p. 119), Stitt (1923 p. 35),
Use: Synthetic whey.
Cunningham (1924 p. 165).
Reference: Besson (1920 p. 208).
933. Wollman's Starch Bouillon 936. Kendall, Day and Walker's Mannitol
Bouillon
Constituents:
1. Water 600.0 cc. Constituents
2. Bouillon 400.0 cc, 1. Bouillon 1000.0 cc.
3. Starch 30.0 g. 2. Mannitol 10.0 g.
(2) Add 400.0 cc. of (1) to 600.0 cc. of (3) Distribute in 100.0 cc. lots.
400.0 cc. of distilled water with the in the proportion of 1:10 and 1:100.
(9) Dilute each of the extracts in (2) Use: To study bacterial nutrition of Bacil-
in the proportions 1:10 and 1:100. lus influenzae. Author reported that
Sterilization: Sterilization of pea or bean growth in unheated potato occurred
extract effected in steps (6) and (7) in equally well without the addition of yeast
preparation. Sterilization of bouillon extract. When the potato was heated at
not given. 120 for 45 minutes no growth occurred
Use: To study bacterial nutrition of unless the yeast extract was added.
Bacillus influenzae. Author reported Medium did not give continued growth.
that the medium supported growth of Initial inoculation made from blood
B. influenzae. Growth accessory ma- broth.
terials resisted boiling for 10 minutes, Variants: The authors omitted the yeast
were destroyed by autoclaving, would extract.
pass thru a Berkefeld filter, but were Reference: Thjotta and Avery (1921
absorbed by charcoal. Broth containing p. 109).
extracts alone, however, did not give
continued growth of the influenza bacilli. 943. Thjotta's Bacterial Emulsion Bouillon
Primary inoculations were made from Constituents
blood broth. 1. Bouillon.
p. 100). Preparation
(1) Prepare bouillon with a reaction of
942. Thjotta and Avery's Potato Bouillon
pH = 7.8.
Constituents: (2) Grow bacteria (mucoid bacillus
1. Distilled water 400.0 cc. (Friedlander's), Bacillus ozoenoe and
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 289
Sterilization: Sterilization of bouillon not (d) Bull and Pritchett added fragments
given. of sterile skeletal muscle of the pigeon
Use To study bacterial nutrition of Bacillus
: or rabbit to 100.0 cc. quantities of
influenzae. Author reported no growth bouillon. They used the medium for
in dilution of 1 to 10,000 of blood cells. the production of toxin by Bacillus
Reference: Thjotta and Averj' (1921 welchii. Medium
incubated in a
p. 109). vacuum from which the oxygen
jar
has been removed. They also used
947. Smith, Brown and Walker's Tissue the above medium with the addition
Bouillon
of 0.0 to 1.0% glucose, 0.2 or 0.3%
Constituents giving the best results.
1. Bouillon. References: Smith, Brown and Walker
2. Tissue, Rabbit or Guinea Pig. (1905-06 p. 196), Wrzosek (1905 pp. 1268-
Preparation 1270), Harrass (1906 p. 2237), Bull and
(1) Prepare ordinary bouillon. Pritchett (1917 pp. 129, 871, 873, 875),
(2) Tube in fermentation tubes. G6zony (1920 p. 566).
(3) Kill a rabbit or guinea pig by chloro-
948. Orr's Glucose Heart Bouillon
form.
(4) Moisten the animal thoroly with Constituents:
water. 1. Bouillon 1000.0 cc.
(5) Reflect the skin and open the abdo- 2. Glucose (1.0%) 10.0 g.
men with sterile instruments. 3. Heart, beef 100.0 g.
(6) Remove the liver, spleen, kidneys and 4. Marble
bits of tissue with sterile forceps. Preparation
(7) Place these bits of tissue, just large (1) Prepare 1.0% glucose bouillon.
enough to pass into the narrow part (2) Add (1) to 100.0 g. of finely minced
of the fermentation tubes. The tis- beef heart.
sue is pushed into this narrow por- (3) Adda quantity of marble chips.
tion, but not necessarily beyond. (4) Adjust to pH = 8.0.
(8) Incubate two or more days to test (5) Cover with a layer of paraffin oil.
sterility. Sterilization: Method not given.
Sterilization: Sterilization of bouillon not Use: Cultivation of B. botulinus and toxin
given. production. Author reported that
Use: Cultivation of anaerobes and flag- M.L.D. varied from 0.0001 cc. to 0.05 cc.
ellates. depending on the strain used.
Variants : Reference: Orr (1920-21 p. 128).
(a) Wrzosek reported that the addition of
949. Park, Williams and Krumwiede's Meat
sterile tissue to a medium permitted
Medium
the growth of obligate anaerobes.
(b) Harrass sterilized at 100 C. for 90 Constituents
to 120 minutes the medium after the 1. Bouillon.
of flagellates. 3. Tissue.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 291
(5) Prepare casein digest according to the NaOH to slightly clarify, but still
Reference: Kahn (1922 p. 174), Torrey and (4) Sterilize (method not given).
Kahn (1923 p. 483). (5) Thoroly mix 1 part egg yolk with
10 parts distilled water.
955. Besredka and Jupille's Egg Bouillon (6) Add 1.0 cc. normal soda solution
(Besson) to 100.0 cc. (5).
Constituents (7) Boil.
1. Bouillon 500.0 cc. (8) Filter.
2.Egg white (10.0% soln.) 400.0 cc.
(9) Sterilize (method not given).
3. Egg yolk (10.0% soln.) 100.0 cc.
(10) Mix 4 parts (4) with 1 part (9)
Preparation: (1) Mix 5 parts of bouillon and 5 parts nutrient bouillon.
with 4 parts of a 10.0% egg white solution
References: Besson (1920 p. 55), Stitt (1923
and 1 part of 10.0% egg yolk emulsion in Klimmer (1923 p. 202).
p. 34),
sterile water.
Sterilization: If the egg whiteand yolk are 956. Robertson's Alkaline Egg Bouillon
not obtained under aseptic conditions,
sterilize by heating at 55C. Constituents
Use: Cultivation of pneumococci, gono- 1. Water 1000.0 cc.
cocci, meningococci, various other highly 2. Eggs 4
parasitic forms as well as saprophytic 3. NaOH (normal) 12.0 cc.
organisms. 4. Bouillon
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 293
(2) Add 1 part (1) to 5 parts bouillon. (b) Wollstein cultivated Pfeiffer bacillus
Sterilization: Method not specified. on a medium prepared as follows:
Use: Cultivation of cholera organism. (1) Boil fresh rabbit blood for 2 minutes
Reference: Park, Williams and Krumwiede in a water bath.
(1924 p. 126). (2) Centrifuge.
(3) Add 0.5 cc. of the pale pink or yel-
957. Piorkowski's Alkaline Egg Albumin low fluid resulting from (2) to
Bouillon
20.0 cc. of bouillon adjusted to
Constituents pH = 7.8.
1. Bouillon 1000.0 cc. (c) Tanner and Besson added 0.5, 0.3 or
2. Glucose (1.0%) 10.0 g. 0.25 the volume of blood (or serum)
3. Peptone (2.0%) 20.0 g. to bouillon.
4. Egg albumin (2.0% dry) (d) Besson prepared a medium for the
5. NaOH cultivation of pneumococci, meningo-
Preparation cocci and gonococci as follows:
(1) Prepare a 1.0% glucose bouillon con- (1) Collect 400.0 cc. of beef blood in a
taining 2.0% peptone. sterile flask containing 30.0 cc. of a
(2) Distribute in 5.0 cc. lots. 1.0% ammonium citrate.
(3) Prepare a 2.0% solution of dry egg (2) Shake thoroly.
albumin in tap water. (3) Store for several hours at room
(4) Add 1.0 cc. (3) and 20.0% of a N/10 temperature.
NaOH solution. (4) Dilute (3) with 3 volumes of sterile
Sterilization: Method not given. physiological salt solution.
Use: Enrichment of streptococci from the (5) Add 1 part (4) to 15 parts sterile
blood. bouillon.
Reference: Piorkowski (1922 p. 69). (e) Thjotta and Avery boiled whole blood
for 10 minutes, centrifuged and
958. Hibler's Blood Bouillon
diluted the supernatant fluid 1 to 10,
Constituents to 1:10,000 with bouillon adjusted to
1. Bouillon. pH = 7.8. He reported no growth
2. Blood, dog. with dilutions of 1:1000 or greater.
Preparation References: Hibler (1899 p. 604), Davis
(1) Collect dog blood in sterile test tubes (1903 p. 405), Wollstein (1919 p. 556),
in about 5 cc. lots. (Blood may be Tanner (1919 p. 47), Besson (1920 p. 31),
collected in beaker and then the finely Thjotta and Avery (1921 p. 109).
divided clot with a corresponding
959. Dieudonne's Alkaline Blood Bouillon
amount of serum, be distributed in
tubes. Constituents:
(2) Add a few drops of bouillon (or 1. Bouillon 500.0 cc.
water). 2. Blood (defibrinated beef).... 250.0 cc.
Sterilization: Sterilize by heating at 58 or 3. KOH(normal) 250.0 cc.
97C. Before use heat the tubes for 15 Preparation
minutes in flowing steam. (1) Add 250.0 cc. of normal KOH to
Use: Cultivation of anaerobes and various 250.0 cc. defibrinated beef blood.
parasitic forms. Author reported that (2) Adjust nutrient bouillon (or peptone
the anaerobes grew better in a medium solution) to neutral point to litmus.
sterilized at 58C. Fresh blood gave Sterilization: Sterilization not specified.
best results. Author mentioned that (1) might be
Variants sterilized in the autoclave.
(a) Davis mixed one-third volume of Use: Enrichment of cholera vibrio.
294 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Variants: Kraus, Zia and Zubrzciky incu- 961. Fildes' Pepsinized Blood Bouillon
bated the flasks for 3 hours at 50C. for Constituents
24 hours at 37C. before use. 1. Bouillon
References Dieudonnc (1909 p. 108), Kraus,
:
2. Saline solution 150.0 cc.
Zia and Zubrzciky (1911 pp. 1084-1085). 3. Blood, defibrinated, sheep... 50.0 cc.
Preparation
960. Orcutt and Howe's Fat Blood Bouillon
(1) Place 150.0 cc. of saline solution, not
distilled water, 6.0 cc. pure HCl,
Constituents
1. Bouillon.
50.0 cc. defibrinated sheep blood and
1.0 g. pepsin (B.P. granulated) in a
2. Blood, whole defibrinated.
250.0 cc. bottle fitted with a well
3. Cream.
Preparation :
ground glass stopper. Add ingre-
dients in order specified.
(1) Distribute sterile bouillon in 1.0 cc.
(6) Add 0.02 cc. of (5) to each tube of (3). (6) Add pure HCl, drop by drop until
Sterilization: Sterilization of bouillon not
pH 7.0 to 7.2 is reached, using phenol
100C. for 20 minutes on each of 3 suc- (7) Add 0.25% chloroform and dissolve
cessive days.
by shaking.
Use: To study the influence of fat on (8) When ready for use, shake the flask
hemolysis by staphylococci. Authors and remove the pepsinized blood with
a sterile pipette. Add from 2.0 to
reported that with the organism studied
hemolysis took place in the presence of 5.0% to bouillon.
whole milk, cream, butter, olive oil and
Sterilization: Not specified.
sions to bouillon so that each cubic (5) Add a sufficient quantity of sterile
cm. of bouillon shall contain 0.002 1.0% Kahlbaum's purified litmus
cc. of fat. solution to impart a distinct color to
Reference: Orcutt and Howe (1922 p. 412). the whole.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 295
(6) Incubate 2 or 3 days to detect any- (1) Mix one part rabbit serum with 2
accidental contamination. parts physiological salt solution.
Sterilization: Sterilize (2), (3) and litmus (2) Prepare a bouillon containing 10.0%
solution separately in the Arnold on each peptone.
of 3 successive days. (3) Add 1.0% of (2) to (1).
Use: To study fermentation by diplococci. (4) Adjust (3) to pH = 7.2.
Reference: Dunham (1906 p. 19). (5) Distribute into 3 to 4.0 cc. lots into
test tubes approximately 1.0 cm. in
963. Shmamine's Liver Serum Bouillon
diameter.
Constituents (6) Inoculate with one drop of infected
1. Bouillon 100.0 cc. blood, or if subcultures are being
2. Sodium nucleate 1.0 to 2.0 g. made, add a drop of fresh rabbit
3. Physiological salt soln. 20.0 cc. blood and rotate the tube gently.
4. Serum, horse 100.0 cc. (This furnishes the necessary fibrin.
5. Liver, rabbit Instead of fibrin, 0.05 to 0.1% agar
Preparation may be used.)
(1) Dissolve 1 to 2.0 g. of the sodium salt (7) Cover with a layer of oil about
of nucleic acid in 20.0 cc. of physio- 1.5 cm. high.
logical salt solution. (8) Incubate at 28-30C.
(2) Boil in the water bath for 15 minutes. (b) Klimmer added one part serum to 3
(3) Mix with 100.0 cc. of sterile clear
(2) to 20 parts bouillon, under aseptic
transparent horse serum. conditions.
(4) Add a small piece of rabbit liver that (c) Stitt cultivated Borrelia recurrentis
has been burned off in the flame. on a medium prepared as follows:
(5) Mix equal parts sterile bouillon (1) Mix three parts of a 1.0% peptone
and (4). bouillon with one part rabbit or
(6) Following final sterilization pass C0> horse serum (ascitic fluid may be
thru the tubes for 2 minutes and then used).
seal with rubber stoppers and para- (2) Tube in tall tubes.
ffin. (The fact that the medium was (3) Cover with a layer of oil not to e.x-
to be tubed and a small piece of rab- ceed 1.5 cm. in height.
bit liver added to each tube was not References: Besson (1920 p. 31), Kligler
specified.) and Robertson (1922 p. 315), Klimmer
Sterilization: Method of sterilization of (1923 p. 200), Stitt (1923 p. 54).
serum or bouillon not specified. Sterilize
965. Veillon's Ascitic Fluid Bouillon
(5) for one hour on each of 3 successive
days at 60 C. Constituents:
Use: Cultivation of spirochetes. 1. Bouillon 1000.0 cc.
Reference: Shmamine (1912 p. 323). 2. Ascitic fluid 1000.0 cc.
Preparation
964. Besson's Serum Bouillon
(1) Adjust the reaction of bouillon to be
Constituents slightly alkaline.
1. Bouillon. (2) Add an equal amount of sterile ascitic
2. Serum. fluid to sterile (1).
Preparation: (1) Mix one-half, one-third or Sterilization: Method of sterilization of
one-fourth the amount of sterile serum bouillon or ascitic fluid not given.
with ordinary sterile bouillon under Use: Cultivation of gonococci and other
aseptic conditions. highly pathogenic organisms.
Sterilization: Not specified. Variants
Use : General culture medium. (a) The author mixed bouillon with its
Variants volume of ascitic fluid.
(a) Kligler and Robertson cultivated (b) Elser and Huntoon isolated meningo-
Spirochaeta obermeieri on a medium cocci from blood or spinal fluid in a
prepared as follows: mixture of 1000.0 cc. of ascitic fluid
296 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
and 2000.0 cc. of bouillon with a re- inoculated thru the liquid vaseline and
action of +0.6 to phenolphthalein. cooled quickly, incubated for 24 hours
(c) Besson mixed ^, 3 or J the volume of and 0.1, 0.2, 0.4 and 0.5 cc. of the culture
sterile ascitic fluid with sterile transferred to 1.0 cc. of a 5.0% suspension
bouillon. of washed fresh sheep erythrocytes. Add
(d) Kligler and Robertson cultivated sufficient physiological salt solution to
Spirochaeta obermeieri in a medium each tube to bring the volume to 20.0 cc.
prepared as follows: and incubate at 37C. for one hour.
(1) Prepare a 10.0% peptone broth Reference: Kahn (1922 p. 198).
(exact method not given).
967. Lyall's Carbonate Ascitic Fluid
(2) Add 1.0% of (1) to undiluted ascitic
Bouillon
fluid.
(6) Cover with a layer of oil about Use: To determine hemolysis by strepto-
cm. high.
1.5 cocci. Grow the organism in the medium
They reported that the spirochetes for 18 hours. Add a definite amount of
grew well at 37C. but degeneration the 18 hour broth culture to 1.0 cc. of a
changes tended to set in early. 5.0% solution of washed red blood cells
After the culture was well started, of a sheep. Incubate in a water bath for
growth proceeded well at room 1 hour at 37.5C. Author reported that
temperature. three degrees of hemolysis are possible (1)
References: Veillon (1898 p. 24), Elser and complete solution of erythrocytes, (2)
Huntoon (1909 p. 382), Besson (1920 p. 31), change of undissolved cell from a bright
Kligler and Robertson (1922 p. 315). red to a dark brown due to a transforma-
tion of oxyhemoglobin to methemo-
966. Kahn's Casein Digest Ascitic Fluid globin, but cells are not dissolved, (3) no
Bouillon hemolysis or change in color of the cells.
Constituents Reference: Lyall (1914 p. 497).
1. Bouillon 1000.0 cc.
968. Roddy's Bile Bouillon
2. Casein (2.0%) 20.0 cc.
3. Ascitic fluid 200.0 cc. Constituents:
4. CaCOs 1. Bouillon 1000.0 cc.
Preparation 2. Ox bile 1000.0 cc.
(1) Preparation of bouillon not given. Preparation: (1) Mix equal parts of ox bile
(2) Mix 1 part ascitic fluid and 5 parts and bouillon.
(1) adjusted to +0.3. Sterilization: Sterilize in the steam steril-
(3) Add CaCOs (amount not given). izer for 20 minutes on each of 3 successive
(This is Lyall's broth.) days.
(4) Add 2.0% casein digest as prepared Use: Culture medium for parasitic and
by Kahn (see 648). saprophytic organisms.
(5) Tube. Reference: Roddy (1917 p. 42).
(6) Seal the tubes with a vaseline cap by
969. Mayer's Mucin Bouillon
boiling the medium and vaseline.
Sterilization: Not specified. Constituents:
Use: To determine hemolysis by spore 1. Bouillon.
dividuals stimulated the growth of some (3) Emulsify 100.0 g. of brewer's yeast
organisms. Saliva treated in a similar in 400.0 cc. distilled water with the
manner did not. reaction of pH = 4.6.
Reference: Kligler (1919 p. 40). (4) Boil over a free flame for 10 minutes.
298 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Allow to sediment at room tem- (10) Dilute the supernatant fluid from
perature. (9) invarying proportions from 1 10 :
Use : To study bacterial nutrition of Bacil- 977. Savini and Savini-Castano's Bacteria
lus influenzae. Authors reported growth Blood Bouillon
in a blood cell dilution of 1:100,000.
Constituents:
Reference: Thjotta and Avery (1921
1. Bouillon.
p. 109). 2. Glycerol.
976. Thjotta and Avery's Yeast Hemoglobin 3. Blood.
Bouillon 4. Staphylococcus aureus.
Preparation
Constituents
(1) Place 5.0 cc. of glycerol in a small
1. Distilled water 410.0 cc.
Erlenmeyer flask containing glass
2. Brewer's yeast 100.0 g.
beads.
3. Bouillon
(2) Wash the growth from 2 or 3 cultures
4. Hemoglobin (crystalline) .... 1.0 g.
of Staphylococcus aureus (24 to 48
Preparation
hours cultures at 37C.) and place in
(1) E.xact method of preparation of
sterile (Bouillon cultures may
(1).
bouillon not given.
be used.)
(2) Adjust the reaction of (1) to pH Place in a paraffin oven at 58 to GO'C.
(3)
= 7.8.
over night until the bacteria are
(3) Emulsify 100.0 g. of brewer's yeast
kUled.
in 400.0 cc. distilled water with the
(4) Add 3.0 to 4.0 cc. of blood freshly
reaction of pH= 4.6.
drawn under aseptic conditions to (3).
(4) Boil over a free flame for 10 minutes. Shake until the blood is coagulated
(5)
(5) Allow to sediment at room tem- completely.
perature.
(6) Heat at 58 to 60C. for about an hour.
(6) Pipette off the clear supernatant
(When heating here and in step (3)
extract and test sterility.
place the cotton stopper in the tube
(7) Store in the ice chest until ready for or flask lightly to allow evaporation
use. Readjust the reaction of the of water.)
extract from pH = 7.3 to 7.5 just
(7) Add sufficient of (6) to sterile bouillon
before use.
until the bouillon takes on a weak
(8) Prepare crystalline hemoglobin by red color.
the method of Walker and William-
Sterilization: Method of sterilization of
son. J. Biol. Chem., 41: 75, 1920.
glycerol not specified. Method of steril-
(9) Prepare a 10.0% solution of crys- ization of bouillon not given.
talline hemoglobin in water by
Use: Cultivation of influenza bacilli.
weight.
Variants: The author prepared a similar
(10))Dilute sterile (9) with sterile bouil-
medium using a hemoglobin solution in-
lon in varying proportions from
stead of blood. The powdered hemo-
1:10 to 1:200,000.
globin was sterilized at 100C. in the hot
(11) Add yeast extract (7) amount not air sterilizer after drying in the desic-
given.
cator. The powdered hemoglobin was
Sterilization: Method of sterilization of
crushed in a sterile mortar with solution
bouillon not given. Sterilization of yeast
I,
obtained in step (3) above. Filter thru
infusion effected by boiling in step (4).
sterile filter paper. This solution was
the hemoglobin solution thru a
Filter
used instead of blood as indicated in
Berkefeld filter to sterilize.
step (6) and (7).
Use: To study bacterial nutrition of Bacil- and Savini-Castano
Reference: Savini
lus influenzae. Authors reported no
(1911 p. 494).
growth of B. influenzae without yeast
extract. Growth when hemoglobin solu-
SUBGROUP I-C. SECTION 14
tion diluted 1:200,000 (with yeast extract
present). Liquid media or basal solutions contain-
Reference: Thjotta and Avery (1921 ing a digest or autolysate other than a com-
p. 109). mercial digest.
300 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Ai. Containing digests of plant origin only. El. Stomach only, digested.
Bi. Yeast digest employed. Martin's Stomach Digest Solution
Ci. No salts added. (Martin's peptone) 998
Kammen's Yeast Digest (Klimmer). 978 E2. Liver digested.
Ayers and Rupp's Yeast Digest Martin's Liver Digest Solution 999
Solution 979 Seallards and Bigelow's Liver Digest
Robertson's Glucose Asparagin Blood Solution 1000
Solution 980 Dubovsky and Meyer's Liver Digest
Kister's Yeast Digest 981 Solution 1001
Cj. Salts added. E3. Spleen digested.
Kligler's Yeast Autolysate Solution. 982 Emery's Spleen Digest Solution 1002
Jotten's Yeast Autolysate Solution. . 983 E4. Blood clot digested.
Abt and Blanc's Yeast Autolysate Stickel and Meyer's Blood Clot
Solution 984 Digest Solution 1003
Robertson and Davis' Yeast Autoly- Bramigk's Peptic Digest Solution. 1004 .
Douglas Trypsin Broth (Hartley).. 1123 (6) Neutralize the liquid by the addition
Ds. Casein or milk digest specified. of NaOH.
Harvey's Basal Trypsinized Casein (7) Heat at 50 until all the liquid has
Solution 1124 evaporated.
Teruuchi and Hida's Trypsinized (8) Heat at 100 to 105C. for one hour.
Casein Solution 1125 (9) Pound to a powder.
Zipfel's Trypsinized Casein Solu- Sterilization: Material remains sterile in
tion 1126 powdered form.
Bacto Tryptophane Broth (Dehy- Use: Substitute for meat peptone.
drated) 1127 Variants
Bacto Peptonized Milk (Dehy- La) Klimmer added the supernatant fluid
drated) 1128 of (2) to the liquid obtained in step
Mueller's Trypsinized Casein Solu- (6) and prepared a powder as indi-
tion 1129 cated above,
Cole and Onslow's Trypsinized Ca- (b) Klimmer dissolved 10.0 g. of the
sein Solution 1130 powder obtained above, 8.0 g. NaCl
Berman and Rettger's Trypsinized and 0.1 g. KNO3 in a liter of water.
Casein Solution 1131 He specified that this medium could
Norris' Trypsinized Caseinogen be used as ordinary bouillon or might
Solution 1132 be used in the preparation of special
Cannon's Trypsinized Casein Solu- media.
tion (Norton and Sawyer) 1133 Reference: Klimmer (1923 p. 170).
De. Digest of a variety of materials spec-
979. Ayers and Rupp's Yeast Digest
ified.
Solution
Duval and Harris' Tryptic Digest
Solution 1134 Constituents
Harvey's Tryptic Digest Solution. 1135 . 1. Water 1000.0 cc.
Stickel and Meyer's Tryptic Digest 2. Yeast 100.0 g.
Solution 1136 Preparation
C3. Both peptic and tryptic digests added. (1) Add 100.0 g. dry fresh yeast to
Stickel and Meyer's Digest Solution 1137 1000.0 cc. distilled water with 1.0 g.
Frieber's Digest Extract Solution. . 1138 pepsin. Add HCl to obtain pH =
A3. Containing digests of both plant and 4.4.
animal origin. (2) Incubate at 40 for 24 hours.
Davis and Ferry's Hydrolyzed Gli- (3) Steam for 30 minutes.
adin Solution 1139 (4) Filter.
Robinson and Rettger's Hydrolyzed (5) Dilute to 1000.0 cc. with distilled
Edestin Casein Solution 1140 water.
(6) Adjust reaction to pH = 7.5.
978. Kammen's Yeast Digest (Klimmer) Sterilization: Method not given.
Constituents Use: General culture medium. Author re-
1. Water 20,000.0 cc. ported that this medium gave very good
2. Yeast 10,000.0 g. growth of the delicately growing strepto-
Preparation cocci.
(1) Autoclave 10,000.0 g. yeast with 20 Reference: Ayers and Rupp (1920 p. 96).
liters of water for 2 hours at 1.5 to 1
980. Robertson's Glucose Asparagin
atmospheres pressure.
Solution
(2) Allow to settle and decant the liquid.
(3) Add pepsin and about 10 liters of Constituents:
0.5% HCl solution to the thick yeast 1. Distilled water (sterile). . . 1000.0 cc.
residue from (2). 2. Asparagin (Merck) 3.4 g.
(4) Incubate (3) for 5 days at 37C. 3. CaCl2 0.1 g.
(5) Allow to settle and decant the liquid 4. Glucose 20.0 g.
or filter. 5. MgS04 0.2 g.
302 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(10) Add water to the sediment and results. It might also be used as a
centrifuge. basis for the preparation of special
(11) Remove the supernatant liquid of media. For the enrichment of
(10) and add to the liquid obtained cholera allow the autolysis to con-
in (8). This finally leaves about 35 tinue for 3 days instead of 2 as indi-
to 40 liters of extract. cated in step (6).
(12) Boil for 90 minutes in the steamer Reference: Jotten (1920 p. 359), Klimmer
and make slightly alkaline to litmus. (1923 p. 171).
(13) Filter thru paper or cotton.
(14) Add 8.0 g. NaCl, 0.1 g. KNO3 and 984. Abt and Blanc's Yeast Autolysate
0.2 g. NasCOg per liter. Solution
Sterilization: Sterilize in the steamer on
Constituents:
each of 3 successive days.
1. Water.
Use: Meat extract substitute in the prepa-
2. NaCl (0.9%).
ration of general culture media.
3. Yeast (Brewers).
Variants
Preparation
(a) The author evaporated (1) to a quick
mass, similar to meat extract, using a (1) Wash yeast in water and decant after
Stir the yeast mass with water. (10) Heat for 15 minutes at 115C.
(5)
Distribute in 5 liter flasks and (11) Filter.
(6)
incubate at 45C. for two days. (12) Evaporate 10.0 cc. of the filtrate to
Pour off the brown colored liquid. dryness to determine the dry ma-
(7)
Thoroly wash the residue with terial. Calculate the dry material
(8)
in 100.0 cc.
water, and centrifuge out the
residue. (13) Dilute (11) so as to have 2.1 or even
0.5 parts of dry material per 100.0 cc.
(9) Mix the liquid from (7) and (8).
(14) Distribute as desired.
Total volume 30 to 40 liters.
Boil for one hour in the steamer. Sterilization: Sterilize at 110 for 15
(10)
Filter thru cotton or paper. minutes.
(11)
(12) Neutralize to litmus. Use: General culture medium for colon-
(13) Add 0.8% NaCl, 0.01% KNO3 and typhoid group, parasitic and saprophytic
0.02% Na2C03 to (12). forms and others.
(14) Sterilize on each of three succes- Variants: Robertson and Davis prepared
sive days. a similar medium as follows:
(15) The liquid from (9) may be evap- (1) Grow yeast on dextrose agar slants.
orated to a thick brown paste, (2) Suspend in sterile distilled water,
and using 15.0 g. per liter to pre- throw down in centrifuge and wash
pare the medium. in sterile distilled water 3 times.
Klimmer reported that the medium (3) Kill yeast by heating at 70 C. for
might be used instead of ordinary 10 minutes. (The suspension con-
nutrient bouillon and give very good tained about 500,000 per cc.)
304 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
to (5). Amount of each not given. (1) Hydrolyze gelatin with 25.0% H2SO4
They used the medium to study the for 24 hours on sand bath. (Tem-
influence of vitamines on bacterial perature not given.)
growth. (2) Add Ba(OH) 2 until alkaline and filter.
Reference: Abt and Blanc (1921 p. 452), (3) Neutralize exactly the Ba(0H)2 with
Robertson and Davis (1923 p. 154). 10.0% H2SO4. Filter.
(4) Test filtrate for presence of free Ba
985. Robertson and Davis' Yeast Autolysate and SO4 ions.
Solution Concentrate to a thick syrup in a
(5)
Constituents vacuum.
1. water to.
Sterile distilled . 1000.0 cc. (6) Dilute to 2.0% solid material with
2. Asparagin (Merck) 3-4 g distilled water
3. CaCU 0.1 g (7) To each liter of (6) add 3, 4, 5, 6, 7
4. Dextrose 20.0 g and 8.
986. Davis and Ferry's Hydrolyzed Gelatin 988. Capaldi and Proskauer's Mannitol
Solution Hydrolyzed Gelatin Solution
Constituents : Constituents
1. Distilled water 1000.0 cc. 1. Water 1000.0 cc.
2. Hydrolyzed gelatin (syrup). 20.0 g. 2. Mannitol (0.1%) 1.0 g.
Preparation (7) Mix 100.0 cc. (6) with 1000.0 cc. water.
(1) Dissolve 2 in 1. (8) Method of sterilization not given.
(2) Boil gelatin in 1.0% HCl, H2SO4, or This medium is inferior to opsine as
HNO3 for 6 hours. a general culture medium.
(3) Add 2.0% of (2) to (1). Reference: Robinson and Rettger (1917
Sterilization: Not specified. p. 364), (1918 p. 219).
Use: Acid production by colon-typhoid
group. 990. Cannon's Hydrolyzed Casein Solution
Reference: Capaldi and Proskauer (1894
Constituents
p. 464).
1. Distilled water 1000.0 cc.
992. Mueller's Hydrolyzed Casein Heart 993. Robinson and Rettger's Extract
Solution Hydrolyzed Casein Solution
Constituents: Constituents:
1. Water 1000.0 cc. 1. Water 1000.0 cc.
2. NaCl (1.0%) 10.0 g. 2. Casein 50.0 g.
3. MgS04 (0.04%) 0.4 g. 3. Meat extract (Liebig's)
4. CaCl. (0.02%) 0.2 g. (0.5%) 5.0 g.
5. K2HPO4 (0.2%) 2.0 g. Preparation
6. Glucose (0.2%) 2.0 g. (1) Boil 50.0 g. of casein with 10.0% HCl
7. Phenol red (0.02% solution). 80.0 cc. under a condenser until the
reflex
8. Heart, beef 1.0 lb. solution no longer responds to the
9. Casein. Biuret test.
Preparation (2) Evaporate on the water bath until
(1) Prepare beef heart infusion by heat- nearly all the HCl is removed.
ing 1.0 pound of beef heart in (3) Neutralize the remaining acid with
500.0 cc. of tap water to boiling. NaOH. This solution is of dark
Strain and filter. brown color and is known as "Ca-
(2) Decolorize by boiling for 25
(1) sein C."
minutes with 10.0% "Norit," a com- (4) Dissolve 50.0 g. of (3) in 1000.0 cc.
mercial grade of wood charcoal. water.
(3) Filter thru paper. (5) Decolorize by filtering thru animal
(4) Prepare casein hydrolysate by boil- charcoal.
ing commercial casein 18 hours with a (6) Add 0.5%Liebig's meat extract.
mixture of 6 times its weight of water (7) Heat on a boiling water bath for
and 3 times its weight of concen- 20 minutes.
trated H2SO4. (8) Adjust the reaction to +0.6 to phenol-
(5) Free from H2SO4 by the addition of phthalein, and filter.
Ba(0H)2. Wash precipitate with (9) Distribute in 20.0 cc. lots in 150.0 cc.
water and concentrate the filtrate Erlenmeyer flasks.
and washings. Sterilization: Sterilize at 12 to 14 pounds
(6) The quantity of casein hydrolysate pressure for 15 minutes.
used in the following preparations is Use: Cultivation of B. diphthcriae and
equivalent to 0.5 g. of the original toxin production.
casein. Reference: Robinson and Rettger (1917
(7) Dissolve 2, 3, 4, 5, 6 and 7 in 1000.0 cc. p. 363).
of water.
994. Groer and Srnka's Hydrolyzed
(8) Prepare the following solutions:
Placenta Solution
[Decolorized infusion (1).. 25.0 cc.
(a) i Glucose salt solution (7) 25.0 . . cc. Constituents:
[Casein hydrolysate (6) 1. Water 4000.0 cc.
to 0.5 g. 2. Placenta 4000.0 g.
[Water 25.0 cc. Preparation
(b) {Glucose salt solution (7).. 25.0 cc. (1) Mix about 4 liters (3 placenta to
[Casein hydrolysate 0.5 g. 1 liter on an average) placenta with
(9) Adjust to pH = 7.8. the blood, cut into medium sized
(10) Filter if necessary and tube. pieces, with 4 liters of water.
Sterilization: Sterilize at 10 pounds pres- (2) Add about 10.0 cc. concentrated HCl
sure for 10 minutes. and mix well.
Use: To study food requirements of strep- (3) brown mix-
Boil for about 2 hours to a
tococci and pneumococci. Author re- ture, adding water.
ported growth with casein hydrolysate (4) Strain and filter thru paper while
present. No growth in other solutions. still warm.
Reference: Mueller (1922 p. 327). (5) Add 10.0% NaOH until phenol-
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 307
phthalein is turned red (hot titra- (7) Neutralize the acid completely and
tion) to the clear light greenish make the medium faintly alkaline to
yellow filtrate. phenolphthalein.
(6) Boil for one hour. (8) Boil (7) for 15 minutes.
the albumin.
(7) Filter off (9) Filter hot thru filter paper.
(8) Readjust the reaction (pH = 7.8). (10) Dilute to 1000.0 cc. by the addition
(9) Distribute the crystal clear fluid into of water.
flasks. (11) Dissolve 3 and 4 in (10).
Sterilization: Method not given. (12) Make the desired final adjustment of
Use : Cultivation of diphtheria bacillus and the reaction.
toxin production. Sterilization: Sterilize in the autoclave.
Reference: Groer and Srnka (1918-19 Use: General culture medium.
p. 334). Variants
(a) The author suggested the steriliza-
995. Boyd's Hydrolyzed Meat Solution tion of solution (9) above by auto-
2. H,S04 (strong) 100.0 cc. steps (10), (11) and (12) as indicated.
5. Lean meat 1.0 lb. (1) Add 500.0 g. lean meat to 40.0 cc. of
(1) To 300.0 cc. of tap water add 100.0 cc. (2) Pound together in a mortar.
strong H2SO4 gradually with con- (3) Mix thoroly and
digest in a water
draw the flask from the boiling water (2) Add to it 80.0 cc. strong commercial
and add calcium hydrate (quicklime, hydrochloric acid of specific gravity
CaO, that has been e.xposed to the 1.16, per kilogramme.
free air for some time is best) until (3) Mix thoroly.
a large precipitate of CaC04 is (4) Keep in water bath 4 days at 70C.
formed. Continue adding lime until (5) Bring the volume up to 1000.0 cc.
the precipitate occupies about 1 of for each kilogramme of minced meat
the fluid in the flask. used.
(6) Filter thru lint and wash thru the (6) Mix 1 part (5) with 2 parts boiling
lint with a little tap water. Con- water.
tinue the neutralization of the fil- (7) Add 75.0 cc. 40.0% sodium hydroxide
trate obtained with more lime, best for each kilogramme of minced meat
carried out in 3 stages so as to avoid used.
an excess of precipitate at any one (8) Make the reaction faintly alkaline to
time. litmus.
308 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(9) Steam 60 minutes. (6) Heat the filtrate and make alkaline
(10) Pour the mixture on to a wet, thick, when the temperature reaches about
clean cloth. 80C.
(11) Collect the fluid which drains thru (7) Filter the fluid thru paper to clarify.
the cloth together with that ob- (8) Heat at 120C.
tained by squeezing the cloth. (9) Filter.
(12) Estimate and adjust the reaction of (10) Distribute in flasks.
the medium to a definite pH value or Sterilization: Sterilize by heating at 115
to faintly alkaline to litmus or 1.0% for 15 minutes.
acid to phenolphthalein. Use: Cultivation of diphtheria bacilli and
(13) Filter thru well-wetted, thick, filter toxin production. The author reported
paper. that the addition of 2.0 g. acetic acid per
(14) Add part of the filtrate to 2 parts
1 liter favored toxin production. Other
boiling water. organisms grew well on this medium.
Sterilization: Sterilize in the autoclave.
Variants
Use: General culture medium. (a) Nicolle, Debains and Jouan culti-
Reference: Harvey (1921-22 p. 69). vated meningococci on a medium
997. Frouin and Ledebt'sHydrolyzed Serum prepared as follows:
Solution (1) Heat one liter of water to about
55C.
Constituents: Add 10.0 cc. of pure HCl (22
(2)
1. Water 1000.0 cc.
Baum's) to (1).
2. NaCl 6.0 g Add 300.0 g. of finely divided hog's
(3)
3. KCl 0.3 g stomach.
4. Potassium phosphate 0.5 g Regulate the temperature to about
(4)
5. MgS04 0.3 g 50 C. and keep at this temperature
6. CaCla 0.15 g
for 7 to 8 hours.
7. Serum albuminoids 10.0 g
(5) Heat to 80 or 90 degrees to destroy
Preparation
the pepsin and stop digestion.
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
(6) The peptone thus obtained may
(2) Hydrolyze 10.0 g. of serum albumi-
be preserved for several weeks.
noids in (1). (Method not given.)
(7) To use the peptone, make the
(3) Neutralize (indicator not specified).
peptone slightly alkaline to litmus
Sterilization: Method not given.
and heat at 120 C.
Use: Cultivation of colon typhoid group.
(8) Filter on a wet filter paper.
Variants: The authors added 2.0% glucose
or 3 to4.0% glycerol to the medium. (9) Add 2.0 g. of glucose.
small pieces. (d) See medium 877, step (1) thru (8)
(2) Mix 200.0 g. of (1), 10.0 g. pure HCl for Park, Williams and Krumwiede's
and 1000.0 cc. water at 50C. method of preparation.
(3) Allow to stand at 50C. for 12 to References: Martin (1898 p. 32), Thoinot
24 hours. and Masselin (1902 p. 22), Nicolle,
(4) Heatatl0OC. (Time not specified.) Debains and Jouan (1918 p. 151), Besson
(5) Strain or filter thru a layer of ab- (1920 p. 28), Harvey (1921-22 p. 98), Park,
sorbent cotton. Williams and Krumwiede (1924 p. 118).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 309
999. Martin's Liver Digest Solution Sterilization: Sterilize (4) in the Arnold.
Method of sterilization of blood not
Constituents given. Sterilize the mi.xture of blood and
1. Water 2000.0 cc.
digest for 15 to 20 minutes in the Arnold.
2. Stomach (hog) 200.0 g.
Use: Cultivation of measles virus from
3. Liver, beef or hog 200.0 g.
patients' blood.
Preparation Reference: Sellards and Bigelow (1920-21
(1) Mi.x chopped hog stomach
200.0 g. of
p. 242).
and 200.0 g. chopped hog or beef liver,
and 20.0 cc. of commercial HCl in 2 1001. Dubovsky and Meyer's Liver Digest
liters of water. Solution
(2) Digest (1) at 50C. for 12 to 24 hours.
Constituents
(3) Boil the liquid, at the end of 24 hours.
1. Water, tap 5000.0 cc.
(4) Allow to cool and settle for 24 hours
2. Stomach, hog 400.0 g.
or less.
3. Liver 400.0 g.
(5) Decant or siphon off the clear liquid.
4. K2HPO4 (0.2%).
(6) Neutralize and make slightly alkaline
5. Heart 1000.0 g.
by the addition of soda. Indicator
Preparation
not specified.
(1) Wash clean and mince finely 5 or
(7) Heat at 120 degrees.
more large hogs' stomachs.
(8) Filter and distribute as desired.
(2) Mince an equal amount of clean
Sterilization: Method not given.
hog or beef liver.
Use: Cultivation of typhoid bacilli.
(3) Mi.x 400.0 g. (1), 400.0 g. (2), 40.0 g.
Author reported that typhoid and para
HCl and 4000.0 cc. tap water at
typhoid bacilli developed rapidly in this
50C.
medium, producing gas, and acid and
(4) Keep in glass or porcelain receptacles
finally the reaction became alkaline.
for 18-24 hours.
References: Martin (1915 p. 261), Harvey
(5) Make Biuret and tryptophan test,
(1921-22 p. 99).
when both are +
the digest is green-
yellowish and contains little un-
1000. Sellards and Bigelow's Liver Digest
digested debris.
Blood Solution
(6) Transfer to large bottles and steam
Constituents: 10 minutes at 100.0C. to stop
1. Water 1000.0 cc. digestion.
2. Veal 100.0 g. (7) Strain the digest thru cotton or
3. Stomach (hog) 150.0 g. preferably store over night in the
4. Liver 200.0 g. ice chest and decant after 24 hours.
5. Blood, horse or rabbit (8) Warm the digest to 70C. and neu-
(10.0%) 100.0 cc. tralize with 2 N Na2C03 using
Preparation litmus.
(1) Prepare apeptone by digesting (9) Filter the desired amount, add 0.2%
hog stomach and
100.0 g. veal, 150.0 g. K2HPO4 and adjust to "pH = 7.4.
200.0 g. liver in a liter of water con- (10) Slowly heat to boiling finely ground
taining 1.0% concentrated HCl, for 1000.0 g. fat-free heart and tap water
19 hours at 53C. 1000.0 cc.
(2) Heat in an Arnold to destroy the pep- (11) Adjust to pH = 8.0 or 8.2.
sin. (12) Cool and carefully skim off the layer
(3) Decant the supernatant fluid and of fat which floats on medium.
neutralize at room temperature first (13) To each liter of (12) add 2 liters
to litmus and then to china blue and of (9).
rosalic acid. (14) Adjust to pH = 7.2 or 7.4.
(4) Again decant and dilute with an equal Sterilization: Sterilize at 18 pounds pres-
volume of water. sure for one hour. Incubate 5 days and
(5) Add 10.0%, horse or rabbit blood to repeat the same sterilization for one hour
sterile (4). at 18 pounds pressure.
310 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Use: Cultivation and toxin production by (10) Transfer to large bottles and steam
B. botulinus. Authors reported toxin for 10 minutes to stop digestion.
production very good in this medium. (11) Strain thru cotton or preferably
Reference: Dubovsky and Meyer (1922 store over night in ice chest and
p. 505). decant after 24 hours.
(12) Warm (5) to 70C. and neutralize
1002. Emery's Spleen Digest Solution with 2 normal Na2C02 to litmus.
Constituents (13) Filter into a flask.
1. Water 1500.0 cc. (14) Add the 2.0 g. K0HPO4.
2. Spleen (beef) 1500.0 g. (15) Adjust to reaction using
desired
3. Stomach (hog) 250.0 g. litmus or preferably to a definite
Preparation H-ion concentration (pH = 7.0
1003. Stickel and Meyer's Blood Clot Digest 1004. Bramigk's Peptic Digest Solution
Solution
Constituents:
Constituents 1. Water 4000.0 cc.
1. Tap water 1000.0 cc. 2. Blood clot 1000.0 g.
2. Blood clots 100.0 g. 3. Stomach (hog) 2
3. Pig's stomach (minced) 100.0 g. Preparation
4. K2HPO4 2.0 g. (1) Obtain a bucket of blood clots from
Preparation the slaughter house.
(1) Obtain 10 liters fresh beef blood from (2) Wash the clots free from blood with
the abattoir. running water.
(2) Decant and store the serum (which (3) Press the clots free from water.
has separated on standing) in a (4) To 1000.0 g. clots (contains about
refrigerator. 230.0 g. fibrin) add 3 liters of water
(3) Weigh the blood clots and mix and 15.0 cc. of H2SO4.
100.0 g. with 1 liter tap water. (5) Allow to stand over night.
(4) Place mixture in an enameled pot, (6) Filter thru a sieve and press the
bring slowly to a boil and boil slowly water from the clot or fibrin.
for 5 minutes, stirring constantly. (7) Add the clot to 3 liters water and
(5) Wash and mince pig's stomach. 18.0 cc. H2SO4 heated to about 50C.
(6) Cool (4) to 50C. and add 100.0 g. of (8) Remove the mucous membrane from
minced pig's stomach for each liter two fresh pigs' stomachs.
of (4). (9) Run the stomachs thru a meat chop-
(7) Transfer to glass or porcelain re- ping machine.
ceptable and finally add 1.0% HCl. (10) Mix (9) with 1 liter of water and
(8) Digest at 50C. for 18-24 hours. 8.0 cc. H2SO4.
(9) Make a Biuret and tryptophane, (11) Heat to 35C.
test; when both are +, the digest is (12) Mix (11) and (7) thoroly and place
yellowish green and contains very at 37C. for 48 hours. Stir fre-
little indigested debris. quently. Some fat will settle out.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 311
(13) Filter and neutralize with ammonia. (14) Collect the fluid which drains thru
(14) Distribute in flasks and sterilize. the cloth, together with that ob-
Sterilization: Method not given. tained by squeezing the cloth.
Use: General culture medium and substi- (15) Filter the fluid collected thru well-
tute for commercial peptone. wetted, thick, filter paper.
Variants: The author prepared a dry ma- (16) Bring the volume up to 1000.0 cc.
terialby treating the medium obtained (17) Filter thru well wetted, thick filter
above as follows: paper.
(1) Neutralize the digest with Ba(0H)2 (18) Make the reaction neutral to litmus.
(about 40.0 g.) and boil until the (19) Add 75.0 cc. of the decanted serum
upper fluid is clear. (3). _
(2) The reaction should be slightly acid, (20) Steam sixty minutes.
showing a light excess of H2SO4. (21) Allow the clot to form a compact
(3) Allow the digest to settle.
(4) Pour off the clear solution and filter (22) Decant the clear fluid.
the remainder, washing the pre- Sterilization: Sterilize at 100C.
cipitate with water. Use: General culture medium.
(5) This gives a light yellow solution con- Reference: Harvey (1921-22 p. 100).
taining about 10.0% peptone.
This fluid may be evaporated over a 1006. Martin's Stomach Digest Infusion
(6)
Broth
water bath or in a vacuum to a thick
syrupy paste and then drawn into Constituents:
threads and dried in a desiccator. 1. Water 2000.0 cc.
(7) This peptone is equal to Witte's pep- 2. Veal 500.0 g.
tone in the preparation of media. 3. NaCl 5.0 g.
Reference: Bramigk (1921 p. 429). 4. Stomach (hog) 200.0 g.
Preparation
1005. Harvey's Blood Clot Digest
(1) Infuse 500.0 g. of finely ground veal
Solution
with 1000.0 g. water for 20 hours
Constituents: at35C.
1. Water 1000.0 cc. (2) Strain out the meat and add 5.0 g.
2. Blood. NaCl to the fluid.
3. Pig's stomach 100.0 g. (3) Chop the stomachs of 5 hogs into
4. HCl 10.0 g. small pieces.
Preparation (4) Mix 200.0 g. of (3), 10.0 g. HCl (pure)
(1) Procure ox or sheep blood from the and 1000.0 cc. water at 50C.
slaughter house. (5) Allow to stand at 50C. for 12 to 24
(2) Allow to clot. hours.
(3) Decant and store the serum in an (6) Heat at 100C. (Time not spec-
ice chest. ified).
(4) Mince the clot. (7) Strain or filter thru a layer of ab-
(5) Add 100.0 g. clot to 1000.0 cc. tap sorbent cotton.
water. (8) Heat the filtrate and make alkaline
(6) Raise slowly to boiling temperature. when the temperature reaches about
(7) Boil 10 minutes. 80C.
(8) Cool to 50C. (9) Filter the fluid thru paper to clarify.
(9) Add 100.0 g. minced pig's stomach (10) .Mix 1 liter of (9)with 1 liter of (2).
and
10.0 cc. strong hydrochloric acid. (11) Heatto70C.
(10) Digest 20 hours. (12) Filter thru paper.
(11) Make a biuret and a tryptophane (13) Make alkaline (method not given).
test. Sterilization: Method not given.
(12) Boil 10 minutes to stop the digestion. Use: Cultivation of diphtheria bacilli and
(13) Pour the mixture on to a wet, thick, to.xin production. Author reported that
clean cloth. heating the medium decreased the ability
312 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
of the medium to produce toxin. Heat- (g) Klimmer used the stomachs of 5 hogs,
ing only to 70C. and filtering thru a but did not specify the use of 200.0 g.
Chamberland filter gave good results. He carried on the digestion for 24
Other investigators utilized a similar me- hours and then sterilized. Equal
dium for the production of tetanus toxin. volumes of the sterile digest
Variants (1000.0 cc.) and the infusion from
(a) Thoinot and Masselin used the 500.0 g. of veal to 1000.0 cc. of water
stomachs of 5 hogs, but did not were mixed, boiled and neutralized.
specify the use of 200.0 g. They also The reaction was made slightly alka-
used 500.0 g. beef instead of veal. line, or adjusted to any desired pH
(b) BezanQon digested the stomach for value, filtered, tubed and sterilized
24 hours instead of 12 to 24, infused in the usual manner,
beef instead of veal, heated the mix- (h) Park, Williams and Krumwiede in-
ture at 115 for 15 minutes instead of fused the veal and 1000.0 cc. of water
heating at 70 C. in step (11) above for 18 to 24 hours at 35C., heated at
and sterilized in the autoclave at 45 to 48C. for one hour, then boiled
115 for 15 minutes. briskly for 30 minutes and strained
(c) Besson used 250.0 g. hogs stomach thru cheese cloth. This infusion at
instead of 200.0 and added 7.0 cc.
g. 70 C. was mixed with an equal
of normal soda solution to the me- volume of stomach digest at 70C.,
dium after it had been neutralized to prepared according to Martin's
litmus. method, and heated to boiling. The
(d) Wilcox studied the production of reaction was adjusted to -f-0.5 to
tetanus toxin using one of the follow- phenolphthalein at room tempera-
ing combinations, prepared according ture. The medium was sterilized at
to Martin's process: 15 pounds pressure for 30 minutes
(1) (a) 200 g. minced stomach in after the reaction was readjusted
1000.0 cc. water. and filtered thru cotton and paper.
(b) 400 g. minced stomach in They prepared a similar medium with
1000.0 cc. water. a reaction of +1.0 to phenolphthalein
(c) 200 g. minced stomach in and added 1.0% glucose. This me-
1500.0 cc. water. dium was used for the production of
(d) 300 g. minced stomach in tetanus toxin.
1000.0 cc. water. References: Martin (1898 p. 35), Thoinot
(2) Veal infusions prepared from one of and Masselin (1902 p. 23), BezanQon
the following: (1920 p. Ill), Besson (1920 p. 29), Wilcox
(a) 500 g. veal to 1000.0 cc. water. (1921 p. 414), Dopter and Sacquepee (1921
(b) 500 g. veal to 500.0 cc. water. p. 119), Harvey (1921-22 p. 99), Klimmer
(3) Add 1.0% glucose if desired. (1923 p. 200), Park, Williams and Krum-
Author reported that 1 part (1) (a) wiede (1924 p. 133).
to 1 part (2) (a) gave highest toxin
production with organism studied. 1007. Besredka and Jupille's Egg Stomach
(e) Dopter and Sacquepee used beef in- Digest Solution
stead of veal, added 7.0 cc. of normal
soda to the medium after it had been Constituents:
neutralized to litmus and autoclaved 1. Distilled water.
2. Beef or veal 500.0 to 750.0 g.
at 117C. for 15 minutes instead of
heating at 70C. as in step (11) above. 3. Egg.
(f) Harvey neutralized the medium to 4. Stomach (hog).
(9) Distribute in flasks or tubes in (4) Transfer to large bottles and steam
20.0 cc. lots. for 10 minutes at 100C. to stop
(10) Prepare a bouillon from 500 to digestion.
750.0 g. chopped beef or veal with (5) Strain thru cotton or preferably
1000.0 cc. water and Martin's pep- store over night in the ice chest and
tone, by heating to boiling under a decant after 24 hours.
low flame and then boiling for (6) Warm (5) to 70C. and neutralize
30 minutes. See med. (1006). with 2 N Na2C03, to litmus.
(11) Filter and make slightly alkaline. (7) Filter into a flask.
(12) Heat at 120C. to activate pre- (8) AddS.Og. K2HPO4.
cipitation. (9) Adjust to desired reaction using lit-
(13) Filterthru double paper. mus or preferably to a definite H-ion
(14) Distribute in flasks or Rou.x tubes. concentration (pH 7.0 to 7.5).
(15) Add to 500.0 cc. of (14), 400.0 cc. of (10) Heat in steamer at 100 for 15
(4) egg white solution), and
(sterile minutes.
100.0 cc. (9) (sterile egg yolk solu- (11) Correct reaction and filter thru
tion) under aseptic conditions. paper.
(16) Distribute in sterile tubes in 10.0 cc. (12) Distribute in receptacles used for
lots under aseptic conditions. cultures.
Sterilization: Sterilize (4) and (9) at 115C. Sterilization: Sterilize at 100 for 30 min-
for 20 minutes. Sterilize (14) at 115C. utes on 2 successive days or at 10 pounds
for 25 minutes. pressure for 15 minutes.
Use: General culture medium, for highly Use: General inexpensive culture medium.
parasitic and saprophytic forms. To cul- Variants
tivate tubercle bacilli, add cold, and (a) The authors prepared a medium as
under aseptic conditions, 20.0 cc. of indicated from step (1) thru (6).
(4) (egg white solution) and 5 to 20.0 cc. The remainder of the preparation was
of (9) (egg yolk solution) to 100.0 cc. of as follows:
the bouillon, prepared as before but con- (7) Sterilize at 10 pounds pressure for
taining no peptone. 15 minutes in the autoclave or for
Reference: Besredka and Jupille (1913 30 minutes at 100 C. on 2 suc-
p. 1009). cessive days.
(8) Inoculate (7) with 1.0% of a
1008. Stickel and Meyer's Meat Digest 24 hour old broth culture of B.
Solution
saccharolyte or B. coli and incu-
Constituents: bate for 12-18 hours at 37C.
1. Water, tap 4000.0 cc. (9) Steam for 20 minutes.
314 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(10) Adjust to desired reaction. (5) Allow the flask to stand at room
(11) Add 0.2-0.4% K2HPO4 and 2.0% temperature or in the incubator.
of purified talcum. Shake thoroly several times a day.
(12) Filter thru paper. (6) Add 5.0% HCI from time to time.
(13) Distribute for use. Use Congo red as an indicator (blue
(14) Sterilize at 100C. for 30 minutes color).
on 2 successive days or at 10 (7) About 10 to 14 days are required for
pounds pressure for 15 minutes. complete digestion of the fibrin.
(b) Park, Williams and Krumwiede omit- The reaction may be allowed to reach
ted the K2HPO4. acid to litmus at this time.
References : Stickel and Meyer (1918 pp. 78, (8) Allow the digest to stand undis-
79),Harvey (1921-22 p. 99), Park, Wil- turbed for 2 or 3 days.
liams and Krumwiede (1924 p. 119). (9) In order to obtain a fat-free digest,
remove the bottom dark brown
1009. Frieber's Gelatin Digest Solution digest by means of a rubber tubing.
Constituents: The fat floats on the top surface.
1. Water 2000.0 cc. (10) In order to obtain about a 1.0%
2. Gelatin 20.0 g. peptone solution dilute the stock
3. NaCl 5.0 g. solution about 3 with water and add
4. KH2PO4 2.0 g. 5.0% NaOH solution to neutralize
5. MgS04 0.2 g. to litmus.
6. Tryptophane 0.3 g. (11) A heavy flaky precipitate is formed.
Preparation Do not add an excess of NaOH or
(1) Add 20.0 g. of gelatin, 10.0 cc. HCI the precipitate will dissolve.
and 2.0 g. Witte's peptone to 2 liters (12) Boil and filter or allow the pre-
of water and allow to digest for cipitate to settle. This gives a light
several days. yellowish 1.0% peptone solution.
dry material is placed in a flask so (4) Add without filtration 5.0 cc. water
that the flask is about ^ full. free soda (strength solution not
Add to each liter flask about 3 to given), 5.0 cc. toluol, 5.0 cc. chloro-
(2)
5 grams pepsin powder and fill the form, and 2.0 g. pancreaten (Rhe-
flask with water. nania).
(3) For each liter of material add 10.0 cc. (5) Mix well and incubate at 37 for 120
of concentrated (1.19) HCI. hours, shaking occasionally. (Test
(4) Shake thoroly. for tryptophane using bromine
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 315
water.) Add HCI to stop the di- (5) Strain fluid thru cheese cloth and
gestion. pass the residue thru a fruit press,
Sterilization: Method not given. cool to 37C.
Use: Indol production. Use this digest in (6) Make the thick brownish fluid
1.0 or 2.0% solution for indol reaction. slightly alkaline to litmus.
Author reported that the addition of (7) Add 1.0% pancreatic extract and
0.1% of this digest gave equally as good incubate at 37 for 5, 24, 48 hours.
growth when added to nutrient medium (8) When the process is sufficiently ad-
as did 1.0% Witte's peptone. vanced, render slightly acid with
Reference: Bramigk (1921 p. 431). glacial acetic acid and boil slowly
for 15 minutes.
1112. Jensen's Milk Digest Solution
(9) Either filter or decant the clear fluid
Constituents: which results on placing the digest
1. Milk 1000.0 cc. over night in a cool place.
Preparation (10) Adjust the reaction as desired.
(1) To sterilized milk add 10.0 cc. of pure (11) Dissolve 3 in (10).
concentrated HCI per liter and 2.0 g. (12) Heat
for 15-30 minutes in the
of a pure pepsin preparation. steamer at 100 C.
(2) Incubate at 35-37 for 36 to 48 hours (13) Filter again if necessary.
shaking often at first until the casein (14) Clear (13) by adding 5-10.0% of the
has precipitated and to dissolve the decanted beef serum. Steam 45-
pepsin, and then only occasionally. 60 minutes.
(3) Approximately neutralize the acid (15) Remove (14) from steamer and allow
(acid hydrolyzes the lactose when clot to form as a compact mass.
heated) and heat in the autoclave at Decant, or better, centrifuge the
115 to 120 for 10 minutes. medium to remove the clot. * .
(10) When ready for use, use in 40.0% (5) Precipitate out the oxalate by the
strength. addition of calcium acetate.
(11) Sterilization of stock solution not (6) Add 40.0 g. trypsin to the plasma and
specified. allow it to act for 8 days.
References: Stickel and Meyer (1918 p. 81), (7) At the end of this time, boil for a few
Knorr (1921 p. 598), Harvey (1921-22 minutes, allow to stand and filter.
p. 117), Klimmer (1923 p. 176). Method not given.
Sterilization:
Use: Inexpensive medium. Author re-
1114. Spray's Blood Clot Digest Solution ported that this medium contains about
Constituents: 8.0% nutrient materials and should be
1. Water. diluted 1:3 before use.
2. Blood clot. Reference: Loffl (1915-16 p. 109).
3. NajCOa.
Preparation 1116. Gordon et al. Trypsinized Heart
Remove the serum from the blood Solution (Tanner)
(1)
clot and allow to drain. Constituents:
(2) Pass the clot thru a wire gauze. 1. Water 1000.0 cc.
(3) Boil and divide finely again. 2. Heart, beef 500.0 g.
(4) Place one liter of this
semi fluid ma- Preparation
terial in a two liter flask. (1) Free fresh bullock's heart from fat
(5) Add 20.0 g. anhydrous
Na2C03, 5.0 g. and blood vessels.
trypsin, and 15.0 to 20.0 cc. chloro-
(2) Mince (1) very finely and weigh.
form. (3) To each 0.5 kilo add 1 liter of water,
(6) Incubate for 15 days with a second and make faintly alkaline to litmus
addition of 3.0 g. trypsin on the by the addition of 20.0% KOH
tenth day. Chloroform may be solution.
added on the fifth and tenth days. (4) Heat slowly to 75 to 80C. for
(7) Make strongly acid by the addition 5 minutes.
of 50.0 to 75.0 cc. HCl. (5) Cool to 37C. and add 1.0% of liquid
(8) Steam in the water bath to drive off
trypsine comp., and keep at 37 for
the chloroform. 2.5 to 3 hours.
(9) Adjust to pH = 7.4 to 7.5.
(6) When trypsinizing is finished test for
(10) Tube. peptone with copper sulphate and
Sterilization: Sterilize in the autoclave. KOH (Method not given).
Use: The author reported that this blood (7) Render slightly acid by the addition
clot digest may be added in from 5.0 to of glacial acetic acid.
10.0% to melted North's or other agar for (8) Bring slowly to boil and boil for
growth of H. influenzae, pneumococcus 25 minutes.
and streptococcus. When sodium oleate (9) Leave over night in a cool place.
is added H. influenzae grows very
vigor- Siphon off the clear liquid in the
(10)
ously. morning.
Reference: Spray (1927 p. 14). (11) Make faintly alkaline to litmus.
Sterilization: Sterilize in an autoclave at
1115. Loffl's Trypsinized Blood Solution
118 for one hour on each of two days.
Constituents: Use: General culture medium.
1. Water. Reference: Tanner (1919 p. 45).
2. Blood.
Preparation 1117. Harvey's Trypsinized Heart Solution
(1) Allow 9 liters of blood to flow into a
liter of 0.8% ammonium oxalate. Constituents:
Mix well. 1. Water 400.0 cc.
(2)
Centrifuge at 3000 revolutions per 2. Heart, ox 1
(3)
minute. 3. NaCl 1.0 g.
water boiled as above and added (19) Add 20.0 cc. of (18) to each liter of
(1) Free 750.0 g. meat from fascia and (24) Dilute the stock broth as desired
cut in finger-thick pieces. for According to Hottinger
use.
(2) Drop (1), piece by piece, into it may
be diluted 10, 20 or more
1500.0 g. of boiling water, stirring times. Excellent results were ob-
constantly. tained, however, by diluting 1 part
(3) Boil up strongly and take from stock broth with 1 part water,
fire. (b) Park, Williams and Krumwiede pre-
(4) Take out the meat and put thru pared a similar medium as follows:
a chopping machine. (1) Add 300 to 500.0 g. of fat-free
(5) Cool the water to 37C. and add chopped meat to 1000.0 g. of water
1.5 g. NaoCOs per liter. to which 0.4% Na-iCOj has been
(6) Put the chopped meat in flasks added.
(2 liter Erlenmeyer) 550.0 g. in (2) Soak over night.
each flask. (3) Heat at 80 C.
(7) Add (5) (the water) at 37C. to the (4) Cool to 38C. and add 15.0 cc. of
flask, filling them up to the narrow liquid trypsin.
neck. (5) Keep at 38C. for 5 hours stirring
(8) Add 3.0 g. of pancreatin, 10.0 cc. frequently. If kept over night at
chloroform and 10.0 cc. toluol to this temperature add 10.0 cc. of
each flask. toluol (or thymol crystals).
(9) Cork tightly. (6) Add normal HCl to neutralize
(10) Shake thoroly. (indicator not specified).
(11) Incubate at 37 over night. (7) Boil 7 minutes.
(12) Shake the next morning and add (8) Strain.
more pancreatin unless the fluid (9) Adjust the reaction.
shows a yellow color, and particles (10) Boil 30 minutes.
of meat look smaller, indicating (11) Filter.
digestion. (method not given).
(12) Sterilize
(13) Digest for 4 or 5 days at room References: Klimmer (1923 p. 175), Park,
temperature or for 2 or 3 days in Williams and Krumwiede (1924 pp. 118,
the incubator. Shake the flasks 119).
each day. The meat should be in
1120. Peckham's Trypsinized Beef Solution
a finely divided mass, giving off a
very offensive odor, when digestion Constituents
is complete. 1. Water 1000.0 cc.
(14) The medium may be stored in the 2. Beef 500.0 g.
ice box, after acidifying to litmus 3. NaCl 5.0 g.
by the addition of HCl, if neces- 4. Trypsin 4.0 g.
sary. (Hottinger's statement.) Preparation
(15) As soon as the digestion is com- (1) Place 500.0 g. of finely chopped beef
plete decant the liquid thru cheese which is as old as can be obtained
cloth. (This process gives better from the shops, in order that it be
results according to the author.) free from muscle sugar, in 500.0 cc.
(16) Add an equal amount of water of water.
to the residue. (2) Make slightly alkaline with sodium
(17) Shake (16) thoroly. carbonate.
(18) Allow to settle and again decant. (3) Place in a water bath and raise tem-
(19) Place the meat on the cheese cloth perature to 40C. and add trypsin.
and allow to drain. (4) After an hour the mixture must be
(20) Boil the filtrate a few minutes. again made alkaline with sodium
(21) Filter thru absorbent cotton and carbonate.
paper until clear. (5) Allow to digest only from one to Ih
(22) Autoclave at 15 pounds pressure hours, or traces of indol may be
for 30 minutes. detected.
(23) Store as stock broth. (6) Boil and strain thru gauze.
320 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(7) Filter thru wet filter paper when Sterilization: Filter (12) thru a Berkefeld
cold to remove fat. and a Doulton candle in series.
(8) Add and enough water to bring
salt Use: Cultivation of B. tetani and other
the volume to 1 liter. anaerobic organisms. Author reported
(9) Adjust so that 20 to 30.0 cc. of a that B. sporogenes was inhibited.
decinormal NaOH solution will be Variants: Harvey prepared a similar me-
required to bring one liter of the dium as follows:
medium the
to neutral point of (1) Mince finely fat-free beef or rabbit
phenolphthalein. flesh.
(10) Clarify and filter. (2) Add 500.0 g. to 1000.0 cc. water con-
Sterilization: Method not given. taining 5.0g. sodium carbonate.
Use: Indol production. (3) Place in incubator 20 hours.
Reference: Peckham (1897 p. 554). (4) Make faintly alkaline to litmus.
(5) Add trypsin solution to 2 per cent.
1121. Harvey's Trypsinized Meat and (6) Place in the incubator for a further
Kidney Solution 20 hours.
(7) Filter thru well-wetted, thick filter
Constituents
paper.
1. Water 1000.0 cc.
Meat 500.0 g. (8) Make faintly acid to litmus.
2.
Kidney, rabbit (9) Steam or boil 45 minutes.
3.
(10) Filter thru well-wetted, thick filter
Preparation
paper.
(1) Mince meat.
finely, fat-free lean
(11) Bring the volume up to 1000.0 cc.
(2) Add 500.0 g. to 1000.0 cc. tap water.
Heat the mixture 20 minutes at a (12) Make neutral to phenolphthalein.
(3)
temperature not exceeding 50C. (13) Steam 30 minutes.
(14) Filter, while hot, thru well-wetted
(4) Skim on the surface.
off fat floating
thick filter paper. Occasionally it
(5) Raise the temperature rapidly to
boiling point.
may be necessary to filter thru a
Doulton candle.
(6) Boil 10 minutes.
(15) Distribute the filtrate into test
(7) Make faintly alkaline to litmus.
tubes.
(8) Add trypsin solution to 2 per cent.
Place in incubator in an open vessel, (16) Add just before use l/16th part of
(9)
4 days.
fresh sterile rabbit kidney and use as
soon as possible after the addition of
(10) Filter thru well-wetted, thick filter
the kidney.
paper.
Reference: Harvey (1921-22 p. 115).
(11) Bring the volume to 1000.0 cc. by
the addition of water.
1122. Celozzi's Placenta Digest Blood
(12) Make neutral to phenolphthalein at
Solution
room temperature.
(13) Store the sterile medium in sterile Constituents
flasks under layer of paraffin to 1. Ringer-Locke solution 1000.0 cc.
which sodium formate has been 2. Placenta 500.0 g.
added to the extent of 1 per cent of 3. Glucose (0.5%) 5.0 g.
the total volume of the medium. 4. Glycerol (2.0%) 20.0 g.
(14) Test the sterility of the medium 5. Blood
before use by anaerobic culture. Preparation
Incubate at least 7 days. (1) Thoroly wash fresh placenta and pass
(15) Add before use l/16th part of fresh thru a meat chopper.
sterile rabbit kidney to 5.0 cc. me- (2) Mix two parts by weight of Ringer-
dium. With kidney added, incuba- Locke solution with one part (1).
tion may be aerobic. Use as soon (3) Adjust the reaction to that of human
as possible after the addition of the blood and add 1.0% pancreatin and
kidney. 0.5% chloroform.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 321
(anhydrous) and cool to 45^0. (19) Add 3 drops of egg albumin mixed
(3) Add 5.0 cc. of chloroform and 5.0 cc. with bouillon (see variant 848 for
of Cole and Onslow's pancreatic preparation) to each tube. (This
extract. serves as an indicator.)
(4) Incubate at 37C. for 6 hours shaking (20) Place the tubes in boiling water for
frequently. 20 minutes.
(5) Add 40.0 cc. normal HCl and heat in Sterilization: Sterilize (15) in the auto-
the steamer for 30 minutes. clave.
(6) Cool and filter. Use: Determine fermentation of sugars by
(7) Adjust to pH 8.0. anaerobic organisms.
(8) Distribute as desired. Added nutrients: Author added 1.0% glu-
Sterilization: Pass steam thru the auto- cose or any other desired carbohydrate,
clave for one hour then raise the pressure alcohol, etc.
slowly to 10 pounds and turn off the Reference: Harvey (1921-22 p. 111).
steam. For sterilization of larger quan-
tities (one liter in a flask) maintain the
1125. Teruuchi and Hida's Trypsinized
pressure at 10 pounds for 30 minutes. Casein Solution
Use: Preparation of diphtheria to.xin. Constituents:
Reference: Hartley (1922 p. 482). 1. Water 1000.0 cc.
2. Casein 100.0 g.
1124. Harvey's Basal Trypsinized Casein
3. NaCl (0.5%) 5.0 g.
Solution
Preparation
Constituents: (1) Dissolve 100.0 g. of pure casein in
1. Water 1000.0 cc. 1 liter of 0.8% NazCOj (anhydrous).
2. Casein 100.0 g. (2) Add 5.0 to 10.0 g. of pancreatin
3. NaCl 2.5 g. (Gehe and Co.) and shake with
4. CaCl2 0.125 g. chloroform.
322 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(3) Place for 3 to 5 days in the incubator, (6) Digest at 39 C. for 10 days, with
shaking occasionally. daily shaking and addition of more
(4) Each day test the mixture for trypto- toluol if necessary.
phane, by adding a few drops of (7) Add per liter, 100.0 cc. 7.5 per cent
acetic acid to a sample and then hydrochloric acid.
adding bromine water. (8) Steam 20 minutes.
(5) When the tryptophane content has (9) Filter thru well-wetted, thick filter
reached the maximum and the paper.
tjTOsin has separated out as white (10) Make the reaction nearly neutral
clumps, heat the fluid for a short to litmus with 5% sodium hy-
time at 80 C. droxide.
(6) Filter and neutralize with a few cc. (11) Preserve as stock tryptic broth or
of HCl. stock "tryptamine."
(7) Heat moderately in a vacuum if (12) use "tryptamine" 1:
Dilute for
possible and evaporate to a syrupy water 2 = tryptamine bouillon.
thickness. (Possibly may be fil- (c) Harvey prepared a similar medium as
tered again.) follows:
(8) Place in a mortar and knead it with (1) Add very gradually 200.0 g. casein
alcohol (amount not specified). to 1000.0 cc. boiling water contain-
(9) Dry in a vacuum desiccator. ing 20.0 g. anhydrous sodium
(10) This yields a light yellow colored carbonate.
powder, which dissolves quite easily (2) Allow to cool to 45C.
and clear in water. The yield is (3) Add pancreatin 3.0 g. or pan-
nearly quantitative. creatic extract 50.0 cc, chloroform
(11) Add 0.5% NaCl and 1.0% NajCO, to 15.0 cc
a 5.0% watery solution of (9). (4) Place in incubator 5 days, shaking
Sterilization: Not specified. vigorously each day to break up
Use: Enrichment of cholera vibrio, toxin clumps.
production by Proteus vulgaris and gen- (5) Add again pancreatin 3.0 g. or
eral culture medium. pancreatic extract 50.0 cc.
Variants (6) Place in incubator again for 10
(a) Berthelot prepared a pancreatic ca- days.
sein digest, and dissolved 20.0 g. of (7) Add 400.0 cc. N/1 hydrochloric
it in a liter of water. The medium acid.
was sterilized with ether, method not Note: Or 400.0 cc. pure con-
given, and used to produce toxin by centrated hydrochloric acid di-
Proteus vulgaris. luted with 350.0 cc water.
(b) Harvey prepared a "tryptamine" (8) Steam 30 minutes.
medium as follows: (9) Filter, while hot, thru well-wetted,
2. Plasmon (sodium caseinate). 500.0 g. (21) Filter and add H2SO4 to the filtrate
3. KH2PO4 5.0 g. so that it is 5.0% H2SO4.
(3) Autoclave. (Time not specified.) (5) Add 6.4 cc. 5.0% sodium hydroxide
(4) Add asparagin, ammonium lactate, for every 100.0 cc. of 0.5% hydro-
K0HPO4 and MgS04. chloric acid used.
(5) Make up to 1 liter and adjust to +1 (6) Stir well and filter thru folded filter
phenolphthalein. paper.
Sterilization: Final sterilization not speci- (7) Shake up with a little toluol.
fied. (8) Make the reaction less acid by the
Use: To show production of indol by cautious addition of 10.0% sodium
bacteria. hydroxide.
Reference: Norton and Sawyer (1921 (9) Store in a stoppered bottle in a cool,
p. 473). dark place.
(10) Mince finely ox heart or human
1134. Duval and Harris' Tryptic Digest
placenta.
Solution
(11) Add 500.0 g. to 1000.0 cc. tap water.
Constituents: (12) Make faintly alkaline to litmus.
1. Serum, egg albumin, liver or (13) Heat slowly to 75C. and maintain
placenta 200.0 cc. at this temperature 10 minutes.
Preparation (14) Cool to 37C.
human serum,
(1) 200.0 cc. of fresh sterile (15) Add 1.0 or 2.0% of (9) to (14).
egg albumin, or the equivalent in (16) Keep at 37C. for 4 hours. If the
grams of liver or placenta tissue are digestion is extended to 6 hours or
placed in a flask and 20.0 cc. of a longer it is necessary to add chloro-
1.0% physiological salt solution of form or toluene. Control the prog-
added.
sterile trypsin ress of digestion by Biuret and
(2) Digest for 5 days at 37 C., changing tryptophane tests.
the reaction to neutral as occasion (17) Make faintly acid to litmus with
demands. glacial acetic acid.
(3) Heat at 70C. for one hour. (18) Raise slowly to the boiling point.
Sterilization: Filter thru a Berkefeld filter. (19) Boil gently 15 minutes.
Use: Cultivation of leprosy bacilli. The (20) Filter.
author stated that the same materials (21) Adjust the reaction.
might be autolyzed under sterile condi- (22) Add 0.2% di-potassium phosphate.
tions at 40C. for 2 weeks or hydrolyzed (23) Steam 20 minutes.
with a culture of Finkler and Prior's (24) Clarify and filter.
vibrio or some other bacterial proteoly- Sterilization: Sterilize in the autoclave or
zer for 24 to 36 hours, and then neu- steamer.
tralized with normal NaOH. Sterilize as Use: General culture medium.
indicated above. Reference: Harvey (1921-22 p. 114).
Reference: Duval and Harris' (1911 p. 168).
(2) Mix 500.0 g. beef heart or human (6) Transfer to large bottles and steam
placenta with 1000.0 cc. water. for 10 minutes at 100C. to stop
(3) Make faintly alkaline to litmus digestion.
using N KOH or N Na-.COs. (7) Cool to 80C. and make faintly alka-
(4) Heat slowly to 70-80 for 5-10 line to litmus using 2N KOH or
minutes. 2 normal Na2C03.
(5) Cool to 37C. and add 1.0% pan- (8) Cool to 37C. and add 1.0% pan-
creatic extract or "Bacto" trypsin. creatic extract or "Bacto" trypsin.
(6) Incubate at 37C. for 2-5 hours. (9) Keep the mixture at 37C. for 3 to
(7) Control the progress of digestion by 10 hours depending on the reaction
repeated Biuret and tryptophane of the trypsin and the digestion
tests. In case it is necessary to desired. Control the process by
extend the digestion over a period of repeated tests for tryptophane.
6 hours add chloroform or toluene. (10) When trypsinizing is sufficiently ad-
(8) When the process is sufficiently ad- vanced render reaction slightly acid
vanced, render slightly acid with <vith glacial acetic acid, and bring
glacial acetic acid and boil slowly slowly to boiling point for 10
for 15 minutes. ininutes.
(9) Either filter or decant the clear (11) Filter thru paper or keep in cool
fluid which results on placing the place over night and decant the
digest over night in a cool place. clear liquid in the morning.
(10) Adjust the reaction as desired. (12) Add K2HPO4 and adjust the reaction
(11) Dissolve 2, 3, 4, 5 and 8 in (10). faintly alkaline or to the desired
(12) Heat for 15-30 minutes in the H-ion concentration.
steamer at 100 C. (13) Heat in steamer at 100 for 15
(13) Filter again if necessary. minutes.
Sterilization: Sterilize at 100C. on 3 con- (14) Correct reaction and filter thru
secutive days if not to be used at once. paper.
Use: General inexpensive culture medium. (15) Distribute in receptacles used for
(5) Make a Biuret and tryptophane test. (11) Adjust to desired reaction and steam
When both are + the digest is yel- another 15 minutes.
lowish green and contains very (12) Filter thru paper.
little undigested debris. (13) Distribute as desired.
328 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(14) Sterilize at 100 for 30 minutes on 2 (3) Exactly neutralize with 10.0% H2SO4
successive days or 10 pounds for and test for absence of both Ba and
15 pounds in the autoclave. SO4 ions.
References : Stickel and Meyer (1918 p. 80), (4) Concentrate to thick syrup in
Harvey (1921-22 p. 101). vacuum.
(5) Dilute to a final solids content of
1138. Frieber's Digest Extract Solution
2.0% with distilled water.
Constituents: (6) Treat gelatin in the same manner as
1. Water 1000.0 cc. gliadin (1) thru (5) above.
2. Physiological salt solution.. 3000.0 cc. (7) Dissolve 3, 4, 5, 6, 7 and 8 in 500.0 cc.
3. Fibrin of (6).
4. Beef extract, Liebig's 5.0 g. (8) Mix equal (7) and (5).
parts
5. NaCl 5.0 g. (9) Adjust to pH
from 8.0 to 8.2.
Preparation : (10) Steam 15 minutes and check the
(1) Prepare Frieber's Fibrin Digest reaction.
Solution by digesting fibrin with pep- (11) Distribute as desired.
sin and HCl (see medium 1110). Sterilization: Heat at 115C. for 20 minutes.
(2) Add 5.0 g. Liebig's beef extract, Use: Cultivation of Bad. diphtheriae for
5.0 g. NaCl and 7.0 cc. of normal soda toxin production.
solution to 1000.0 cc. of (1). Reference: Davis and Ferry (1919 p. 232).
ported that the cultures were nearly Ab. Containing organs of flowering plants
non-toxic. or their derivatives.
Variants: The authors added 0.1% sterile Bi. Leguminous plants specified.
glucose or lactose to the sterile medium. Ci. Commercial extracts used.
Reference: Robinson and Rettger (1917 Buchanan's Basal Legume Extract
p. 364). Solution 1156
Buchanan's Salt Legume Extract
SUBGROUP I-C. SECTION 15 Solution 1157
C2. Commercial extracts not used.
Liquid media or basal solutions not con- Di. Leaves and stems employed.
taining digests; containing plant deriva- Buchanan's Basal Clover Infusion
tives of unknown chemical composition. Solution 1158
Ai. Containing yeast derivatives. Buchanan's Vetch Infusion Solution 1159
Bi. Basal solutions; employed with the D2. Flowers employed.
addition of other nutrients. Wilhelmi's Clover Flower Infusion
Henneberg's Basal Yeast Infusion Solution 1160
Solution 1141 D3. Pods employed.
Bj. Complete media. Reed and Cooley'a Bean Pod Infu-
Ci. Containing additional organic carbon of sion Solution 1161
known chemical composition. D4. Seeds or their derivatives employed.
Di. Additional carbon supplied as carbo- Maze's Sucrose Bean Infusion Solu-
hydrates. tion 1162
Heinemann's Glucose Yeast Infu- DeRossi's Glucose Bean Infusion
sion Solution 1142 Solution 1163
Gassner's Lactose Yeast Infusion Kaufmann's Jequirity Infusion Solu-
Solution 1143 tion 1164
Korf's Sucrose Yeast Infusion Solu- Tanner's Pea Flour Infusion Solu-
tion 1144 tion 1165
Bottger's Nitrate Yeast Infusion Stutzer's Basal Legume Seed Infu-
Solution 1145 sion Solution 1166
Dj. Additional carbon supplied as alcohols. B2.* Non-leguminous plants or their deri-
Janke's Alcohol Yeast Infusion Solu- vatives specified.
tion 1146 Ci. Tubers or roots employed.
Bertrand's Sorbitol Yeast Infusion Di. Containing potato or derivatives.
Solution 1147 Smith's Potato Infusion Broth 1167
Ci. Not containing additional organic car- Eisner's Hydrochinone Potato Infu-
bon of known chemical composition. sion Solution 1168
Thoinot and Masselin's Yeast Infu- Robertson and Davis' Potato Infu-
sion Solution 1148 sion Solution 1169
Aj. Containing bacteria or derivatives. Lubinski's Glycerol Potato Infusion
Stoklasa's Arabinose Azotobacter Solution 1170
Solution 1149 Berthelot's Vegetable Infusion Solu-
A3. Containing fungi (fleshy) or their tion 1171
derivatives. Migula's Potato Juice 1172
Lanken and Meyer's Fungus Infu- Winogradsky's Gypsum Root Solu-
sion Solution 1150 tion 1173
A4. Containing peat, moss, etc., or their Da. Not containing potato or derivatives.
derivatives. Tanner's Carrot Infusion Solution. 1174
.
Ampola and Garino's Nitrate Peat Robertson and Davis' Carrot Infu-
Solution 1151 sion Solution 1175
Revis' Peat Infusion Solution 1152 Cs.t Leaves or stems employed.
Brussoff's Iron Peat Infusion Solu- Di. Hay or straw.
tion 1153
Schmidt's Hydrolyzed Peat Solution 1154 * See Bj next page.
Schmidt's Peat Solution 1155 tSee C3 and C4 next page.
330 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Migula's Hay Infusion Solution 1176 Di. Extracts or infusions of grains (not
Winogradsky's Ferric Hydroxide including malts, beer worts, etc.).
Hay Infusion Solution (Molisch). 1177 Harvey's Wheat Flour Solution 1207
Jensen's Nitrate Straw Solution. . . . 1178 Omeliansky's Flax Stem Solution.. 1208
Wolbach and Binger's Glucose Hay Speakman and Phillips' Maize Mash
Infusion Solution 1179 Solution 1209
Sherman's Soil Hay Infusion Solu- D2. Malt extract, beer wort, beer, etc.,
tion 1180 employed.
D2.* Cabbage. El. Beers used.
Smith's Cabbage Infusion Solution. 1181 Lafar's Beer Solution 1210
Conrad's Glucose Cabbage Infusion Waterman's Sucrose Beer Solution. 1211
Solution 1182 E2. Malts used.
Will's Cabbage Juice 1183 Wurtz's Malt Infusion Solution... 1212
D3. Other leaves or stems. Beijerinck's Malt Wort Solution... 1213
Reed and Cooley's Basal Spinach Peklo's Malt Infusion Solution 1214
Infusion Solution 1184 Bokorny's Malt Infusion Solution. 1215 .
(b) Henneberg prepared a 10.0% yeast (e) Besson prepared the medium as
infusion solution and added 2.0 to follows:
5.0% of any desired carbohydrate, (1) Boil 100.0 g. of beer yeast in
alcohol, etc. 1000.0 cc. of water.
(c) Bobilioff-Preisser added one of the (2) Filter thru paper.
following materials to yeast infusion: (3) Add 5.0% of glucose or sucrose.
dextrin glucose (4) Add a small amount of phosphoric
galactose maltose acid and then add sufficient lime
levulose raffinose water to give a slightly alkaline
lactose sucrose reaction.
The media were sterilized in the (5) Heat for 5 minutes at 116 to IH^C.
steamer. (6) Filter.
(d) MUller-Thurgau and Osterwalder pre- (7) Tube.
pared the basal solution as follows: (8) Sterilize at 115C.
(1) Add water (amount not given) to References: Henneberg (1903 p. 8), Bobi-
1000.0 g. pressed yeast and boil. lioff-Preisser (1916 p. 387), Muller-
(2) Filter. Thurgau and Osterwalder (1918 p. 2),
(3) Dilute the filtrate to 10 liters by the Besson (1920 p. 35).
addition of water.
(4) Acidify by the addition of 1.0% 1142. Heinemann's Glucose Yeast Infusion
malic acid. (Do not add acid here Solution
when adding an acid as a carbon
source.) Constituents:
(3) After pouring off the last water add ness of alcohol or ether have dis-
18 liters of water to the remaining appeared.
washed yeast cells. (15) Prepare a 10.0% solution of (14) in
(4) Boil in the autoclave or steamer as in distilled water.
the preparation of meat bouillon. (16) Mix about 10.0% of (15) with 10.0%
(5) Allow to stand for a suitable length of (9).
of time and remove the liquid from (17) Distribute in 150.0 cc. lots in fer-
the sediment or filter thru filter paper. mentation flasks.
(6) Add 0.3 or 0.5% lactose to the filtrate Sterilization: Sterilize in streaming steam.
or supernatant fluid. Use: To study fermentation by yeast.
(7) Adjust to slightly alkaline to litmus. Waterman used a similar medium to
Sterilization: Not specified. study the inversion of sucrose by bacteria.
Use: General inexpensive culture medium. Variants
Variants The author used the medium with
: (a) Syr(e prepared a similar medium as
constituents of double strength, with or follows:
without 0.5% NaCl. (1) Wash brewers yeast in a decanta-
Reference: Gassner (1916-17 p. 311). tion funnel with distilled water
until the wash water, to which
1144. KorfE's Sucrose Yeast Infusion some yeast has been added, boiled
Solution
and filtered, will give no reaction
Constituents: with Fehling's solution.
1. Water 2000.0 to 3000.0 cc. (2) Boil 1000.0 g. of the washed yeast
2. Yeast 1000.0 g. with 2 liters distilled water for
3. Sucrose. 2 hours.
Preparation (3) Filter.
(1) Mix 1000.0 g. of yeast with water and (4) Boil the filtrate once more.
decant. Continue this until the (5) Filter.
liquid is colorless and does not re- (6) Estimate the nitrogen content by
duce Fehling's solution. Kjeldahl's method and dilute so
(2) Pour the yeast in a porcelain funnel that the nitrogen content is
(11) Filter while hot in the hot water The reaction was made neutral with
funnel into a large porcelain dish. soda. The medium was used for the
(12) Add 2 liters of warm absolute alco- cultivation of lactic acid bacteria.
hol, stirring continually. (c) Will added 6.0% sucrose to yeast
(13) Pour the cool alcohol from the sugar water (preparation not given).
and wash the sugar with absolute (d) Waterman added 4.0% sucrose to
alcohol and then with ether. yeast water (preparation not given)
(14) Dry at 60C. until all traces of damp- to study the inversion of sucrose by
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 333
Variant: Author used 0.17, 0.41, 0.83, 1.67 (4) Make slightly alkaline by the addi-
tion of soda.
or 3.33% NaNO, instead of KNO3.
Reference: Bottger (1921 (5) Filter.
p. 224).
(6) Distribute as desired.
Sterilization: Sterilize in the autoclave at
1146. Janke's Alcohol Yeast Infusion
115 for 15 minutes.
Solution
Use: General culture medium.
Constituents: Variants
1. Water 900.0 cc. (a) Henneberg used a 2.5, 5.0 or 10.0%
2. Yeast (pressed) 100.0 g. yeast water solution but did not give
3. Alcohol (3.0%) 30.0 cc. the method of preparation. He culti-
Preparation vated lactic acid bacteria.
(1) Boil 100.0 g. of pressed yeast with (b) Heinemann prepared the medium
900.0 cc. water. as follows:
(2) Filter.
(1) Boil one pound of pressed yeast or
(3) Add 3.0% by volume of absolute alco- one liter of washed yeast with
hol after sterilization. 2 liters of water forone hour.
Sterilization: Method of sterilization of (2) Neutralize to phenolphthalein.
(2)not given. Filter the solution until clear.
(3)
Use: Cultivation of acetic acid bacteria the Arnold on 3 con-
(4) Sterilize in
found in beers. secutive days.
Reference: Janke (1916 p. 6). (c) Gassner prepared a similar medium
as follows:
1147. Bertrand's Sorbitol Yeast Infusion
(1) Place about 10 liters of brewers
Solution
yeast in a flask and wash with
Constituents: water. Allow to stand for 30 min-
1. Yeast infusion 1000.0 cc. utes and pour off the liquid.
2. KH2PO4 0.1 g. (2) Repeat the washing process until
3. Sodium phosphate 0.1 g. the wash water is no longer brown
334 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Sterilization: Sterilize thoroly (method not References: Lanken and Meyer (1921
given). p. 511), Wiegert (1922-23 p. 110), Klim-
Use: Decomposition of organic nitrogen mer (1923 p. 204).
products by Bacillus mycoides.
1151. Ampola and Garino's Nitrate Peat
Reference: Stoklasa (1911 p. 470).
Solution
(6) Add 2 or 3 pieces of sterile iron 1156. Buchanan's Basal Legume Extract
filings to sterile (5) under aseptic Solution
conditions.
Constituents:
Sterilization: Sterilize (5) in streaming
1. Water 1000.0 cc.
steam for 6 hours. Sterilize 2 or 3 pieces
2. Legume extract (Park Davis & Co.).
of iron filings dry heat at 150 for one
Preparation
hour.
(1) Boil 1000.0 cc. of one of Park Davis &
Use: Cultivation of Ferribacterium duplex.
Company's standard legume fluid ex-
Author reported that after 5 or 10 days a
tract until all the alcohol has evapo-
slight yellow membrane was formed.
rated. The legume extracts used
Reference: Brussoff (1916 p. 549).
were those of clover, tolu, catechu,
physostigma, red clover blossom,
1154. Schmidt's Hydrolyzed Peat Solution
glycyrrhiza, kino, baptisia, senna and
Constituents scoparius.
1. Water 1000.0 cc. (2) Make up to 1000.0 cc. by the addition
2. Peat 20.0 g. of water.
3. Salts. (3) Filter.
Preparation (4) Prepare 10.0, 5.0, 1.0 and 0.1% solu-
(1) Add 1000.0 g. of 0.3% HCl to 20.0 g. tions of these extracts with water.
of peat and hydrolyze for 1 hour at In case of the clover extract, do not
2.5 atmospheres pressure. dilute. In case of the glycyrrhiza
(2) Filter. prepare only 1.0 and 0.1% solutions.
(3) Neutralize and add an excess of (5) Add one of the added nutrients to
CaCOs. extract solutions.
(4) Add the usual salts (amounts or kinds Sterilization: Sterilize for 20 minutes on
not given). The salts are to be nitro- each of 3 successive days in streaming
gen free. steam.
Sterilization: Not specified. Use: To study gum formation by Bacillus
Use: To study nitrogen assimilation by radicicola. The author reported that
soil forms. Author reported that azoto- the extract of catechu, physostigma, kino
bacter formed as a scum on the surface of and scoparius inhibited growth. The
the medium in several days. remaining legume extracts favored growth
Reference: Schmidt (1920 p. 283). and gum production.
Added nutrients: The author added 2.0%
1155. Schmidt's Peat Solution sucrose or 1.0% maltose to the extracts
Constituents prepared as indicated in step (4) above.
1. Water 1000.0 cc. Reference: Buchanan (1909 p. 391).
2. NH4CI (0.25%) 2.5 g.
K2HPO4 (0.05%) 1157. Buchanan's Salt Legume Extract
3. 0.5 g.
Solution
4. CaCOs (2.0%) 20.0 g.
5. Peat 20.0 g. Constituents
Preparation 1. Water 1000.0 cc.
(1) Dissolve 2, 3 and 4 in 1. 2. Legume extract (Park Davis & Co.).
(2) Add 20.0 g. of peat to (1). Preparation
Sterilization: Not specified. (1) Boil 1000.0 cc. of one of Park Davis &
Use: Cultivation of and horse manure
soil Company's standard legume fluid
forms. Azotobacter. Author reported extract until all the alcohol has
that azotobacter formed a membrane on evaporated. The legume extracts
the surface after several days. used were those of clover, tolu, ca-
Variants: The author used moss instead of techu, physostigma, red clover blos-
peat. som, glycyrrhiza, kino, baptisia,
Reference: Schmidt (1920 p. 284). senna and scoparius.
CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS 337
(2) Make up to 1000.0 cc. by the addition the bacteroids, either unmodified or upon
of water. the addition of asparagin or peptone.
(3) Filter. Added nutrients: The author added one of
(4) Heat distilled water containing 0.2% the following:
KH2PO4 and 0.01% MgS04 to boiling. KH2PO4 0.2%
(5) Cool (4) and filter. MgS04 0.1%
(6) To each 100.0 cc. of (5) add 0.1%, Ammonium phosphate 0.5%
1.0%, 2.0%, 5.0% or 10.0% of one of Asparagin 1.0%
(3). (Filtered extract solution.) Sodium asparaginate 1-0%
(7) Incubate at room temperature for one Peptone 0.1, 0.5, 1.0,
week to test sterility. 2.0 or 5.0%
for 20 minutes on
Sterilization: Sterilize [Sucrose 2.0%
each of 3 successive days in streaming \ [0.005, 0.01,
steam. [KNO3 i0.05, 0.1, 0.2
Use: To study gum formation by Bacillus [0.5 or 1.0%
radicicola. Author reported that tolu, Glucose 2.0%
red clover blossoms, glycyrrhiza, kino, Reference: Buchanan (1909 pp. 62, 386,
baptisia and senna favored production. 391).
Cathechu, physostigma, scoparius and
kino in high concentrations were un-
1159. Buchanan's Vetch Infusion Solution
suitable. Constituents:
Variants: The author added 1.0% maltose. 1. Water 2000.0 cc.
Reference: Buchanan (1909 pp. 72, 386). 2. Vetch stems and leaves 200.0 g.
Preparation:
1158. Buchanan's Basal Clover Infusion
(1) Boil 200.0 g. of dry vetch stems and
Solution
leaves in 2 liters of water.
Constituents: (2) Filter.
1. Water 4000.0 cc. (3) Concentrate to ^ its volume by heat.
2. Clover leaves and stems (4) Add several times its volume of
{Trifolium pratense) 200.0 g. alcohol.
Preparation: (5) Filter.
(1) Extract 200.0 g. of the leaves and (6) Evaporate the filtrate to dryness.
stems of clover (Trifolium pratense) (7) Wash the precipitate with absolute
with 4 liters of boiling tap water. alcohol, several times and evaporate
(2) Filter until clear. to dryness.
(3) Add one of the added nutrients to (2). (8) Dissolve each of these extracts in
(4) Heat to boiling. 100.0 cc. of distilled water.
(5) Cool and filter. (9) Filter.
(6) Incubate for one week at room tem- (10) The first of these is called alcoholic
perature to test sterility. extract and the second the aqueous
for 20 minutes on
Sterilization: Sterilize extract.
each of 3 successive days in streaming (11) Prepare 0.01, 0.1, 0.2 or 1.0% solu-
steam. tions of the aqueous extract in dis-
Use: To study gum formation by Bacillus tilled water and 0.1, 0.5, 1.0, 2.0 or
radicicola, and cultivation of Bacillus 5.0% solutions of the alcoholic ex-
radicicola bacteroids. Author reported tract in distilled water.
that clover extracts alone favored the (12) Tube.
production of gum. Peptone, and to a (13) Incubate for one week at room tem-
less degree, asparagin and sodium aspara- peratures to test sterility.
ginate inhibited the production of gum. Sterilization: Sterilize in streaming steam
Nitrates except in large amounts did not for 20 minutes on each of 3 successive
effect growth and gum production. days.
Clover extract did not constitute a Use: To study gum formation by Bacillus
favorable medium for the development of radicicola and cultivation of Bacillus
338 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
given). paper.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 339
Added nutrients and variants: (2) Add a little distilled water (amount
(a) The author added one of the fol- not given).
lowing :
(3) Allow to stand in the cold for
Asparagin 2.0% 24 hours.
Glucose 1.0, 5.0 or 10.0% (4) Press thru a towel.
Sucrose 1.0, 5.0 or 10.0% (5) Sterilize until there is no more co-
starch 2.0% agulum formed (time not specified).
flour 2.0% (Filtration not specified.)
inulin 2.0% (b) Tanner prepared a similar medium as
oil small amount follows:
(b) The author prepared an infusion from (1) Wash and grind a few potatoes.
clover seeds in the following manner: (2) To 30.0 g. of (1) add a liter of dis-
(7) Autoclave one hour. (8) Add various amounts of (7) to each
(8) Filter thru cotton (very tedious). tube of sterile (4).
(9) Store in flasks and sterilize in the Sterilization: Autoclave (3) at 20 pounds
autoclave for 30 minutes at 15 pressure for 30 minutes.
pounds pressure. Use: To study influence of vitamines on
References: Smith (1905 p. 42), Peklo (1910 bacterial growth. Authors reported that
p. 551), Tanner (1919 p. 60), Besson (1920 yeast grew very luxuriantly.
p. 35), Dopter and Sacquepee (1921 Reference: Robertson and Davis (1923
p. 120), Robertson and Davis (1923 p. 154), p. 154).
Park, Williams and Krumwiede (1924
1170. Lubinski's Glycerol Potato
p. 122).
Infusion Solution
(6) Pour the mixture on a clean, thick (9) Allow to stand for 24 hours in the
cloth. cold or better on ice.
(7) Collect the fluid which drains thru (10) Filter thru paper.
the cloth together with that ob- (11) Distribute as desired.
tained by squeezing the cloth. Sterilization: Sterilize at 115 for 20
(8) Filter the fluid collected thru minutes.
thick filter paper. Use: General culture medium for sapro-
(9) Add the filtrate to an equal quan- phytes and pathogenic forms.
tity of distilled water. Variants: Tanner gave the following
(10) Steam 60 minutes. method of preparation:
(11) Add glycerol to 4 per cent. (1) Peel the potatoes.
(12) Mix well. (2) Wash the carrots and turnips.
(13) Filter. (3) Cut (1) and (2) in small pieces and
(14) Distribute into test tubes. place in cold water.
(15) Sterilize in the autoclave or (4) Boil 4 hours.
steamer. (5) Strain.
(b) Harvey prepared a medium similar (6) Make weakly alkaline.
to the above as follows: (7) Heat in autoclave at 120C.
(1) Grate finely washed, peeled po- (8) Allow to stand in a refrigerator
tatoes. over night.
(2) Add 2 parts 4 per cent glycerol to (9) Filter thru paper.
1 part potato gratings. (10) Final sterilization not given.
(3) Boil. References: Berthelot (1917 p. 131), Tan-
(4) Filter. ner (1919 p. 46).
(5) Use the filtrate as potato extract.
1172. Migula's Potato Juice
References: Lubinski (1895 p. 126), Harvey
(1921-22 pp. 118, 119). Constituents:
1. Potato.
1171. Berthelot's Vegetable Infusion
Preparation:
Solution
(1) Pass peeled potatoes thru a sieve, or
Constituents: grind real fine.
1. Water 4000.0 cc. (2) Press the juice thru a linen towel.
2. Potato 300.0 g. (3) Distribute in tubes.
3. Carrots 150.0 g. Sterilization Boil for 20 minutes on each of
:
after 5 to 7 days. The slime at the bot- (d) Draper gave the following method of
tom became black. The liquid became preparation. The medium was used
opalescent and surface 3 to 6 weeks a for the cultivation of Oidium albi-
membrane of sulfur formed on the surface. cans.
Reference: Winogradsky (1888 p. 11). (1) Chop raw carrots into fine particles
and allow to extract in sterile dis-
1174. Tanner's Carrot Infusion Solution tilled water at about 10C. for
Constituents: 48 hours.
1. Distilled water 1000.0 cc. (2) Filter.
(4) Filter.
1175. Robertson and Davis' Carrot Infusion
(5) Boil the filtrate.
Solution
(6) Filter again if necessary.
(7) Tube. Medium identical with medium 1169 but
(8) If the reaction is too acid, adjust carrot used instead of potato.
as usual.
Sterilization: Method not given.
1176. Migula's Hay Infusion Solution
(2) Boil with a little water. Tausz and Peter (1919 p. 509), Tanner
(3) Cut the grass into small pieces and (1919 p. 57), Besson (1920 p. 35), Dopter
boil for 20 minutes with 3.5 liters and Sacquepee (1921 p. 120), Harvey
of water, (1921-22 pp. 120, 121).
(4) Filter. The filtrate is dark brown.
(5) Neutralize by the addition of sev- 1177. Winogradsky's Ferric Hydroxide Hay
eral drops of KOH. Infusion Solution (Molisch)
(6) Sterilize by steaming on each of 3
Constituents:
successive days for 30 minutes.
1. Water.
They cultivated Bacterium alipha- Hay.
2.
ticum, Bacterium aliphaticum lique-
3. Ferric hydroxide.
faciens, paraffin bacteria.
Preparation
(d) Tanner, Besson, and Dopter and
(1) Place a handful of macerated hay
Sacquepee. that has been well extracted with
(1) Macerate 15.0 to 20.0 g. of finely
water in a glass cylinder 50 cm. high.
chopped hay in 1000.0 g. of water
(2) Sprinkle some freshly precipitated
for one or two hours.
ferric hydroxide on the hay,
(2) Boil a few minutes.
(3) Fill the cylinder with well water.
(3) Filter. Sterilization: Not specified.
(4) Tube. Use: Cultivation of iron bacteria, Chlamy-
(5) Sterilize at 115C.
dothrix {Leptothrix) ochracea.
(6) The infusion may be neutralized if
Reference: Molisch (1910 p. 32).
the reaction is a little acid.
tion being about 0.2% acid to plienol- 1182. Conrad's Glucose Cabbage Infusion
phthalein. Solution
(2) Decant the water and add 1.0% Constituents:
glucose. 1. Water 200.0 cc.
(3) Adjust the reaction to neutral to 2. Cabbage 100.0 g.
phenolphthalein. 8. Glucose 3.0%
(4) Tube after sterilization. 4. NaCl 0.5%
Sterilization: Sterilize by filtration thru Preparation:
Chamberland F filter.
(1) Prepare an extract of cabbage (Weiss-
Use: Cultivation oi Spirocheta elusa (free kraut abkochung) in the ratio of 1:2.
living). Author reported that the me- Method not given.
dium was clouded by growth in about (2) Add 3.0% glucose and 0.5% NaCl
48 hours. Medium not so satisfactory if to(l)._
sterilized by repeated steaming or in the (3) Place in a large flask.
autoclave. Sterilization: Sterilize on 3 successive days
Reference: Wolbach and Binger (1914 p. 9). in the steamer.
Constituents:
or higher NaCl concentrations, m '
(7) Adjust the reaction. (a) Used tartaric acid instead of malic.
(8) Filter thru well-wetted, thick filter (b) Used 0.3% tartaric acid and 0.3%
paper. malic acid instead of malic acid
(9) Distribute into flasks or test tubes. alone.
References: Tanner (1919 p. 59), Besson (c) Heinemann prepared a similar me-
(1920 p. 36), Harvey (1921-22 p. 119). dium as follows:
(1) Dilute wine must with 4 times its
1194. Turner's Quince Seed Infusion
weight of water.
Solution
(2) Dissolve 0.5% ammonium tartrate
Constituents: in (1).
1. Distilled water. (3) Macerate in the water bath for
2. Quince seed. 1 hour.
Preparation (4) Filter.
(1) Boil quince seeds in distilled water. (5) Sterilize in the Arnold for 3 con-
(2) Pass the thick glutinous mass thru a secutive days.
sieve. References: Schukow (1896 pp. 608, 610),
(3) Dilute with distilled water to obtain Heinemann (1905 p. 129).
the desired consistency.
1197. Perold's Wine Medium
Sterilization: Not specified.
Use: Cultivation of Euglena. Constituents
Reference: Turner (1917 p. 239). 1. Spatburger wine.
Preparation: (1) Neutralize completely fer-
1195. Miiller-Thurgau and Osterwalder's
mented Spatburger wine of an acidity of
Grape Juice
9.15 g. wine acid per liter, to 5.0% acetic
Constituents acid by the addition of 21.28% KOH.
1. Grape juice. Sterilization: Method not given. After
Preparation: (1) Preparation of grape juice sterilization the medium had the follow-
not given. ing composition:
Sterilization: Not specified. Total acid 5.0% acetic acid.
Use: To study fermentation by Bacterium Alcohol content 8.5% by volume.
mannitopeum, Bacterium intermedium, Volatile acid 0.39% acetic acid.
Bad. Gayoni, Mannite bacteria from Use: Cultivation of bacteria found
in wine.
winft. Bacterium gracile, Micrococcus Variants
acidovorax, Micrococcus variococcus (a) Miiller-Thurgau and Osterwalder
Reference: Miiller-Thurgau and Oster- used red or white wine. Wine may
walder (1918 p. 17). or may not be sterilized. Used to
study fermentation of Bacterium
1196. Schukow's Grape Must Solution
mannitopoeum Bacterium interme-
Constituents: dium, Bact. Gayoni mannitol bac-
1. Water 300.0 cc. teria from wine.
2. Grape must 100.0 cc. (b) Tanner and Besson neutralized the
3. Malic acid (9.0 to wine before sterilization.
10.0%) 36.0 to 40.0 g. References: Perold (1909 p. 18), Muller-
Preparation Thurgau and Osterwalder (1918 p. 19),
(1) Dilute one part grape juice with 3 Tanner (1919 p. 57), Besson (1920 p. 36).
parts water.
1198. Bierberg's Currant Must Solution
(2) Add 9.0 to 10.0% of malic acid to (1).
(3) Distribute into fermentation flasks Constituents:
(sealed with H2SO 4). 1. Water 4000.0 cc.
Sterilization: Sterilize in a steamer. 2. Currant must 1000.0 cc,
Use: To study the utilization of acid by 3. NH4CI 20.0 or 40.0 g.
yeast. Preparation
Variants: The author gave the following (1) Add 1 liter of currant must (800
variants Oechsle) to 4 liters of water.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 349
(2) Dissolve 20.0 or 40.0 g. of NH4CI short time, however, the alkali was
in (1). neutralized and development stopped.
Sterilization: Not specified. Melon juice was superior to cucumber
Use: To study fermentation by yeast. juice.
Author reported that the addition of Variants : The author substituted cucumber
ammonium salts seemed to hasten the juice for melon.
COj production. The higher amount of Reference: Ottolenghi (1911 p. 370).
(2) Neutralize the juice with 3.0% Preparation: (1) Preparation of pear juice
Na.COa and add 10.0%. not given.
(3) Flask. Sterilization: Not given.
(4) Sterilize in the autoclave. Use: To study fermentation by Bacterium
(5) Filter. mannitopoeum, Bacterium, intermediutn,
Sterilization: Sterilize in the autoclave Bad. Gayoni, mannitol from wine.
following filtration. Variants: The authors specified that malic
Use: Enrichment media for cholera acid or alcohol might be added to the
vibrio. The author reported that in pear juice.
melon juice the cholera vibrio developed Reference: MuUer-Thurgau and Oster-
much like in peptone water. After a walder (1918p. 48).
350 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1. Grape must.
1207. Harvey's Wheat Flour Solution
Preparation
(1) Prepare grape must of 16.8 Balling. Constituents:
(2) Distribute into fermentation flasks 1. Water 1000.0 cc.
(sealed with H2SO4). 2. Glucose 15.0 g.
Sterilization: Sterilize in a steamer. 3. KNO3 1.0 g.
0.25 g. acid from currant must, and 0.05 g. (Further preparation not given.)
acid from apple must. Acid determined Sterilization: Not specified.
of amoba.
1208. Omeliansky's Flax Stem Solution
Variants: Author used the following in-
stead of grape must: Constituents:
(a) Currant must of 14.4 Balling. 1. Distilled water 1000.0 cc.
(b) Currant must of 14.4 Balling diluted 2. Ammonium phosphate 1-0 g.
(c) Dopter and Sacquepee prepared a ported that at the end of 5 days there was
medium in the same manner as vari- 35 mg. H.iS formed per liter.
ant (a) above, but used only 50.0 g. Reference: Beijerinck (1895 p. 54).
of malt dust instead of 100.0 g.
(d) Harvey cultivated yeast, molds and
1214. Peklo's Infusion Solution
trichophyta on a medium prepared
as follows: Constituents
(1) Prepare: Crushed malt 1 part; dis- 1. Water to 1480.0 cc.
tilledwater at 70C. 4 parts. 2. Malt
(2) Keep 60 minutes at 60C. in a flask 3. K2HPO4 8.0 g.
closed with a rubber cork. 4. K2CO3 6.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 353
(6) After several sterilizations (times or 1217. Reddish's Malt Extract Solution
method not specified) and filtering ofi
Constituents:
the formed precipitate, distribute in
1. Distilled water 900.0 cc.
sterileErlenmeyer flasks.
2. Malt extract 100.0 g.
(7) To 700.0 cc. of (6) add 780.0 g. of dis-
Preparation
tilled water, 8.0 g. K2HPO4 and 6.0 g.
(1) Dissolve 100.0 g. dry extract of malt
of K2CO3.
in 900.0 cc. distilled water.
(8) The reaction is strongly alkaline.
(2) Make to 8 Kaiser (saccharometer)
Sterilization decreases the alkalinity
by adding distilled water (approxi-
somewhat but the reaction still re-
mately 100.0 cc).
mains alkaline.
(3) Adjust the reaction to +1.5 (1.5%
Sterilization: Sterilize (7); method not
agar may be added and dissolved).
given.
(4) Autoclave for 15 minutes at 15 pounds
Use: Cultivation of plant actinomyces.
pressure.
Reference: Peklo (1910 p. 470).
(5) Filter thru folded filter paper.
(6) Tube.
1215. Bokorny's Malt Infusion Solution
Sterilization: Sterilize at 15 pounds for
Constituents 10 minutes or 10 pounds for 15 minutes.
1. Water 500.0 cc. Use: Substitute for beer wort.
2. Malt infusion 500.0 cc. Reference: Reddish (1919 p. 6),
3. Maltose (2.0%) 20.0 g.
Preparation 1218. Park, Williams and Krumwiede's
(1) Preparation of malt infusion not Basal Beer Wort Solution
given.
Constituents
(2) Mix (1) with equal parts of water and
1. Beerwort.
add 2.0% maltose.
Preparation
Sterilization: Not specified.
(1) Obtain hopped beerwort from the
Use: Cultivation of yeast.
brewery.
Variants: The author used the following:
(2) Autoclave.
(a) Undiluted malt infusion without any
(3) Cool.
maltose.
(4) Filter.
(b) Dilute malt infusion with 3 times
(5) Dissolve 2.0% of one of the added
its volume of water and add 1.0%
nutrients in the filtrate.
maltose.
(6) Tube.
Reference: Bokorny (1920 p. 30).
Sterilization: Sterilization not specified.
1216. Bacto Malt Extract Broth Use: Cultivation of yeast and molds.
(Dehydrated) Added nutrients: The authors added 2.0%
of any desired carbohydrate, alcohol, etc.
Constituents:
Reference: Park, Williams and Krumwiede
1. Distilled water 1000.0 cc.
(1924 p. 134).
2. Malt e.xtract, dehydrated,
Bacto 15.0 g.
1219. Peklo's Beer Wort Solution
Preparation:
(1) Dissolve 15.0 g. Bacto dehydrated Constituents
malt extract in 1000.0 cc. of distilled 1. Distilled water 450.0 cc.
water. 2. Beer wort 550.0 cc.
354 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
3. K2HPO4 6.0 g.
2. K2HPO4 6.0 g.
4. K2CO3 4.5 g.
3. K2CO3 4.5 g.
5. Calcium tartrate 24.0 g.
4. Beer wort 450.0 cc.
Preparation
(c) 1. Distilled water. 200.0 cc.
.
Preparation: (1) Prepare a 12.0%, unhopped Preparation: (1) Add 0.7% asparagin to
(3) Heat in autoclave a few minutes. Liquid media or basal solutions not con-
(4) Filter thru filter paper. taining digests, but containing ingredients
(5) Tube. of animal origin of unknown chemical
Sterilization: Method not given. composition.
Use: To show acid production by silage Ai.* Only one constituent of unknown
organisms. chemical composition present.
Reference: Sherman (1916 p. 449). Bi. Materials of animal origin, exclusive of
infusions and extracts employed.
1226. Buchanan's Silage Infusion Solution
Ci. Containing animal tissues, cells or
Constituents their derivatives.
1. Water, tap 1000.0 cc.
2. Silage 500.0 g. See page 358 for A2 and page 357 for B2
356 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Zotta's Brain Infusion Solution 1233 Toyoda's Glucose Serum Solution. 1261 .
Fi. Basal solutions; employed with the Besson's Defibrinated Blood 1274
addition of other constituents. Row's Defibrinated Blood Solution
Hollande and Fumey's Basal Al- (Harvey) 1275
bumin Solution 1246 Carpano's Defibrinated Blood
Waksman's Basal Albumin Solution . 1247 Solution 1276
F2. Complete media. Ej. Blood derivatives used.
Kent's Glycerol Albumin Solu- Waksman's Basal Fibrin Solution 1277
. .
El. Basal solutions; employed with the Ottolenghi's Nitrate Bile Solution.. 1284
addition of other materials. Jackson's Lactose Bile Solution 1285
Hiss' Basal Serum Solution 1253 Meyerstein's Glycerol Bile Solution. 1286
Leuch's Basal Serum Solution D4. Other body fluids employed.
(Klimmer) 1254 Thoinot and Masselin's Aqueous
E2. Complete media. Humor Medium 1287
Fi. Not containing additional organic Chira and Noguchi's Glucose Ascitic
carbon. Fluid Medium 1288
Lorrain-Smith's Alkaline Serum Sinton's Glucose Body Fluid
Solution (Heinemann) 1255 Solution 1289
Klein's Basal Alkaline Serum Solu- C3. Containing animal secretions or ex-
tion (Leuch) 1256 cretions.
Martin, Pettit and Vaudremer's Di.f Secretions employed.
Serum Solution 1256a
* See D3 and D4.
* See C3 next column. t See page 357 for Da
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 357
El. Skim or whole milk used. Doerr's Mannitol Nutrose Solution. 1318
Fi. No indicators added. F2. Nutrose not specified.
Tanner's Milk Powder Solution 1290 Whittaker's Lactose Caseinogen
Brown and Howe's Milk Solution. . 1291 Solution 1319
Boekhout's Sucrose Milk Solution . . . 1292 Waksman's Basal Casein Solution.. 1320
Reinsch's Milk Medium 1293 Seliber's Basal Casein Solution
Fj. Indicators added. (Harvey) 1321
Gi. Litmus or its salts used. Laxa's Lactic Acid Casein Solution. 1322
Hiss' Basal Litmus Milk Solution. . 1294 Baginsky's Casein Solution 1323
Tanner's Azolitmin Milk Powder Harvey's Alkaline Casein Solution. 1324
Solution 1295 D2. Excretions employed.
Bacto Litmus Milk (Dehydrated) 1296 . . El. Urine used.
Calandra's Picric Acid Litmus Milk Geilinger's Basal Urine Solution... 1325 .
Thoni and Allemann's Nutrose Harde and Hauser's Fish Infusion.. 1346
Solution 1314 Pergola's Mussel Infusion 1347
Ficker and Hoffmann's Cafiine Nu- Besson's Chicken Infusion (Tanner). 1348
trose Solution 1315 Cj.* Extracts employed.
LoefHer's Lactose Nutrose Solution. 1316
Hill's Artificial Milk Medium 1317 See C3 page 358.
358 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
D3. Ascitic or other serous fluids used. Use : Cultivation of obligate anaerobes.
Noguchi's Ascitic Fluid Tissue Reference: Harrass (1906 p. 2237).
Medium 1367
1231. Moon's Brain Medium
Kligler and Robertson's Ascitic
Fluid Egg Medium (Stitt) 1368 Constituents:
C2. Animal fluids not added. 1. Ringer's solution.
Bacto Egg-Meat Medium (De- 2. Brain, dog.
hydrated) 1369 Preparation
Rettger's Egg-Meat Medium 1370 (1) Obtain brain material from normal
Besredka's Egg Meat Infusion dog under the most careful aseptic
Medium (BezanQon) 1371 precautions.
CULTURE MEDIA FOR CULTIVATION' OF MICROORGANISMS 359
(5) Just before use, place the tubes in (8) Tube the pieces of heart and just
flowing steam for 30 minutes and cover them with the extract, taking
cool rapidly. care to wash down any pieces of
(6) Seed with a pipette, swab or needle tissue which adhere to the sides of
and mix material with meat the tube.
particles. (9) Sterilize in the autoclave.
(c) Bachmann studied the effect of spices References: Migula (1901 p. 24), Holman
on the growth of Clostridium botu- (1918 p. 125), Bachmann (1918 p. 237),
linurn, using the following medium: Burke (1919 p. 557), Besson (1920 p. 55),
(1) Grind hamburger and add enough Cunningham (1924 p. 166).
water to make 100.0 cc.
1240. Nevin's Glucose Veal Medium
(2) Add one of the following, cloves,
cinnamon, all-spice, ginger, nut- Constituents:
meg, cayenne pepper, white mus- 1. Water 1000.0 cc.
tard, black mustard in 1.0, 2.0 or 2. Chopped veal 1000.0 g.
2.5 g. quantity. 3. NaCl 10.0 g.
(3) Mix thoroly. 4. Glucose 40.0 g.
(4) Distribute into 6 inch test tubes. Preparation
(5) Sterilize in the autoclave. (1) Mix equal parts chopped veal and
(6) Inoculate when cool. water.
He reported that the organisms were (2) Add 0.5% NaCl and 2.0% glucose.
not affected by any of the spices used (3) Adjust reaction to 0.3% to 0.5%
in amounts up to 2.0%. 2.5% alkaline to phenolphthalein.
retarded the growth of some Sterilization: Method not given.
organisms. Use: Cultivation of B. botulinus.
(d) Burke isolated B. botulinus in a Reference: Nevin (1924 p. 228).
medium prepared as follows:
1241. Hueppe's Egg Meditim.
(1) Chop 500.0 g. of beef heart or other
lean beef with 1000.0 cc. of water. Constituents:
(2) Bring slowly to a boil, stirring 1. Eggs.
constantly. Preparation
(3) Neutralize. (1) Wash the eggs thoroly.
(4) Tube. (2) Sterilize the shell by dipping in sub-
(5) Sterilize at 15 pounds pressure for limate solution.
30 minutes. (3) Wash in sterile water and dry with
(e) Besson cultivated Vibrion septique sterile cotton.
on a medium prepared as follows (4) Make opening in the top of the
a fine
(1) Add 500.00 to 600.00 g. of finely egg with an instrument heated to
chopped beef to a liter flask. glowing.
362 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Inoculate thru the hole with a plati- (4) Autoclave at 15 pounds pressure for
num wire or loop. 20 minutes.
(6) Shake well to mix. (5) Some of the albumin will settle to the
(7) Cover the opening with a small piece bottom of the tubes. This does not
of sterile paper. detract from the value of the medium.
(8) Seal completely with a colloidion (6) The tubes are sealed with a vaseline
membrane. cap.
Sterilization: Given under preparation. Reference: Kahn (1922 pp. 175, 192).
Use: General culture medium.
1243. Harvey's Egg Solution
Variants: Investigators have used slightly-
different methods in sterilization of the Constituents
shell, and several have specified that the 1. Water 300.0 cc.
egg be thoroly shaken before use. The 2. Egg
differences are not of sufficient impor- Preparation
tance to warrant a separate discussion for (1) Add the contents of one egg to 300.0
each investigator. cc. water.
References: Hueppe (1888 p. 80), Heim (2) Shake to mix.
(1891 p. 430), Hammerl (1894 p. 155), (3) Raise the temperature slowly to
Abel and Draer (1895 p. 65), Roux and boiling point with frequent shaking.
Rochaix (1911 p. 129), Ball (1919 p. 83), (4) Distribute into test tubes.
Besson (1920 p. 54), Harvey (1921-22 Sterilization: Sterilize in the autoclave or
p. 85). steamer.
Use: Cultivation of wound organisms and
1242. Robertson's Egg Solution (Kahn) other anaerobes. Also used to cultivate
Constituents other microorganisms.
1. Water 500.0 cc. Variants Klimmer prepared the medium as
:
(1) Beat the yolk of one and the whites of flask containing glass beads.
2 eggs in a beaker. (2) Add 300.0 cc. of distilled water to (1).
(5) Filter thru cotton, wool and muslin. Reference: Harvey (1921-22 p. 85), Klim-
(6) The reaction at this point is about mer (1923 p. 229), Stitt (1923 p. 35),
pH = 8.2. Park, Williams and Krumwiede (1924
(7) Tube. p. 126).
Sterilization: Autoclave at 15 pounds pres-
1244. Nastiukoff's Egg Yolk Solution.
sure for 20 minutes.
Use: Cultivation of spore forming ana- Constituents:
erobes. The tubes were sealed with a 1. Distilled water 1000.0 cc.
line cap. 2. Egg yolk 100.0 cc.
Variants: The author reported better Preparation
growth in a more acid medium (pH = (1) Separate the yolk of egg from the
7.2) prepared as follows: white by Bunge's method (allow the
(1) Add gradually the yolk of one and the whites to roll from a blotting paper
whites of 2 eggs to 500.0 cc. of tap containing the egg).
water. (2) Add 0.5 cc. of a 10% NaOH solution
(2) Titrate the mixture so that the re- and 100.0 cc. of egg yolk to 1000.0 cc.
action is pH = 7.2. distilled water.
(3) Heat very slowly to 95 and keep at (3) Place in a fiask and steam for 2 hours
this temperature for an hour. in a steamer.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 363
Added nutrients: The authors used any Sterilization: Sterilization of Na^COs solu-
desired carbohydrate, alcohol, etc. tion not given. Sterilize the egg albumin
Reference: Hollande and Fumey (1917 at 125 to 130C.
p. 836). Use: Cultivation of anaerobic bacteria
found in milk. The author added 0.1 cc.
1247. Waksman's Basal Albumin Solution
to 8.0 cc. of Na2C03 solution, and then
Constituents: added the sterile egg albumin. The air
1. Water 1000.0 cc was removed.
2. Glycerol 30.0 g Reference: Barthel (1910 p. 6).
3. K2HPO4 1.0 g
4. KCl 0.5 g 1250. Bainbridge's Albumin Solution
5. MgSOi 0.5 g Constituents:
6. FeS04 0.01 g
1. Distilled water 1000.0 cc.
7. Egg albumin (powdered).. 5.0 g
2. Egg albumin 1.0 to 5.0 g.
Preparation
3. NaCl 5.0 g.
(1) Dissolve 2, 3, 4, 5, 6 and one of the
4. Na2S04 1.0 to 2.5 g.
added nutrients in 1.
5. CaClz trace
(2) Dissolve powdered egg albumin in
6. Potassium phosphate.. trace
N/10 NaOH, and add to (1).
Preparation
(3) Tube in 10-12 cc. lots.
(1) Dissolve 2 (proteins used were egg
Sterilization: Sterilize at 15 pounds for 15
albumin, serum protein and alkali-
minutes.
albumin) 3, 4, 5 and 6 in 1.
,
Use: To study metabolism of actinomy-
(2) Add N/100 H2SO4 until the medium is
cetes.
very faintly alkaline to extremely
Added nutrients: The author added 2.0 g.
sensitive red litmus paper.
of one of the following
(3) After sterilization, distribute in 5.0
NaNOs (NH 4)2804
cc. quantities.
NaN02 (NH4)2C03
Sterilization: Sterilize by passing thru a
Reference: Waksman (1920 p. 3).
Berkefeld filter.
Preparation:
1252. Hogue's Ovomucoid Medium (Hegner
Prepare a 2.0% Na-COs solution.
and Becker)
(1)
(2) Add 8.0 cc. of (1) to Gruber tubes Constituents:
containing sterile egg albumin. 1. Water 600.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 365
(g) Roddy prepared the medium as (4) Add 1.0% of a 5.0% aqueous solu-
follows: tion of litmus to (3).
(1) Mix 300.0 cc. distilled water with (5) Add 1.0% of any desired carbo-
100.0 cc. of beef blood serum. hydrate, alcohol, etc., to (4).
(1) Mix one part blood serum with 3 (3) Prepare 10.0 or 20.0% solutions of
parts distilled water. any desired carbohydrate, alcohol,
etc.
(2) Neutralize (indicator not specified).
(3) Heat in an Arnold steamer until the (4) Heat (3) in small containers in the
mixture becomes opalescent. Arnold sterilizer for 30 minutes on 3
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 367
1254. Leuch's Basal Serum Solution 1256. Klein's Basal Alkaline Serum
(Klimmer) Solution (Leuch)
Constituents: Constituents:
1. Water 800.0 cc. 1. Water 400.0 cc.
2. Serum 180.0 cc. 2. Serum 90.0 cc.
3. NaCl (0.5%) 5.0 g. 3. NaOH (15.0%) 10.0 cc.
4. NaOH (15.0% soln.) 20.0 cc. 4. Litmus
Preparation 5. NaCl (0.5%) 2.5 g.
(8) Add (7) to 1000.0 cc. of (4). while both are warm.
(9) Tube. (10) Tube.
Sterilization: Method of sterilization of Sterilization: Sterilize on each of three
(4) not specified. Sterilize the medium successive days for ten minutes each day.
following distribution into tubes on each Use: Detection of typhoid-paratyphoid
of 3 successive days for 10 minutes. and cholera organisms.
368 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Added nutrients: The author used 1.0% (4) A pH = 7.0 gave the best results.
glucose, 1.0% lactose, 2.0% sucrose, 2.0% (5) Cover with a layer of paraffin oil.
mannitol, etc. (f) Stitt cultivated Balantidium coli
Variants: The author suggested the use of intestinal protozoa and amoeba in a
Czaplewski's alkaline serum (10.0 cc. medium prepared as follows:
normal NaOH per 100.0 cc. serum) instead (1)Mix one part inactivated human
of the alkaline serum given above . Czap- blood serum with 16 parts 0.5%
lewski's mixture may be sterilized in the salt solution.
autoclave. (2) The reaction is faintly alkaline to
Reference: Leuchs (1920 p. 1415), Klein litmus.
(1920, p. 297). (3) Place 8.0 cc. in tubes having a
diameter of 10 mm. and a length of
1256a. Martin, Pettit and Vaudremer's
150 mm. giving the medium a depth
Serum Solution.
of about 100 mm.
Constituents: not specified.
(4) Sterilization
1. Physiological salt solution. 1000.0 cc. Inoculate with 0.1 cc. of undiluted
(5)
2. Serum, bovine 100.0 cc. feces containing mucus, by means
Preparation of the tube capillary pipette into
(1) Dilute bovine serum 1 to 10 with the bottom of the tube.
physiological salt solution. References Martin, Pettit and Vaudremer
:
Sterilization: Not given in the abstract. (1917 p. 197), Griffith (1919-20 p. 60),
Use: Cultivation of Spirochaeta ictero- Harvey (1921-22 p. 79), Hogue (1922 p.
haemorrhagiae. 619), Stitt (1923 p. 52).
Variants
(a) Rabbit serum diluted 1:16 proved 1257. Davis' Serum Medium.
to be a better medium. Constituents:
(b) Griffith cultivated Spirochaeta 1. Serum, rabbit.
icterohaemorrhagiae and other or- Preparation
ganisms on a medium prepared by (1) Tube sterile rabbit's blood serum into
diluting one part beef serum with two sterile test tubes.
parts physiological salt solution and Sterilization: Method not given.
heating at 70C. until the mixture Use: Used in uncoagulated form by Davis
became slightly viscous. He covered to cultivate the Ducrey bacillus (chan-
the medium with a thin layer of croid bacillus). Other investigators cul-
paraffin oil after inoculation. tivated spirochetes, pneumococci,
(c) Harvey mixed 5 parts sterile 0.85% streptococci, etc., in similar media.
NaCl solution with one part rabbit Variants
serum heated at 56C. for 30 minutes. (a) Longcope cultivated pneumococci
(d) Harvey mixed 1 part ox serum heated and streptococci in a medium pre-
to 56C. for 30 minutes with 9 parts pared as follows:
sterile 0.85% NaCl solution, or a (1) Collect 20.0 cc. of blood from the
sterile solution obtained by dissolv- arm vein and allow to clot in a cool
ing 9.2 g. NaCl, 0.05 g. NajCOa, place.
0.1 g. KCl, 0.1 g. CaCh and 10.0 g. (2) Draw off the serum after from 24 to
sodium citrate in a liter of water. 48 hours.
This solution is Locke's solution. (3) Use from 2 to 5.0 cc. serum as
(e) Hogue cultivated Spirochaeta eury- culture medium.
grata on a medium prepared as (b) Schereschewsky heated horse serum
follows: at 60C. until it was brought to a
(1) Dilute serum (best results obtained gelatinous consistency, then incu-
with pig serum) 1:4. bated at 37C. until partial autolysis
(2) Tube 0.85%, sterile salt solution in was effected. The medium was used
15.0 cc. lots in sterile tubes. for the cultivation of the syphillus
(3) AddO.3 cc. of (1) to each tube of (2). spirochaete.
369
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Wang tubed serum (ox) in 2.5 cc. 1260. Sinton's Glucose Serum Solution
(c)
quantities and heated at 56C. for Constituents:
3 or more successive days until the 1. Serum, horse 1000.0 cc.
serum became syrupy. The medium 2. Glucose (50.0% soln.) 15.0 cc.
was used in the diagnosis of diph- Preparation
theria. (1) Add 1.5 cc. of a 50.0%, sterile glucose
(d) Harvey cultivated spirochaetes in solution to each 100.0 cc. of asepti-
sterile rabbit serum heated 30 min- cally collected horse serum.
utes at 58 to 60C. covered with a Sterilization : Not specified.
layer of sterile paraffin oil. Use: Cultivation of Spirochaeta carteri
References: Davis (1903 p. 405), Longcope causing Indian relapsing fever.
(1905 p. 627), Schereschewsky (1909 p. Reference: Sinton (1923-24 p. 826).
35), Wang (1919 p. 233), Harvey (1921-
1261. Toyoda's Glucose Serum Solution
22 p. 80).
Constituents:
1258. Marmier's Serum Solution. 1000.0 cc.
1. Serum, dog
Constituents 2. Glucose 10-0 g.
(2) Make slightly alkaline by the addition glucose solution and 0.3 cc. of a
of soda. 2.0% sterile sodium citrate in 0.85%
Sterilization: Sterilize at 115 in the co- NaCl solution.
agulator (time not specified). (2) Pipette the serum in small narrow
Use: Anthrax toxin production by anthrax test tubes 5 cm. high and inactivate
bacilli. the serum at 45C. for one hour.
Reference: Marmier (1895 p. 569). Sterilization: Not specified.
Use: Cultivation of Babesia cannis
1259. Boeck's Glucose Serxim Solution. Reference: Toyoda (1913 p. 76).
Constituents: Serum Solution
1262. Marbais' Lactose
1. Distilled water 1000.0 cc.
cilli. Author reported that typhoid (5) Sterilize (4) at 15 pounds pressure
cause coagulation after 24 hours, color for 15 minutes.
was blue-violet. Paratyphoid A clouded (6) Mix 1 part (5) with 10 parts (3).
the medium, and the color was lilac. (7) Sterilize on each of 3 successive
Paratyphoid B gave a red color after 5 days for 20 minutes at 100C.
or 6 hours incubation, then changed to Reference: Hiss (1901-05 pp. 324, 330),
original color. B. coli coagulated and Harvey (1921-22 p. 112).
decolorized the medium.
1264. Legroux's Formol Serum Solution
Reference: Marbais (1918 p. 602).
Constituents:
1263. Hiss' Inulin Serum Solution 1. Distilled water 1200.0 cc.
2. Serum (beef or horse) 600.0 cc.
Constituents:
3. Formol 1.0 cc.
1. Distilled water 200.0 cc.
Preparation:
2. Beef serum 100.0 cc.
(1) Mix 600.0 cc. of beef or horse serum
3. Inulin (pure) (1.0%) 3.0 g.
with 1.0 cc. of commercial formol.
4. Litmus (5.0% soln.) 3.0 cc.
(2) Dilute (1) with 1200.0 cc. distilled
Preparation
water.
(1) Mi.x two parts distilled water with one
(3) Mix well.
part fresh clear beef serum.
(4) The reaction is acid to methyl red.
(2) Add 1.0% pure inulin, and 3.0 cc. of
(5) Distribute in tubes or flasks.
5.0% solution Merck's highly purified Sterilization: Sterilize in the autoclave at
litmus.
112 to 115 for varying lengths of time
Sterilization: Sterilize at 100C. for 10
depending on the size of the container.
minutes (if inulin contains heat resisting
Use: Enrichment of meningococci. The
spores, autoclave at 10 to 15 pounds
author reported that the formol kept the
pressure for 15 minutes.)
medium from coagulating during steriliz-
Use: Differentiation of pneumococci and
ation. It also aided in giving a clear
streptococci. Author reported that
medium.
pneumococci. cultures coagulated the
Reference: Legroux (1920 p. 466).
serum, streptococci did not.
Variants : 1265. Marzinowsky's Citrated Blood
(a) Hiss omitted the litmus. Solution
(b) Hiss prepared a similar medium as Constituents:
follows: 1. Water 100.0 cc.
(1) Add 4.0 g. powdered starch to 400.0 2. Sodium citrate (10.0%) 10.0 g.
cc. water, and boil for 30 minutes. 3. Blood, horse
Allow to stand over night. Preparation:
(2) Obtain, next morning, the clear (1) Prepare a 10.0% watery solution of
fluid by pipetting off the water sodium citrate.
from the starch particles. (2) Distribute in 1.5 to 2.0 cc. lots in test
(3) Add one part serum to two parts of tubes.
the supernatant fluid from (2). (3) Add10.0 cc. of horse blood to each
(4) Sterilize at 68C. for 1 hour on 6 tube of sterile (2). The blood is
consecutive days. taken directly from a vein.
(c) Hiss heated the beef serum and water Sterilization: Sterilize (2); method not
at 100C. for several minutes and given.
then added the inulin and litmus. Use: Cultivation of Piroplasma equi and
(d) Harvey prepared the medium as other piroplasma.
follows: Reference: Marzinowsky (1908-09 p. 419).
(1) Mix 1 part ox serum with 3 parts
water. 1266. Griffith's Citrated Blood Solution
(2) Steam 15 minutes. Constituents
(3) Add sufficient litmus solution to 1. Blood, citrated (horse, beef,
give a blue color. rabbit) 100.0 cc.
(4) Prepare a 10.0% solution of inulin. 2. Physiological salt solution... 200.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 371
Constituents: Preparation:
1. Water 1000.0 cc.
(1) Prick the extensor surface of the
2. NaCl 8.0 g. thumb just back of the base of the
3. Sodium citrate 15.0 g. nail, the skin having been previously
4. Blood cleansed with soap and water and
Preparation alcohol.
(1) Dissolve 8.0 g. NaCl and 15.0 g. of
(2) Applying a tourniquet at the base of
sodium citrate in 1000.0 cc. of water. the thumb just tight enough to
(2) Mix 20.0 cc. of sterile (1) with 80.0 cc. obstruct the venous flow, a consider-
of blood obtained under aseptic able quantity of blood may be
conditions. obtained.
Sterilization: Sterilize (1); method not
The blood is drawn up by capillary
(3)
given attraction into a small glass tube,
Reference: Besson (1920 p. 34). one end of which has been drawn out
Blood (Stitt) to a very fine caliber over the Bunsen
1268. Rogers' Citrated
flame, and the whole tube sterilized
Constituents: by the same means.
1. Blood. When about 0.2 cc. of blood has
(4)
2. Sodium citrate (10.0%).
flowed into the tube the capillary end
3. Citric acid.
may be sealed in the flame, and the
Preparation other end plugged with cotton, giving
(1) Place 1.0 or 2.0 cc. of a sterile 10.0%
a miniature culture tube.
sodium citrate slightly acidified with
(5) The medium is nearly always sterile
citric acid into the barrel of a syringe
when obtained in the manner in-
and aspirate splenic blood directly dicated.
into it.
Sterilization: Given in the preparation.
Sterilization: Not specified.
Use: Cultivation of Ducrey bacillus (chan-
Use: Cultivation of Leishmania.
croid bacillus), pneumococci and strep-
Reference: Stitt (1923 p. 52).
tococci.
1269. Buchner's Blood Solution Variants: Stitt reported that pneumococci
and streptococci maintained their viru-
Constituents:
1000.0 cc. lence in a medium prepared as follows:
1. Water
100.0 cc. (1) Obtain rabbit or human blood under
2. Blood
aseptic conditions and preserve
3. Sucrose (10.0%) 100.0 g.
(whole blood) in small test tubes.
Preparation
cane Heat for 30 minutes at 56C. to
(1) Prepare a 10.0% solution of
(2)
grew first on the surface of the medium, one to two inches thick. This gives a
then on the bottom; after several days the column of serum Ho 1 inch thick.
whole tube became turbid and was finally Sterilization: Not specified.
References: Bass, Foster and Johns (1912 (3) Remove blood under aseptic condi-
p. 570), Harvey (1921-22 p. 72), Stitt tions from the jugular vein of a
(1923 p. 53), Park, Williams and Krum- horse.
wiede (1924 p. 133). (4) Defibrinate (3) and store in the ice
box if not to be used at once.
1274. Besson's Defibrinated Blood (5) Add 9.0 cc. of (4) to each tube of
sterile (2) under strictly aseptic
Constituents:
conditions.
1. Blood.
(6) Mix well and place in an incubator at
Preparation:
25C. for 24 hours. At the end of
(1) Collect blood under aseptic condi-
this time the mixture is clear.
tions in a sterile flask containing glass carefully.
Sterilization: Sterilize (2)
beads.
(Method not given.)
(2) Shake thoroly for 10 minutes. and
Use: Cultivation of Babesia caballi
(3) Remove the liquid from the fibrin by
Nuttallia equi. To inoculate carefully
asparating.
place the end of a sterile fine pointed
(4) Distribute the fluid in sterile test
pipette containing infected blood into
tubes.
the red blood cells in the bottom of the
Sterilization: Not specified.
tube, and allow the infected blood to run
Variants: Harvey did not separate the fluid
into the tube.
from the fibrin and incubated for 48 hours
Reference: Carpano (1914 p. 43).
to test sterility.
References: Besson (1920 p. 34), Harvey 1277. Waksman's Basal Fibrin Solution
(1921-22 p. 73).
Constituents:
1. Water 1000.0 cc.
1275. Row's Defibrinated Blood Solution 30.0
2. Glycerol g.
(Harvey)
3. K2HPO4 10 g.
Constituents: 4. KCl 0.5 g.
1. Distilled water 1000.0 cc. 5. MgS04 0.5 g.
2. Blood, defibrinated 100.0 cc. 6. FeS04 0.01 g.
3. NaCl (1.2% solution) 2200.0 cc. 7. Fibrin g. 5.0
Preparation Preparation
(1) Defibrinated human or rabbit blood. (1) Dissolve 2, 3, 4, 5, 6 and one of the
(2) Add 10 times its volume distilled added nutrients in 1.
water. (2) Tube in 10-12 cc. lots.
(3) Add 1 volume of the laked blood thus (3) Add small pieces of fibrin to each
obtained to 2 volumes 1.2% sterile tube.
sodium chloride solution. Sterilization: Sterilize at 15 pounds for 15
Sterilization: Not specified. minutes.
Use: Cultivation of Leishmania. Use: To study metabolism of actino-
References: Harvey (1921-22 p. 72), Stitt mycetes.
(1923 p. 52). Added nutrients: The author added 2.0 g.
of one of the following:
1276. Carpano's Defibrinated Blood NaNOa (NH5)2S04
Solution (NHOiCOa
NaN02
Constituents Reference: Waksman (1920 p. 3).
1. Distilled water 100.0 cc.
1278. Stoklasa's Glucose Fibrin Solution
2. NaCl (c.p.) 7.0 g.
3. Sodium citrate (c.p.) 7.0 g. Constituents:
4.Defibrinated horse blood 1. Water 1000.0 cc.
finished medium in the steamer, using the causes the bile acid to precipitate out
fractional method. in silky crystals so-called crystalline
Use: To study decomposition of organic N bile.
materials by soil forms. Author reported (5) Dry and pulverize the crystals.
little change in total nitrogen content (6) Add one or two knife points of (5)
2. Physiological salt solution . . 1000.0 cc. the patients blood to each tube of (6).
Preparation: Variants: The following authors have pre-
(1) Collect pigeon blood under aseptic pared media in the manner indicated.
conditions and add to an equal (a) Kayser
volume of physiological salt (0.85%) (1) Secure normal beef bile immedi-
solution. ately after the death of an animal in
(2) Mix well and centrifuge, or allow to large sterile flasks.
stand in the cold for 24 hours. (2) Distribute in amounts of at least
(3) Thoroly wash the red residue twice 5.0 cc. in test tubes.
more with sterile NaCl solution. (3) Sterilize at 110.
(4) Dissolve the hemoglobin with a trace (4) Put 2.5 cc. of blood of patient into
of ether. this at bedside.
(5) Evaporate the ether at 30C. (b) Bezangon.
(6) Separate the hemoglobin from the (1) Collect bile at a slaughter house.
stromata by passing thru a bacterial (2) Sterilize at 100C.
filter. (3) Allow to settle.
Sterilization: Method of sterilization of (4) Decant the clear liquid.
salt solution not specified. (5) Tube in 3.0 cc. quantities in sterile
Use: This hemoglobin solution is added to tubes.
other media which is used for the cultiva- (6) Sterilize at 105C. for 20 minutes.
tion of pneumococci, vibrio, etc. One (c) Klimmer.
cubic centimeter of this solution is added (1) Puncture the gall bladder of a beef
to 8.0 cc. of media, or this solution is with a knife, and collect the bile.
streaked on solid media. (2) Boil (1) in a steamer for 15 minutes.
Variants: The author also used commercial (3) Distribute in 5.0 cc. quantities in
hemoglobin. This solution was prepared test tubes.
as follows: (4) Steam on each of 3 successive days
(1) Mix 10.0 g. of commercial hemoglobin for 20 to 30 minutes.
with 90.0 cc. of distilled water. References: Meyerstein (1906 p. 1864),
(2) Add about 10.0 cc. of 10.0% KOH. Kayser (1906 pp. 823-826), Harvey
(3) Sterilize in the steamer. (1921-22 p. 89), Bezangon (1920 p. 122),
Reference: Klimmer (1923 p. 196). Klimmer (1923 p. 202), Stitt (1923 p. 47).
When ready for use, decant or filter. Use: Cultivation of anthrax bacilli.
(2)
Add 1.0% lactose that has previously Reference: Thoinot and Alasselin (1902
(3)
been dissolved in small (amount not p. 28).
Constituents: Constituents:
1. Distilled water 1000.0 cc. 1. Milk.
cipitate appears when heated but disap- (3) Seal the flask with a rubber stopper
pears when cool). and shake thoroly.
Use: General culture medium. (4) Allow to stand for several days.
Variants: The author used 4.0 g. sodium (5) Distribute in sterile tubes.
oxalate instead of 4.0 g. sodium citrate. (6) Incubate for 48 hours.
Citrated milk was found to be more (b) Smith prepared the medium as
satisfactory than oxalated milk. follows:
Reference: Brown and Howe (1922 p. 512). (1) Obtain fresh milk and free from fat
by running the milk thru a separa-
1292. Boekhout's Sucrose Milk Solution tor or by allowing the cream to rise
Constituents: spontaneously.
1. Milk 1000.0 cc. (2) Test the reaction. It should not
2. Sucrose (8.0%) 80.0 g. be more than +2.
Preparation: (3) Tube.
(1) Dissolve 8.0% sucrose in milk. (4) Sterilize at 100C. by the fractional
Sterilization: Not specified. method.
Use: Cultivation of dextran formers. A large number of investigators have
{Streptococcus hornensis). Organism prepared milk in a manner very
may be isolated from flowers, honey and similar to that of Smith. Some
bees. sterilized the milk in the autoclave.
Reference: Boekhout (1900 p. 162). The differences in preparation are not
378 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Dialyze (method not given). or azolitmin with 52.7 parts milk powder
(6) Dilute the milk one fifth with water. or 1 part purified azolitmin with 49.6
References: Reinsch (1892 p. 31), Migula parts milk flour. One part of this mix-
(1901 p. 19), Smith (1902 p. 104), Thoinot ture was dissolved in 9.5 parts water, and
and Masselin (1902 p. 27), Committee the medium was sterilized in the
A.P.H.A. (1905 p. 109), Smith (1905 p. 46), autoclave.
(1905-06 p. 202), Abel (1912 p. 29), Lohnis References: Tanner (1918 p. 83), Hamilton
(1913 p. 20), Tanner (1914 p. 72), Ball (1921 pp. 43, 44).
(1919 p. 83), Besson (1920 p. 32), Percival
1296. Bacto Litmus Milk (Dehydrated)
(1920 p. 57), Abbott (1921 p. 138), Dopter
and Sacquepee (1921 p. 122), Harvey Constituents:
(1921-22 p. 94), Cunningham (1924 p. 16), 1. Distilled water 1000.0 cc.
Park, Williams and Krumwiede (1924 2. Litmus milk, Bacto 105.0 g.
p. 120). Preparation:
(1) Dissolve 105.0 g. of Bacto litmus milk
1294. Hiss' Basal Litmus Milk Solution
(dehydrated) in 1000.0 cc. distilled
Constituents: water.
1. Milk 1000.0 cc. Sterilization Sterilize in the usual manner.
:
2. Litmus.
medium was decolorized if reduction
occurred.
Preparation:
Variants: The author used the following
(1) Obtain fresh milk and free from fat
variants:
by running the milk thru a separator
(a) Dissolved 1.0 g. of Kahlbaum's indigo
or by allowing the cream to rise
carmine in 1000.0 water
cc. distilled
spontaneously.
should not be and used instead of methylene blue
Test the reaction. It
(2)
solution.
more than +2.
(b) Dissolved 1.0 g. of GriJbler's neutral
(3) Tube.
red in 1000.0 cc. of distilled water and
(4) Add sufficient sterile litmus solution
used instead of the methylene blue
to give a distinct pale blue tinge to
solution.
the milk, to sterile (3).
the Reference: Sherman and Albus (1918 p.
Sterilization: Sterilize at 100C. using
167).
fractional method.
Use: General culture medium. 1301. Harvey's Brom Cresol Purple Milk
Variants: Various investigators have pre- Solution
pared media in a number of similar ways.
Constituents:
Some investigators used azolitmin instead 1000.0 cc.
of litmus, and some sterilized the
medium I ^^li\\^
Raise to boiling point and boil 5 (4) Neutralize by the addition of soda
(4)
(indicator not specified).
minutes.
Adjust the reaction by bringing the Sterilization: Not specified.
(5)
color, with the addition of alkali,
Use: Cultivation of the causitive agent of
the foot and mouth disease. Other in-
to a pale grey.
vestigators employed the medium as a
(6) Distribute into test tubes containing
general culture medium.
gas tubes which should project above
the surface. Variants
Sterilization: Sterilize in the autoclave.
(a) The author added Na2C03 to obtain
1303. Bacto
following manner:
Purple Milk, (Dehydrated)
(1) Add 10.0 cc. of distilled water
Constituents: containing 8 to 10 drops of com-
1. Distilled water 1000.0 cc. mercial rennet extract to 1000.0 cc.
2. Purple milk Bacto 105.0 g. of skimmed or separated milk.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 381
(2) Stir briskly for a few seconds. (14) Distribute in test tubes.
(3) Allow to coagulate. (15) Incubate two days in the incubator
(4) When the curd has formed cut with and 8 days at room temperature to
a knife, and leave it for an hour so test sterility.
(2) Separate the casein from the whey adulteration is slightly warmed and
by straining thru a straining cloth. clotted by means of rennet.
(3) Filter the whey thru filter paper (2) Drain the whey and hang the clot
off
(4) Distribute in sterile flasks. (3) Neutralize the turbid or yellow whey
(5) Sterilize by the addition of 1.5% with 4.0% citric acid solution, using
chloroform. litmus as an indicator.
References: Stutzer and Hartleb (1897 p. (4) Heat upon the water bath for 30
403), Percival (1920 p. 58), Klimmer (1923 minutes to coagulate the proteins.
p. 203). (5) Filter and add litmus to obtain a
suitable color.
1306. Emile-Weil's Litmus "Whey Solution Distribute in 10.0 cc. quantities if
(6)
Constituents desired.
1. Whey 1000.0 cc. Sterilization: Sterilize 100C. If not
at
(4) Allow to coagulate for 30 minutes. (1) Precipitate casein from milk with
(5) Cut the curd into large pieces. rennet extract.
(6) Filter thru a thin cloth. (2) Neutralize the whey with 4.0%
(7) Make the filtrate alkaline to phenol-
citric acid solution.
phthalein by the addition of soda. (3) Heat on the water bath for 30
(8) Add 2.0 g. CaCh per liter.
minutes.
(9) Heat at 110 for 15 minutes. (4) Filter.
(10) Filter thru paper until the filtrate is (5) Add litmus solution until a decided
clear. blue color is obtained.
(11) The reaction should be a deep violet (6) Sterilization not specified.
to litmus. (b) Harvey prepared a similar medium
(12) Add 2.0% litmus solution to obtain as follows:
the desired shade. (1) Add rennet to fresh milk.
(13) Filter thru a candle. (2) Keepat60C.
382 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(3) Strain the separated whey thru a 1309. Bronfenbrenner, Davis and Mori-
thick, clean cloth. shima's China Blue Rosolic
(4) Make the reaction of the fluid Acid Whey Solution
neutral to litmus by means of an
Constituents:
organic acid, such as citric acid.
1. Water 200.0 cc.
(5) Steam 60 minutes.
while hot thru well-wetted,
2. Whey 100.0 cc.
(6) Filter
thick filter paper.
3. MnCU (10.0% soln.) 2.5 cc.
4. China blue, rosolic acid
(7) Add
litmus solution to give a deep
indicator 3.0 cc.
purple red color.
Preparation
(8) Distribute into flasks or test tubes,
(1) Allow fresh milk to stand and syphon
(c) Klimmer gave the following method
the milk from under the cream.
of preparation.
(2) Bring it to boiling and add 2.5 cubic
(1) Coagulate the casein in milk by
the addition of rennet by heating
centimeters of 10% MnCU solution to
each 100 cubic centimeters of milk.
at 40C.
(3) Cool the mixture as soon as a clot is
(2) Filter.
Boil the filtrate for 2 hours.
formed and filter thru a single layer
(3)
of cloth.
(4) Neutralize.
(4) Titrate an aliquot portion hot and
(5) Filter.
The whey should be water clear adjust the bulk of medium to neutral
(6)
and slightly yellow. reaction (1 X 10"').
(7) Add 5.0 cc. of sterile litmus solu- (5) Bring quickly to boiling, cool and
tion to each 100.0 cc. of whey. filter thru paper.
(8) Add acid or alkali until the me- (6) Dilute the filtrate with double its
dium is a violet color. volume of water and add 1 cubic
(9) Filter.
centimeter of china blue rosolic acid
(10) Sterilize (method not given). indicator, see medium 535, under
References: Durham (1900-01 p. 379), variants, for each 100 cubic centi-
Heinemann (1905 p. 128), Abbott (1921 meters of medium. (At this point,
p. 140), Harvey (1921-22 p. 95), Klimmer the medium will have an intensely
(1923 p. 207). blue color).
(7) Distribute into sterile tubes con-
1308. Giltner's Sour Whey taining inverted fermentation tubes.
Constituents: Sterilization: Autoclave at 15 pounds pres-
1. Whey. sure for 10 minutes. Immediately after
Preparation autoclaving the medium will have a pink
(1) Inoculate sweet milk with a pure color. On cooling it will become colorless
active culture of Bad. lactis acidi or properly neutralized.
if it is
Preparation Variants
To 1 liter of milk add 10.0 cc. of 15 (a) Migula prepared a similar medium as
(1)
follows:
Baume (equivalent to 25.0 g. crystal-
water) Mix equal volumes of fresh milk
lineCaCh per 100.0 cc. (1)
neutral to litmus by the addition (4) Tube in about 10.0 cc. lots and
of dilute sodium carbonate sterilize in flowing steam for J hour.
Bolution. (5) Add 1 of (4) to (2).
(5) Filter, while hot thru well-wetted, (c) Hiss dissolved 10.0 g. nutrose, 4.0
thick filter paper. cc. of normal NaOH 10.0 cc. of 5.0%
(6) Add litmus solution to give a deep litmus solution and one of the follow-
purple red color. ing in 1000.0 cc. distilled water.
(7) Distribute into test tubes or flasks. Glucose Dextrin
(8) Sterilize. Maltose Mannitol
References: Grimbert and Legros (1901, p. Sucrose
913), Migula (1901 p. 21), Abel (1912 p. (d) Elser and Huntoon prepared the
1. Distilled water 1000.0 cc. (4) Add 30.0 cc. of Kahlbaum's Litmus
2. Nutrose., 10.0 g. Solution.
3. Lactose 20.0 g. (5) Distribute into flasks or fermentation
4. Malachite green (2.0% soln). 50.0 cc. tubes.
Preparation Sterilization: Sterilize in streaming steam
(1) Dissolve 2 and 3 in 1.
for 15minutes on two successive days.
(2) Add 5.0% of a 2.0% malachite green Use: Differentiation of colon typhoid and
solution to (1). dysentery group. The author reported
Sterilization: Not specified. that Flexner's strains gave red coloration
Use: Enrichment of typhoid bacilli from after 24 hours. Shiga group caused no
faeces. change. Typhoid and coli forms gave
Variants: Klimmer prepared the medium the same reaction as in dextrose-nutrose
as follows: medium, coli causing a precipitation of
(1) Dissolve 10.0 g. nutrose in 1 liter hot the nutrose in 24 hours while the typhoid
distilled water. organisms required a longer period to
(2) Add 20.0 g. lactose to (1). produce precipitation.
(3) Flask in 100.0 cc. quantities. Variants Lehman prepared the medium as
:
(4) Add 10.0 g. lactose and 0.1 g. CaCh (2) Suspend 30.0 g. of casein in (1).
to (3). Sterilization: Method not given.
(5) Make the solution up to 1000.0 cc. Use : Cultivation of oidium.
by the addition of distilled water. Reference: Laxa (1901-02 p. 129).
(6) Neutralize and make 0.3 with + 1323. Baginsky's Casein Solution
normal HCl, using phenolphthalein
as an indicator. Constituents:
Sterilization: Sterilize in the autoclave at 1. Water 1000.0 cc.
107 for 20 minutes. 2. Casein 1000.0 g.
Use: Substitute for milk. General culture Preparation
medium. (1) Add 20.0 g. of casein to 20.0 cc. of
Reference: Whittaker (1912 p. 162). water (or nutrient solution, composi-
tion not given).
1320. Waksman's Basal Casein Solution
Distribute into sterile flasks.
(2)
Constituents: Sterilization: Sterilize in the steamer.
1. Water 1000.0 cc. Use: To study fermentation by B. lactis.
2. Glycerol 30.0 g. Reference: Baginsky (1888 pp. 43'^462).
3. K2HPO4 1.0 g.
1324. Harvey's Alkaline Casein Solution
4. KCl 0.5 g.
5. MgS04 0.5 g. Constituents:
6. FeS04 0.01 g. 1. Water 1000.0 cc.
7. Casein 5.0 g. 2. Casein 20.0 g.
Preparation: 3. NaOH (normal) 10.0 cc.
(1) Dissolve 2, 3, 4, 5, 6 and one of the Preparation
added nutrients in 1. (1) Mix 1, 2 and 3.
(2) Dissolve casein in N/10 NaOH and Sterilization: Not specified.
add to (1). Use: Cultivation of anaerobic organisms.
(3) Tube in 10 to 12 cc. lots. Author specified that nutrose or any other
Sterilization: Sterilize at 15 pounds for 15 casein product could be used instead of
minutes. casein.
Use: To study metabolism of actinomy- Reference: Harvey (1921-22 p. 96).
cetes.
Added nutrients: The author added 2.0 g.
1325. Geilinger's Basal Urine Solution
of one of the following: Constituents:
NaXOa (NH4)2S04 1. Urine, cow.
NaXOj (NH 4)200, Preparation:
Reference: Waksman (1920 p. 3). (1) Mix one of the added nutrients with
sterile cow's urine.
1321. Seliber's Basal Casein Solution
Sterilization: Sterilize cow's urine by heat-
(Harvey)
ing for one hour at 0.5 atmosphere pres-
Same as medium 743 but not containing sure or preferably by filtering thru a
peptone. Chamberland clay candle filter.
Use: Cultivation of organisms capable of
1322. Laxa's Lactic Acid Casein Solution
splitting urea, Bac. urea.
Constituents: Added nutrients and variants
1. Water 1000.0 cc. (a) The author added 1.0 cc. of one of the
2. NaCl 5.0 g. following to each 20.0 cc. of sterile
3. CaClj 0.1 g. urine.
4. MgCIa 0.2 g. The added nutrients were prepared as
5. Potassium phosphate follows:
(neutral) 2.5 g. (a) Straw infusion.
6. Lactic acid 3.0 g. (1) Mix 9 parts by weight of
7. Casein 30.0 g. water with 1 part by weight
388 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
of finely chopped straw and (g) Neutralize urine with K2HPO4 and
heat in steamer for one dilute 5 times with water.
hour. Reference: Bokorny (1920 p. 27).
(2) Filter.
(3) Sterilize for 45 minutes at 1327, Besson's Urine Medium (Tanner)
0.5 atmosphere pressure. Constituents
(b) Feces infusion. 1. Urine.
(b) The author used cow's urine without given by the following authors:
any additions. (a) Besson
Reference: Geilinger (1917 p. 246). (1) Collect urine under aseptic condi-
tions in sterile flasks.
1326, Bokorny's Sucrose Urine Solution (2) Distribute in tubes.
(3) Incubate for 24 hours at 37C. to
Constituents
test sterility.
1. Urine 1000.0 cc.
(b) Besson filtered fresh urine thru a
2. Sucrose SO.O g.
Chamberland filter.
Preparation
(c) Besson
(1) Add 80.0 g. sucrose to 1000.0 cc. of
(1) Boil fresh urine.
undiluted and not neutralized urine.
(2) Neutralize to litmus by the addition
Sterilization: Not specified.
of tartaric acid if necessary.
Use: To study the growth of yeast. The
(3) Filter.
author reported that neutralized urine Tube.
(4)
showed better growth than non-neutral- 115C.
(5) Sterilize at
ized. The effect of diluting the urine was (d) Harvey
that of diluting the food materials.
(1) Collect urine fresh.
Growth best if diluted urine be neutral-
(2) Boil.
ized.
(3) Filter.
Variants : The author added 80.0 g. of sucrose (4) Sterilize in the steamer or auto-
to 1000.0 cc. of urine prepared in the clave.
following manner: (e) Harvey
(a) Undiluted urine neutralized with Distribute into test tubes freshly
(1)
K5HPO4. passed urine diluted to specific
(b) Undiluted urine partially neutral- gravity 1.010.
ized. (2) Sterilize in the steamer or auto-
(c) Unneutralized urine mixed with an clave.
equal volume of water. References: Tanner (1919 p. 58), Besson
(d) Neutralize urine with K2HPO4 and (1920 p. 33), Harvey (1921-22 p. 84).
then add an equal amount of water.
1328. Burri and Stutzer's Nitrate Feces
(e) Neutralize urine with K2HPO4 and
Solution
dilute with 3 volumes of water.
(f) Dilute unneutralized urine with 5 Constituents:
times water. 1. Water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 389
[KNO3 0.2 g.
(4) Make up to 500.0 cc. by the addi-
for the culti- tion of tap water if necessary.
These media were used
(5) Place (4) in a sterile liter Erlen-
vation of Bact. diphtheriae and toxin
meyer flask.
production by it.
(6) Heat in the autoclave at 120C.
(e) Waksman studied the metabolism of
for 30 minutes.
actinomyces in media prepared as
(h) Harvey
follows:
water, filter and (1) Mince finely fat-free beef.
(1) Boil beef in
Note: Veal, chicken, ox or
sterilize.
horse heart, horse flesh, rabbit
(2) Inoculate with B. coli communor,
flesh, fish, blood, placenta, liver,
incubate at 37 for 24 hours.
spleen, kidneys, brain and vege-
(3) Boil for 10 minutes, adjust to pH
table materials, such as yeast,
= 7.6 to 7.7. Boil again, filter and
wheat, etc., may serve to furnish
sterilize.
any desired carbo- the extract used as basis for the
(4) Add 1.0% of
medium.
hydrate, alcohol, etc., and 1.0%
(2) Add 500.0 g. to 1000.0 cc. distilled
of Andrade indicator.
water or clear tap water.
(5) Distribute in fermentation
tubes.
minutes to (3) Heat the mixture 20 minutes over
(6) Steam in Arnold for 30
a free flame, at a temperature not
sterilize.
exceeding 50C.
The author reported that the actin-
Note: Or simply keep in a cool
omycetes developed in the infusion
place over night.
alone without the addition of other
(4) Skim off fat floating on the surface.
nutrients.
(5) Raise the temperature to boiling
(f) Besson
point.
(1) Pour 1000.0 cc. of water on 500.0 g.
(6) Boil 10 minutes.
of finely chopped beef, and place
(7) Pour the mixture on to a wet,
in the ice box for 12 hours.
thick, clean cloth.
(2) Stir the mixture well.
(8) Add sodium chloride 5.0 g. to the
(3) Filter thru a cloth and press
the
filtrate.
meat free from juice. Steam 45 minutes.
(9)
(4) Filter the juice thru paper. Bring the volume up to 1000.0 cc.
(10)
(5) Add 5.0 g. NaCl. by the addition of water.
(6) Boil. (11) Estimate and adjust the reaction.
(7) Make slightly alkaline or neutral- (12) Steam 30 minutes.
ize to litmus by the addition of
(13) Filter, while hot, thru well-wetted,
soda. thick filter paper.
(8) Heat at 115 to 117C. for 5 minutes. References: Dunham
(1887 p. 338), Migula
(9) Filter until clear. (1901 p. Abel (1912 p. 13), Linde
13),
(10) Make up to 1000.0 cc. by the addi- (1913 p. 386), Davis and Ferry (1919 p.
tion of distilled water. 235), Waksman (1919 p. 316), Besson
(11) Distribute as desired. (1920 p. 28), Dopter and Sacquepee
110 to 115C. for 20 (1921 p. 120), Giltner (1921 p. 27), Harvey
(12) Sterilize at
minutes. (1921-22 p. 94).
(g) Giltner
1331. Stutzer and Hartleb's Nitrite Infusion
(1) Add 500.0 g. of tap water to 500.0
Solution
g. finely chopped fresh lean beef in
ware pail.
3.5 liter agate Constituents
1. Water... 2000.0 cc
(2) Mix thoroly and allow to stand in
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 391
(6) Decant carefully and strain thru a (5) Make up to the original volume,
straining cloth. adjust the reaction as desired in
(7) Filter thru paper. the same manner as for meat
Sterilization: Not specified. extract.
Use : Use as meat water in the preparation (6) The bouillon may be filtered until
of media. clear.
Reference: Klimmer (1923 p. 172). Usually 0.5% glycerol is added.
(7)
not specified.
(8) Sterilization
1336. Szasz's Blood Clot Infusion
(b) Stefanopoulo cultivated Spirochaeta
Constituents: icterohemorragiae on a medium pre-
1. Distilled water 2000.0 cc. pared as follows:
2. Blood clot , 1000.0 g. (1) Separate the serum from the clot
Preparation of coagulated horse blood.
(1) Take 1000.0 g. of blood from which the (2) Press the clot thru a fine wire gauze.
serum has been separated, coagulated (3) Dilute with two times its volume
blood or blood clots and add 2000.0 with physiological salt solution
cc. of distilled water. prepared by dissolving 8.0 g. of
(2) Boil for a short time. (Time not NaCl in 1000.0 cc. of water.
specified.) (4) Heat for 15 minutes at 80C.
(3) Separate the clot into pieces the size (5) Filter thru paper, and then on a
of a nut and boil longer. Do not cut Chamberland filter.
the clot before heating. Take care (6) Distribute in sterile tubes and cover
that the blood clot does not sink to with a layer of sterile vaseline.
the bottom of the kettle and burn. References: Szasz (1914-15 p. 491), (1915-
Place the kettle or container in which 16 p. Ill), Stefanopoulo (1921 p. 813).
the boiling is taking place over a free
flame. It may be stirred with a 1337. Wellman's Placenta Infusion
wooden spoon. A linen towel may Constituents
be placed in the bottom of the con- 1. Distilled water 1000.0 cc.
tainer and extend part way up the 2. Placenta, human 1000.0 g.
side walls or all the way up and be Preparation
fastened with a cord. This is the (1) Grind fresh human placenta thoroly
simplest way to prevent burning. in a meat chopping machine after
(4) Filter thru a large linen towel. first washing out the blood by running
(5) The bouillon may be clarified with sterile salt solution thru the attached
egg white. vessel.
Sterilization: Not specified. (2) To each kilogram of macerated
Use: Inexpensive medium. Meat infusion placental tissue add 1 liter of distilled
or extract substitute. water.
Variants (3) Infuse for 48 hours in the ice box.
(a) The author prepared a similar me- (4) Tube sterile (3).
dium as follows: (5) Store the medium at 40-41 C. for 2
(1) Divide the blood clots into pieces days before use to inactivate the
the size of a walnut or hazel nut by complement.
means of a piece of wood or hands. Sterilization: Pass thru a No. N Berkefeld
(2) To the clot obtained from every filter that will hold back ordinary bac-
kilogram of blood, add 1.5 liters teria. To facilitate this fill the cylinder
of water. of the filter with a clean fine sterile sand
(3) Mix well and allow to stand in the until the cylinder is completely covered.
cold for 20 to 24 hours, stirring Use: Cultivation of parasitic bacteria.
several times. Author used Bacillus leprae and tubercle
(4) Filter thru a coarse linen cloth and bacilli.
boil the filtrate until brown clumps Variants
are formed and the liquid is yellow. (a) Groer and Srnka studied the produc-
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 393
tion of toxin by diphtheria bacilli, (2) Mix equal parts of (1) and distilled
and cultivated them on a medium water.
prepared as follows: (3) Shake vigorously.
(1) Mix 4 liters of medium fine placenta (4) Filter thru Buchner filters and then
with 4 liters of water. thru Berkefeld filters (finest grade).
(2) Boil for about 3 hours with the (5) Distribute to fermentation tubes.
addition of water. (6) Incubate for 24 hours to determine
(3) Filter. sterility.
(4) Concentrate the filtrate to 3 liters. Sterilization: Sterilization given under
(5) Add N/1 10.0% NaOH until phenol- preparation.
phthalein is turned weakly red. Use: Cultivation of a spore bearing an-
(Hot titration). aerobe from a dog's liver. The medium
Distribute into flasks and sterilize.
(6) obtained at first is rather viscous, red and
(Method not given). slightly acid in reaction. After 48 hours
(b) Park, Williams and Krumwiede pre- incubation proteins are precipitated.
pared a similar medium as follows: Reference: Wolbach and Saiki (1909 p.
(1) Add 500.0 g. of ground up tissue 270).
(placenta) to a liter of water.
Soak for 90 minutes.
1340. Brieger's Thymus Gland Infusion
(2)
(Besson)
(3) Strain thru cheese cloth and
squeeze by twisting the cloth or Constituents:
use a meat press. 1. Distiled water 1000.0 cc.
(4) Filter thru paper or sand. 2. Thymus gland (beef) 1000.0 g.
(5) Sterilize by filtration thru a filter Preparation
candle. (1) Remove the thymus glands from dead
References: Wellman (1912 p. 143), Groer beef.
and Srnka (1918-19 p. 334), Park, Williams (2) Chop into a pulp and add an equal
and Krumwiede (1924 p. 125). weight of distilled water.
(3) Allow to soak for 12 hours.
1338. Moon's Brain Infusion
(4) Filter thru a gauze, pressing out the
Constituents: liquid.
1. Physiological salt solution.. . 900.0 cc. (5) Add anequal weight of water to the
2. Brain (dog) 100.0 g. turbid viscous liquid.
Preparation (6) Make slightly alkaline by the addition
(1) Remove brain matter from normal of a 1.0% solution of sodium bicar-
dog under the most careful aseptic bonate.
precautions. (7) Heat at 100C. for 15 minutes in the
(2) Add 900.0 cc. of physiological salt autoclave or salt bath.
solution to 100.0 g. of (1). (8) Filter thru a fine linen cloth.
(3) Emulsify by shaking with glass beads. (9) Distribute in sterile tubes.
Sterilization: Filter thru a coarse Berke- Sterilization: Sterilize at 100 for 15
feld filter. minutes on two days.
Use: Cultivation of Negri bodies. Author Use: General culture medium. Besson re-
reported that growth was poor, both ported that the cholera vibrio did not
anaerobically and aerobically. develop in this medium unless it was
Reference: Moon (1913 p. 233). diluted with 5 or 6 volumes of sterile
water before use.
1339. Wolbach and Saiki's Liver Infusion
Reference: Besson (1920 p. 30).
Constituents:
1. Distilled water 1000.0 cc.
1341. Nencki, Sieber and Wyznikiewicz's
2. Liver (dog) Salivary Gland Infusion
1000.0 g.
Preparation Constituents:
(1) Pass fresh dog's liver thru the meat 1. Distilled water 1000.0 cc.
grinder. 2. Salivary glands 200.0 g.
394 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
containing material contained round cells 1343, Robertson and Davis' Heart Infusion
after two days. Medium
Variants
Constituents:
(a) The author added 3.0% NaCl or 2.0
1. Water, sterile distilled 1000.0 cc.
to3.0% NaCl plus 0.2 to 0.5 g. NaOH
2. Asparagin (Merck) 3.4 g.
or KOH per liter.
1345. Proca's Spleen Infusion Solution Use: Cultivation of typhoid, cholera and
other intestinal forms. Author reported
Constituents:
that the cholera vibrio lost its character-
1. Ringer solution 1000.0 cc.
istic appearance on this medium.
2. Spleen 400.0 g.
Preparation: Variants
(1) Add 400.0 g. of finely chopped fresh (a) The author used oysters instead of
solution (see medium ^180). (b) The medium was prepared as follows:
(2) Heat at 115C. for 30 minutes. (1)Remove oysters or mussels from
(3) Filter while hot thru paper. their shell.
(4) Distribute in tubes or flasks. (The (2) Work (1) into a pulp and mix with a
filtrate may be solidfied with 1.4% double weight of sterile phyiologi-
agar or 10.0% gelatin). cal salt solution.
Sterilization: Sterilize in the autoclave. (3) Allow the mixture to stand for 24 to
Use: General culture medium. 48 hours at 15C.
Reference: Proca (1924 p. 1164). (4) Pour off the liquid, filter thru cotton
then thru paper and finally thru a
1346. Harde and Hauser's Fish Infusion
Berkefeld candle.
Constituents: (5) This gives a yellowish amber fluid.
1. Water 1000.0 cc. The author reported that the organ-
2. Fish (Whiting) 500.0 g. isms failed to develop on this
Preparation: medium.
(1) Boil 500.0 g. of chopped whiting fish
Reference: Pergola (1912 p. 171).
with 1 liter of water for 20 minutes.
(2) Filter thru paper.
1343. Besson's Chicken Infusion (Tanner)
(3) Reaction is neutral to litmus.
Sterilization: Sterilize at 120^ for 20 Constituents:
minutes. 1. Water 1000.0 cc.
Use: Substitute for beef media and as a 2. Chicken 500.0 g.
general culture medium.
Preparation:
Reference: Harde and Hauser (1919 p. lean chicken meat in
(1) Soak 500.0 g. of
1259).
1000.0 cc. water.
Constituents
Sterilization: Not specified.
1349. Biisgen and Hoflich's Meat Extract Reference: Stutzer (1901 p. 83), Tanner
Solution (Linde) (1919 p. 45).
Constituents:
1. Water 1000.0 cc. 1351. Zikes' Basal Glucose Meat Extract
2. Meat
extract (0.5%) ........ 5.0 g. Solution
Preparation Constituents
(1) Dissolve 2 in 1. 1. Water 1000.0 cc.
Sterilization: Not specified. 2. Meat extract (0.5%) 5.0 g.
Use : Enrichment of Cladothrix, Cladothrix 3. Glucose (0.25%) 2.5 g.
dichotoma. Cultivation of other Preparation
organisms. (1) Dissolve 2, 3 and one of the added
Variants nutrients in 1.
(a) The author added a concentrated Distribute as desired.
(2)
Na2C03 solution to obtain an alkaline Sterilization: Not specified.
reaction. Use: Author used the medium to study the
(b) Zikes cultivated cladothrix and nitrogen sources suited to the development
Sphaerotilus natans in a medium of Cladothrix dichotoma and Cladothrix
containing 0.5, 0.25, 0.125, 0.063 or natans. He reported that Cladothrix
0.031% meat extract. dichotoma grew well on any nitrogen
(c) Besson prepared the medium as source but Cladothrix natans grew only
follows: when peptone or asparagin was added.
(1) Dissolve 0.5% Liebig's meat ex- Bloch studied the nitrogen sources suited
tract (or 20.0 g. Cibils meat extract) for the development of Zoogloea rami-
in 1000.0 cc. of water. gera. He reported that asparagin pep-
(2) Make alkaline if necessary. tone, ammonium sulphate and then
(3) Autoclave at 115 to 117 for 5 nitrate was the order of the materials as a
minutes. nitrogen source for Zoogloea ramigera.
(4) Filter while hot thru a wet filter. Added nutrients: Zikes as well as Bloch
(5) Tube. added 0.25% of one of the following:
(6) Sterilize at 110 to 115C. (NH 4)280 4 asparagin
Reference: Linde (1913 p. 372), Zikes KNO3 peptone
(1915 p. 530), Besson (1920 p. 30). Reference: Zikes (1915 p. 542), Bloch (1918
p. 51).
1350. Stutzer's Nitrate Meat Extract
Solution
1352. Stutzer's Glucose Meat Extract
Constituents:
Solution
1. Water
2. KNO3 Constituents
3. Liebig's Meat Extract 1. Water.
Preparation 2. Glucose.
(1) Dissolve 2 and 3 in 1. 3. KNO3
(2) Adjust the reaction to a slight 4. Liebig's ]\Ieat Extract.
alkalinity. Preparation
(3) Distribute in 10.0 cc. lots. (1) Dissolve 2, 3 and 4 in 1. (Peptone
Sterilization: Method not given. may be added).
Use: To study denitrification by B. agilis, (2) Reaction to be slightly alkaline.
B. niirovorus, B. Stuizeri, B. Hartlebi. (3) Distribute in 10.0 cc. lots.
Variants Sterilization: Method not given.
(a) The author used Schiilke and Meyer Use: To study denitrification by B. agilis,
or Cibils meat extract instead of B. niirovorus, B. Stutzeri, B. Hartlebi.
Liebig's. Variants
(b) Tanner dissolved 10.0 g. c.p. NaNOa (a) The author substituted Schiilke and
and 3.0 g. of Liebig's meat extract in Meyer's meat extract or Cibils meat
1000.0 cc. of water. extract for Liebig's.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 397
Constituents
1. Water 1000.0 cc.
2. Meat e.xtract, Cibils 100.0 to 200.0 cc.
3. Ammonium succi-
nate 10.0 g.
Preparation
(1) Thicken 100 to 200.0 cc. of Cibils
meat extract on the water bath.
(2) Transfer the viscous mass to a cru-
cible and carbonize and ash under the
hood.
(3) Take 3.0 g. of the grey white ash thus
obtained and dissolve in 1 liter of
water by boiling an hour.
(4) Filter repeatedly thru cotton.
(5) Add 10.0 g. of ammonium succinate
and neutralize with dilute ammonia.
(6) Distribute the slightly opalescent
medium in tubes.
Sterilization: Sterilize (method not given).
The medium becomes clear.
Use: Cultivation of typhoid and inter-
mediate group.
Reference: Hurler (1912 p. 356).
Constituents:
1. Water 1000.0 cc.
2. Meat extract (1.0%) 10.0 g.
3. Asparagin (0.25%) 2.5 g.
Preparation:
(1) Dissolve 2, 3 and 0.25% of one of the
added nutrients in 1.
(2) Tube or flask.
Sterilization: Not specified.
Use: Cultivation of Zoogloea ramigera.
Author reported that growth was gener-
ally better without carbohydrates.
Added nutrients: The author added 0.25%
of one of the following:
d-glucose lactose
d-levulose starch
d-mannose raffinose
398 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Add 0.5% Cyanamide to (2). (4) To obtain best results it is well to add
(3)
(4) Distribute in Erlenmeyer flasks. some albuminous material. Peptone
Sterilization: Method not given. may be added but a material from the
Use: To study cyanamide decomposition bone jelly gives equally good results.
by bacteria. Mix equal parts of water, bone jelly
Reference: Kappen (1909 p. 392). and HCl (strength not specified) and
heat in a water bath for 24 hours.
1358. Homer's Tryptophane Gelatin Neutralize with soda, clarify and
Solution filter.
Preparation :
1361. Berman and Rettger's Gelatin
(1) Add 1.0% gelatin and 0.15%, tryopto-
Solution
phane to a solution containing neces-
Constituents:
sary nutrient salts.
Sterilization: Not specified. 1. Water 1000.0 cc.
2. Gelatin 2.5 g.
Use: Indol production.
Variants: The author added 1.0% glucose.
3. NaCl 5.0 g.
Preparation
Reference: Homer (1906 p. 402).
(1) Dissolve 2 and 3 in 1. (2.5 g. Liebig's
1359. Remy and Rosing's Gelatin Solution meat extract may be added.)
(2) Tube in 10.0 cc. lots.
Constituents
Sterilization: Not specified.
1. Water 1000.0 cc.
Use: To study bacterial nutrition.
2. Gelatin (0.8%) 8.0 g.
Reference: Berman and Rettger (1918 p.
Preparation
381).
(1) Add 0.8% gelatin to 1000.0 cc. water.
(2) Neutralize with soda. 1362. Fleming's Blood and Minced Meat
(3) Distribute into small Erlenmeyer Medium
flasks. Constituents:
steam
Sterilization: Sterilize in streaming 1. Meat
by means method.
of the fractional 2. Blood.
Use: To study decomposition of organic N Preparation
materials by soil forms. (1) Add a little blood to a minced meat
Reference: Remy and Rosing (1911 p. 39). medium such as is commonly used to
grow anaerobes.
1360. Standfuss and Kallert's Bone Jelly
Sterilization: Not specified.
Solution
Use: Preservation of B. influenzae.
Constituents Author reported that subcultures from
1. Water. this medium after 6 weeks gave growths as
2. Bone jelly. copious as after two days.
Preparation Variants: Harvey added a little whole
(1) Bone jelly is obtained from a factory blood to the meat medium as prepared in
where fresh bones are rendered under medium 1239.
a pressure of 2 to 4 atmospheres in an References: Fleming (1919 p. 139), Harvey
autoclave. The
rendered material (1921-22 p. 72).
is evaporated or condensed to a jelly-
Constituents 3. Kidney
1. Veal infusion solution 100.0 cc. Preparation
2. Blood, defibrinated, rabbit (1) Draw blood from the vein of a normal
or dog 10.0 cc. horse into tall glass cjdinders and
3. Egg yolk 5.0 cc. allow the serum to separate.
Preparation (2) Pipette 4.0 cc. of the serum into tubes
(1) Mix 1, 2 and 3. having a diameter of 1.5 to 1.7 cm.
Sterilization: Not specified. (3) To each tube add 8.0 cc. of physiologi-
Use: Cultivation of tubercle bacilli. cal salt solution and mix well. The
Reference: Kolle and Wassermann (1912 p. contents of the tube are about 6.5 to
413). 7.0cm. high.
(4) Place in a water bath at 58C. raising
1365. Noguchi's Serum Tissue Medium the temperature gradually until it
Constituents: reaches 70 or 71 C. in three hours.
1. Distilled water 300.0 cc. (5) Heat for 30 minutes at 7rC. The
2. Serum 100.0 cc. contents present a translucent milky
3. Tissue, (rabbit) appearance with a semi-solid surface.
Preparation (6) Immerse one or two small pieces of
(1) Tube a mixture of one part serum rabbit kidney in each tube. It may
(sheep, horse or rabbit) and three be necessary to push the kidney to
parts distilled water in test tubes 20 the bottom by means of a sterile glass
cm. high and 1.5 cm. wide to a depth rod.
ofabout 16 cm. Sterilization: Not specified.
(2) Add to each sterile tube a small piece Use: Cultivation of Spirochaeta recurrens.
of freshly removed sterile rabbit Inoculate the medium with infected
tissue kidney or heart
(testicles, blood by means of a sterile capillary tube.
muscle) (liver being unsuitable). Author reported that in the kidney
(3) Incubate at 37 for 2 days to test medium growth appeared after 24 hours
sterility. and reached a maximum on the 4th day.
Sterilization: Sterilize (1) at 100C. for 15 In the buff coagulum medium growth was
minutes on 3 successive days. at a maximum at the end of the 5th to
Use: Cultivation of Treponema pallidum 7th days. The number seemed to be less
400 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents: Constituents:
1000.0 cc. 1. Serum (beef) 200.0 cc.
1. Water
2. Sputum 100.0 cc.
2. Beef 1.0 lb.
3. Glycerol (2.0%) 6.0 g.
3. Egg white
CaCOa (0.5%) 5.0 Preparation:
4.
(1) Mix two parts sterile
beef serum with
Preparation:
Thoroly mix 1 pound of lean chopped one part sterile tuberculous or bron-
(1)
beef with 500.0 cc. of water, chitus sputa.
(2) Neutralize (1) with NasCOs. (2) Add 2.0% glycerolto (1).
bonate to (8).
Preparation:
to 112C. (1) Place 5.0 cc. of sterile beef bile into a
Sterilization: Sterilize at 110
sterile test tube.
in the autoclave for 30 minutes.
Add about 2.5 cc. of the patient's
Use: To study putrefaction by B. coli (2)
communis, B. lactic aerogenes, B. putri- blood that has been removed from a
bacillus of malignant oedema, finger tip or an ear under aseptic
ficus,
conditions.
bacillus of symptomatic anthrax. The
Sterilization: Method of sterilization of
author replaced the air in the flask with
hydrogen gas. bile not specified.
402 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Use: Enrichment medium for typhoid and Reference: Berman and Rettger (1918 p.
paratyphoid used in diagnosis by blood 382).
culture method,
Reference: Kayser (1906 p. 186), Klimmer 1376. Adams' Coke Milk Medium
(1923 p. 214). Constituents:
Milk.
1.
1374. Noguchi's Blood Serum Solution
Coke.
2.
Constituents: Preparation
1. Serum...., 100.0 cc. (1) Add sterile pieces of coke to skimmed
2. Ringer's Solution 300.0 cc. milk.
3. Plasma, citrated, rabbit 150.0 cc. Sterilization: Method not given in the
Preparation abstract.
(1) Mix 1 part rabbit serum with 3 parts Use: Cultivation of butyric acid bacilli.
Ringer's solution (see medium 180) Before use the oxygen was removed by
and 0.5 part of citrated rabbit plasma. heating and after inoculation with the
Sterilization: Not specified. fecal material the tubes were again
Use: Cultivation of Spirochaeta ictero- heated for 2 to 3 minutes at 70C. to
haemorrhagiae, Leptospira icterohaemor- eliminate the less resistant organisms.
rhagiae and other spirochaeteceae. The Reference: Adams (1921 p. 59). Taken
medium was covered with a layer of from (1921 p. 319).
paraffin oil following
inoculation with
suspected blood material. SUBGROUP I-C. SECTION 17
1375. Berman and Rettger's Casein Meat Stoklasa's Nitrate Soil Infusion II.. 1383
Extract Medium
.
Di. Organic nitrogen not added. moment over the flame or heat in
Gutzeit's Mannitol Soil Infusion the autoclave for 20 minutes or
Medium, 1393 more at 10 pounds steam pressure.
Jensen's Nitrate Soil Infusion This medium will vary faintly
Medium 1394 alkaline to phenolphthalein to
alkaline -0.6 according to the
1377. Harrison and Barlow's Basal Wood amount of ash used.
Ash Medium (5) If desired acid potassium phosphate
Water
404 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(3) Acidify the filtrate with HCl. soilwith a liter of tap water for 30
(4) Wash the precipitate until the wash minutes in the autoclave under pres-
water shows a very weak acid sure of 1 atmosphere or boil with 2
reaction. liters of water over a free flame.
(5) Dissolve the filtrate in weak NaOH (2) Pour the turbid liquid.
off
Dissolve 1.0% (NH 4)2804 and 0.5% 1384. Lohnis and Pillai's Basal Soil Infusion
(6)
K2HPO4 in (5). Constituents
(7) Add chalk after inoculation with soil.
1. Water 1000.0 cc.
not specified.
(8) Sterilization 2. Soil 1000.0 g.
Reference: Gutzeit (1906 p. 370), Lohnis 3. K2HPO4 (0.5%) 5.0 g.
(1913 p. 110). 4. CaCOs (1.0 to 2.0%). 10.0 to 20.0 g.
Preparation
1382. Stoklasa's Nitrate Soil Infusion I
(1) Add 1000.0 g. soil to 1 liter of water.
Constituents: (2) Place in a covered pot and autoclave
1. Distilled water for 30 minutes at 1 atmosphere pres-
2. Soil 9000.0 g. sure.
3. NaNO, 1-5 g.
(3) Filter. One obtains about 600.0 cc.
4. K2HPO4 10 g-
(4) To add 0.5% K2HPO4 and 1.0%
(3)
5. MgSOi 10 g- of the added nutrients.
Preparation (5) Add 1.0 to 2.0% CaCO,.
(1) Extract 9000.0 g. of soil with distilled Sterilization: Not specified.
water at 60 C. (Amount of water or Use: To study N assimilation. Author
time not specified.) reported that more nitrogen was assim-
(2) Concentrate the extract (19 liters) ilated with mannitol than glucose,
to 1 liter. This liter contains 2.2 g. of CaCOa favored the nitrogen assimilation
carbon in the form of soluble organic with sugar but not with the acids.
compounds. Variants: The author omitted the CaCOs.
(3) Add 1.5 g. NaNOa, 1.0 g. KjHPO* Added nutrients: The author added 1.0%
and 1.0 g. MgS04
to 600.0 cc. of (2). of one of the following:
Sterilization: Sterilize four times in the mannitol
steamer. glucose
Use: To study denitrification by Bad. tartaric acid (neutralized with soda)
Hartlebi. Author reported no deni-
Reference: Lohnis and Pillai (1907 p. 88).
trification.
1385. Buchanan's Basal Soil Infusion
Reference: Stoklasa (1907 p. 32).
Constituents
1383. Stoklasa's Nitrate Soil Infusion II 1000.0 cc.
1. Water
Constituents 2. Soil 100.0 g.
1. Distilled water Preparation
2. Soil 2500.0 g. (1) Boil 100.0 g. of rich garden soil
with 1
Variants infusion.
Ai. Water and agar only. ^^^- Cultivation amoebae, algae, para-
of
Added nutrients and variants: The author (3) Pour off as much water as possible by
prepared one of the following media, placing a piece of cheese cloth over
using water and varying amounts of agar the top of the flask.
as a base: (4) Add make up
fresh distilled water to
bacter chroococcum in solution (c) and (d). or dried in air and used as ordinary
Reference: Pringsheim (1910 p. 230). agar. In this case use 1.0% of the dry
material to obtain a medium having
Preparation :
(9) Cool by placing the tins in cold
(1) Dissolve 30.0 to 40.0 g. of agar in water. The agar sets into a very
200 to 400.0 cc. of water. firm jelly which is cut into slices
(2) Add slowly, 700-800 cc. of alcohol which are broken up by a meat
that has been acidified with mincing machine with a very fine
CH3COOH. bore.
(3) Shake the mixture thoroly and allow (10) Place the small vermicular threads
the precipitated agar to settle. obtained from (9) in trays, and
(4) Pour off the alcohol and place the agar desiccate in a drying oven at a
precipitate on a Biicher funnel. moderate heat. When the threads
(5) Wash the precipitate until the wash are dry they have a light brown
water is neutral. color.
(6) The agar may be washed on a piece of Use: A purified agar. A 1.0% solution of
strain cloth instead of a Biicher the agar gives a clear firm medium.
funnel. Variants: The threads may be milled into
(7) After the alcohol has been removed a fine powder.
from the agar, the agar is dried at Reference: Cunningham (1918-19 p. 561).
100C.
(8) The dried agar is a snow white
1401. Harvey's Purified Agar.
powder.
Use: A purified form of agar. When this Constituents
powdered agar is used in the preparation 1- Water 200.0 g.
of various media, it requires no filtration. 2. Agar loO.O g.
Reference: Dominikiewicz (1908 p. 668). Preparation
(1) Mix 1.0 part fibre agar, 0.05 part
1400. Cunningham's Purified Agar
glacial acetic acid and 2.0 parts water.
Constituents (2) Allow the agar to soak in the acidified
1. Water. water 15 minutes.
2. China grass. (3) Remove the fibre agar and wash
Preparation thoroly with water until quite free
(1) Weigh a quantity of china grass from any trace of acid reaction to
and place in a vessel containing suffi- litmus paper.
cient dilute acid (0.01% H2SO4 gives (4) Squeeze in a cloth to get rid of excess
the best results) to completely cover of water.
the fibre. Use: Used in the preparation of media, to
(2) Soak in the acid for 10 minutes. give a 3% solution of agar, calculating
(3) Remove and wash in running water from the weight of the agar in the original
until all traces of acid have been dry condition.
removed. Both the fibre and wash- Variants Harvey treated the agar obtained
:
(7) Spread the vermicular threads ob- (Pure or impure sugars may be added.)
tained in shallow layers on trays. Sterilization: Not specified.
(8) Desiccate in a drying oven at Use: To produce chlamydospores by Sporo-
moderate temperature. thrix schenckii. Smith and Smillie used
(9) Reduce the desiccated material to a similar medium to obtain coccidia
powder. spores from sparrow feces. Teague and
(10) Preserve in the dry state. Deibert studied the constituents neces-
(11) Use for the preparation of media in sary for the growth of Unna-Ducrey's
a strength of one per cent. bacillus and reported no growth on the
Reference: Harvey (1921-22 p. 66, 67). medium used.
Variants: Smith and Smillie, and Teague
SUBGROUP II-B and Deibert dissolved 2.0% agar in a
0.5% aqueous NaCl solution.
Agar Media with All Other Constit- References: Davis (1914 p. 485), Smith and
Smillie (1917 p. 415), Teague and Deibert
uents Inorganic
(1922 p. 70).
Basal or complete media containing agar; Agar
1403. Beijerinck's Thiosulphate
all other constituents inorganic.
Constituents
Key to the Sections of Subgroup II B 1. Water 1000.0 cc.
2. NaoSaOs, 5H2O 5.0 g.
Ai. Nitrogen present as free or elementary
3. K2HPO4 0.1 g.
nitrogen.... Section 1 (Med. 1402-1410)
4. NaHC03 0-2 g.
A2. Nitrogen supplied as ammonium salts
5. Agar 20.0 g.
Section 2 (Med. 1411-1424)
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1.
A3. Nitrogen supplied as nitrites or ni-
Sterilization: Not specified.
trates Section 3 (Med. 1425-1430)
Use: Isolation of Thiobacillus denitrificans
and T. thioparus.
SUBGROUP II-B. SECTION 1
Reference: Beijerinck (1903-04 p. 599).
Basal or complete media containing agar
1404. Gordon's Basal Salt Agar
with inorganic salts. Nitrogen present as
free or atmospheric nitrogen only. Constituents:
Distilled water 1000.0 cc.
Ai. Containing salts of monovalent cations 1.
Carrel and Burrows' Locke's Solu- thoroughly washed and then dried
tion Agar 1405 agar in 1.
Omeliansky's Basal Carbonate Agar. 1406 (2) Dissolve one of the added nutrients
Ashby's Salt Agar (Murray) 1407 in (1).
Winogradski's Salt Agar (Murray) . . 1408 Sterilization: Not specified in the abstract.
Added nutrients: Gordon added various Use: To study nitrogen fixation and to
nitrogen compounds. The compounds determine number of organisms in soil.
used or concentration not specified. Used both anaerobically and aerobically.
Reference: Gordon (1917 p. 371) taken from Reference: Murray (1916 p. 608).
(1917 p. 299).
1408. Winogradski's Salt Agar (Murray)
1405. Carrel and Burrows' Locke's Solution
Constituents:
Agar
1. Water 1000.0 cc.
Constituents: 2. K.HPO4 1.0 g.
1, Locke's solution 1000.0 cc. 3. MgS04 3.0 g.
'
2. Agar (2.0%) 20.0 g. 4. NaCl 0.01 g.
Preparation: (1) Dissolve 2.0% agar in 5. MnS04 0.01 g.
Locke's solution (see medium 6 for 6. CaCO, 10.0 g.
Locke's solution). 7. FeCIa (10% soln.) 2 drops
Sterilization: Not specified. 8. Agar 15.0 g.
Use: Cultivation of tissue in vitrio. Preparation: (1) Dissolve 2, 3, 4, 5, 6, 7
Author reported that the medium did not and 8 in 1.
support growth nearly as well as did Sterilization: Not specified.
plasma. Use: lo study nitrogen fixation and to
Reference: Carrel and Burrows (1911 determine the number of bacteria in the
p. 245). soil. Used anaerobically and aerobi-
cally.
1406. Omeliansky's Basal Carbonate Agar Reference: Murray (1916 p. 608).
Constituents
Water 1409. Liot's Basal Salt Agar
1. 1000.0 cc.
2. Agar 15.0 g. Constituents:
3. NaoCOs 1.0 g. 1. Double distilled water 1000.0 cc.
4. K2HPO4 trace 2. Sodium phosphate 5.0 g.
Preparation: Dissolve 2,
(1) 3, 4 and one of 3. MgS04 2.5 g.
the added nutrients in 1. 4. Agar 25.0 g.
Sterilization: Not specified. Preparation
Use: To study oxidation of sulphites and (1) Macerate 25.0 g. of commercial
phosphites. The author reported that agar in 750.0 cc. of double distilled
neither the phosphite nor sulphite were water for 48 hours.
oxidized. (2) Dissolve in the autoclave at about
Added nutrients: The author added 0.3 g. 100C.
of one of the following: (3) Filter while hot on a Chardin filter
KNO2, KNO3, Peptone. paper.
Variants: The author added 0.0 or 2.0 g. (4) Dissolve 5.0 g. sodium phosphate in
of NaoSOa or NaaHPOj. 125.0 cc. of double distilled water.
Reference: Omeliansky (1902 p. 64). (5) Dissolve 2.5 g. MgS04 in 125.0 cc.
double distilled water.
1407. Ashby's Salt Agar (Murray)
(6) Add sterile (4) to (3). Mix well.
Constituents (7) Add sterile (5) to (6). Mix well.
1. Water 1000.0 cc. (8) Tube in 10.0 cc. quantities.
2. K2HPO4 0.2 g. (9) When ready for use add 0.5 cc. of a
3. MgS04 0.2 g. 1.0% sterile solution of one of the
4. NaCl 0.2 g. added nutrients to each sterile tube.
5. CaS04 0.2 g. (The sodium phosphate and MgS04
6. CaCOj 5.0 g. may be omitted, leaving a non-
7. Agar 15.0 g. mineral agar.)
Preparation: (1) Dissolve 2, 3, 4, 5, 6 and 7 Sterilization: Sterilize (4) and (5) at 115
in 1. for 15 minutes. Sterilize (8) in the usual
Sterilization: Not specified. manner. (Method not given.) Method of
413
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
sterilization of the added nutrients not Fremlin's Ammonium Sulphate Agar. 1417
Nencki, Sieber and Wynikiwicz's
given.
Ammonium Sulphate Agar 1418
Use: To study pigment production by
pyocyanius bacilli. Gowda's Ammonium Sulphate Agar. 1419
Heinemann's Ammonium Sulphate
Added nutrients: The author added 0.5 cc. 1420
1.0% solution of one of the following Agar
of a
Committee S. A. B. Basal Ammo-
to each 10.0 cc. of the medium:
nium Phosphate Agar 1421
Ammonia salts of organic acids
A Ammonia
4. present as salts of phos-
Amides
Monobasic acids phoric acid.
Beijerinck's Sodium Ammonium
Polybasic acids
Phosphate Agar. - 1422
Amines
Ammonia salts of inorganic acids Stutzer's Ammonium Magnesium
Phosphate Agar 1423
Polyatomic alcohols
Monosaccharides Carper's Basal Ammonium Phos-
phate Agar 1424
Polysaccharides
Monoses 1411. Beijerinck's Thiosulphate Ammonium
Reference: Liot (1923 p. 238). Chloride Agar
ride.
mum pH was between 3.0 and 4.0.
Thiosulphate Ammo- Reference: Waksman (1922 p. 607).
Beijerinck's
nium Chloride Agar 1411
1413. Beijerinck and van Delden's Ammo-
Waksman's Thiosulphate Ammo- nium Chloride Agar
1412
nium Chloride Agar
Constituents:
Beijerinck and van Delden's Ammo- 1000.0 cc.
1413 1. Distilled water
nium Chloride Agar 15.0 g.
as ammonium 2. Agar
A2. Ammonia present 0.1 g.
3. K2HPO4
nitrate. 0.1 g.
Ammonium Agar. 1414 4. NH4CI
Munter's Basal .
.
Dissolve 2, 3 and 4 in 1.
Ammonium Nitrate Preparation: (1)
Beijerinck's
.--"IS Sterilization: Not specified.
Agar oligocarbophilus.
present as ammonium Use: Isolation of Bacillus
Ammonia
Author reported that colonies were
A3. dry,
sulphate. agar
Ammonium vSulphate snowv white or rose colored. If the
Pesch's Basal with straw or an
1416 plate" be inoculated
Agar
414 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents:
1. Distilled water 1000.0
2. Agar
3. NH4NO3
4. Potassium phosphate..,
5. MgS04
6. CaCh
Preparation
(1) Wash agar with distilled water for
some time to separate the soluble
material.
(2) Dissolve 3, 4, 5 and 6 in 1.
(3) Dissolve 2.0% (dry) (1) in (2).
(4) Pour into plates.
Sterilization: Not specified.
415
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
2. FeS04 0.4 g.
(1910 p. 15).
3. MgSOi 0.5 g.
1418. Nencki, Sieber and Wyzniklewlcz's 4. K2HPO4 10 g.
Ammonium Sulphate Agar 5. NaCl 2.0 g.
6. (NH4)2S04 10 g.
Constituents 20.0 g.
1000.0 cc.
7. Agar
1. Water 10.0 g.
10.0 to 15.0 g.
8. CaCOa,
2. Agar
Preparation
3. K2HPO4. 0.5 g.
(1) Dissolve 2, 3, 4, 5 and 6
in 1.
NajCOa 1.0 g.
4.
(2) Dissolve 20.0 g. of agar
in (1).
(NH4)2S04 2.5 g.
5.
(3) Add 10.0 g. precipitated
CaCOs to (2).
6. NaCl 5.0 to 10.0 g.
(4) Shake well.
Preparation
(5) Tube.
Soak 10.0 to 15.0 g. agar with 2 or 3
(1)
Sterilization: Sterilize in the autoclave.
times the volume of distilled water.
Use: To study nitrification.
(2) Dissolve the agar in 1000.0 cc. of hot
Reference: Heinemann (1922 p. 38).
water.
(3) Adds, 4, 5 and 6 to (2). Committee S. A. B. Basal Ammonium
1421.
(4) Filter. Phosphate Agar
Sterilization: Sterilize in the autoclave.
Constituents:
Use Study cause of cattle plague. Author
:
(3) Pour distilled water over the solidified 3. Ammonium phosphate 3.0 g.
agar and allow to stand for several 4. Agar 30.0 g.
days. Pour off this water and add Preparation
fresh distilled water from time to (1) Dissolve 2, 3 and 4 in 1 by boiling.
time. This process removes all solu- (2) Neutralize to litmus by the addition
ble material from the agar. of NaOH.
(4) Add 0.2 to 0.5% NH4XaHP04 and (3) Add one of the added nutrients listed
0.05% KCl to the agar, also pre- below.
cipitated CaCOs (amount not given). Sterilization: Sterilize in the autoclave.
(5) Boil. (This serves to sterilize.) Use: Cultivation of tubercle bacilli.
(6) Distribute into sterile dishes. Added nutrients: The author added one of
Sterilization: Sterilization is accomplished the following:
in step (5) above. (a) Glycerol 5.0%
Use: To show nitrite formation by Amoeba (b) Glycerol 5.0%
nitrophil. Author reported that the Beef extract (Liebig's) 0.3%
colonies producing nitrites formed a (c) Glycerol 5.0%
clear ring around the colonies due to the Peptone (Witte) 1.0%
action of nitrites on CaCOa. Killer culti- (d) Glycerol 5.0%
vated protozoa and other soil forms on a Beef extract (Liebig's) 0.3%
similar medium. Peptone (Witte) 1.0%
Variants: Killer specified the use of 0.2% (e) Glycerol 5.0%
NH,NaHP04 4H2O and omitted the
Add 1.0% defibrinated rabbit blood
CaCOs. at 43C. and inspissate on each of
References: Beijerinck (1896 p. 258), Killer 3 successive days for 90 minutes.
(1913 p. 523). (f) Same as (e) but using laked blood.
(g) Same as (e) but using cow's milk,
1423. Stutzer's Ammonium Magnesium Same
(h) as (e) but using 10.0% casein.
Phosphate Agar
(i) Same as (e) but using whole egg.
Constituents: (j) Same as (e) but using egg white.
1. Water ^,
1000.0 cc. (k) Glycerol 5.0%
2. Agar
'.
10.0 g. Add 1.0% sodium nucleinate, pre-
3. K2HPO4 1.0 g. pared by neutralizing yeast nu-
4. NaCl 5.0 g. cleic acid in warm water by means
5. FeS04 0.5 g. of Na2C03 solution. Inspissate as
6. Ammonium magnesium in (e).
phosphate 20.0 g. (1) Glycerol 5.0%
7. MgCOa 20.0 g. Adda suspension of yeast nuclein
Preparation: make a 1.0% suspen-
sufficient to
(1) Dissolve 2, 3, 4 and 5 in 1. sion. Inspissate as in (e).
(2) Mix equal amounts of 6 and 7 in a (m) Glycerol 5.0%
mortar. Add 1.0% of a mush of testes from
(3) Add about a half teaspoon of (2) to rabbits. Inspissate as in (e).
test tubes. (n) Same as (m) but use liver from rab-
(4) Add 10.0 to 15.0 cc. of melted (1) to bits instead of testes.
each tube. (o) Same as (m) but use brain from rab-
Sterilization: Method not specified. bits instead of testes.
Use: To study nitrification by soil forms. Reference: Corper (1919 p. 463).
Reference: Stutzer (1901 p. 173).
Agar
Basal or complete media containing agar
Constituents with inorganic constituents. Nitrogen sup-
1. Water, tap 1000.0 cc. plied as nitrites or nitrates.
2. Salt (sea salt) 5.0 g. Ai. Nitrogen supplied as nitrites.
418 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
4. Potassium phosphate
5. Agar agar 15.0 g.
Preparation:
(1) Dissolve 2, 3 and 5 in 1.
Di. Containing salts of monovalent cations, Bs. Ammonia supplied as salts of phos-
only. phoric acid.
Beijerinck's Mannitol Agar 1451 Ci. Basal media, employed with the addi-
Dj. Containing salts of monovalent and tion of other nutrients.
other cations. C2. Complete media.
Lohnis' Basal Glj'cerol Agar 1452
Di. Only one source of organic carbon
Sackett's Mannitol Salt Agar 1453
added.
Ashby's Mannitol Agar (Jones) .... 1454
El. Carbon supplied as carbohydrates.
Beijerinck's Mannitol Agar (Omeli-
Bengis' Glucose Ammonium
Phos-
ansky and Ssewerowa) 1455
phate Agar 1475
Shunk's Mannitol Agar 1456
Ayers and Rupp's Fuchsin Sulphite
Krainsky's Mannitol Agar 1457
Agar 1476
Heinemann's Mannitol Agar 1458
Cunningham's Cellulose Ammonium
B3.* Carbon supplied other than alcohols or
Phosphate Agar 1477
carbohydrates.
E2. Carbon supplied as organic acids or
Lieske's Acetate Agar 1459
their salts.
Sohngen's Petroleum Agar 1460
Bengis' Lactate Ammonium Phos-
A2. Nitrogen present as ammonium salts.
phate Agar 1478
Bi. Ammonium chloride employed.
Tausz and Peter's Napthenate Am-
Ci. Carbon supplied as carbohydrates.
monium Phosphate Agar 1479
Sohngen's Manganese Oxide Agar. 1461 .
their salts.
Sohngen's Organic Acid Agar 1465 Be. Ammonium salts of organic acids em-
Krainsky's Malate Agar 1466 ployed.
B2. Ammonium nitrate employed. Bengis'Ammonium Lactate Agar.. 1483
Noyes' Starch Ammonium Nitrate Cohn's Ammonium Tartrate Agar
Agar 1467 (Klimmer) 1484
B3. Ammonium
sulphate employed. Nelson's Ammonium Succinate Agar 1485
Carbon supplied as carbohydrate. Dolt's Lactose Lactate Agar 1486
Ammonium
Sulphate Agar 1468 Fischer's Nitrate Tartrate Agar. . . . 1488
Higgins' Glucose Nitrate Agar 1469 As. Nitrogen supplied as nitrates (e.xclusive
C2. Containing disaccharides. of (NH3NO3).
Mortensen's Sucrose Ammonium Bi. Only one source of organic carbon
Sulphate Agar 1470 added.
C3. Containing polysaccharides. Ci. Organic carbon supplied as carbo-
Kellerman and McBeth's Polysac- hydrate.
charide Ammonium Sulphate Sackett's Glucose Nitrate Agar 1489
Agar 1471
Conn and Breed's Carbohydrate Ni-
Sanborn's Ammonium Sulphate Cel- trate Agar 1490
lulose Agar 1472 Groenewege's Sucrose Nitrate Agar. 1491
Vierling's Cellulose Ammonium Sul- Czapek's Sucrose Nitrate Agar
phate Agar 1473 (Conn) 1492
B4. Ammonium
carbonate employed. Pinoy's Dextrin Nitrate Agar 1493
Proskauer and Beck's Glycerol Am- Giltay's Glucose Nitrate Agar
monium Carbonate Agar (Klim- (Giltner) 1494
mer) 1474
C2. Organic carbon supplied as organic
* See also B4, Bs and Be. acids or their salts.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 421
Bj. More than one source of organic car- Added nutrients: The author added 2.0%
bon added. of one of the following:
Conn and Breed's Double Sugar Ni- plasmon sanatogen
trate Agar 1495 tropon nutrose
roborat
1431. Milburn's Basal Glucose Agar Reference: Zipfel (1911 p. 128).
Glucose Agar
2. Agar 15.0 g.
1432. Zipfel's Basal
3. NaCl 5.0 g.
Constituents: 4. Glucose 20.0 g.
1. Water 1000.0 cc. Preparation: (1) Dissolve 2, 3 and 4 in 1.
2. Agar (3.0%) 30.0 g. Sterilization: Not specified.
3. Glucose (1.0%,) 10.0 g. Use: To study chromogenesis by sporo-
4. Malic acid tricha. Author reported that if maltose
Preparation be substituted for glucose, growth and
(1) Dissolve 2, 3 and one of the added pigment production was more luxuriant.
nutrients in 1. Using glucose, pigment formation was
(2) Acidify slightly by the addition of slight. Growth without glucose about
malic acid. the same as when glucose (or impure glu-
Sterilization: Not specified. cose) was employed.
Use : Cultivation of nodule bacteria. Reference: Davis (1915 p. 178).
422 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(10) Same as (9) but omit the dextrose Sterilization: Sterilize in the autoclave.
from the agar. Reaction is 0.5% Use: To study the assimilation of atmos-
acid (Brown Artificial humus pheric nitrogen.
agar E). Reference: Heinemann (1922 p. 39).
(11) Substitute 10.0 cc.humus ex-
of
tract neutralized with HCl for 1442. Miinter's Basal Salt Agar
peptone (Brown Artificial humus Same as medium
but solidified by the
164,
agar F). addition of 1.2% agar. Also solidify me-
(12) Substitute 25.0 cc. of humus ex- dium 140 by the addition of 1.2% agar.
424 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1443. Groenewege's Basal Sucrose Agar hosts. Various authors used the same
or similar media for a variety of purposes.
Constituents:
Variants
1. Water (tap) 1000.0 cc.
(a) Peklo cultivated plant actinomyces
2. Agar to solidify
on a medium solidified with agar con-
3. Sucrose 20.0 g.
taining 0.5% KH,P04, 0.2% MgS04
4. K2HPO4 0.5 g.
and did not specify the use of 1.0%
Preparation: (1) Dissolve 3, 4 and one of
agar as did Buchanan.
the added nutrients in tap water agar
(b) Tanner, citing Moore, cultivated Ps.
(preparation not given).
radicicola on a medium containing
Sterilization: Not specified.
0.1% K2HPO4, 0.05% MgS04, 1.0%
Use: Cultivation of Phytobacter lycoper-
sucrose and 1.0% agar.
sicum n. sp. causing tomato rot. Author
(c) Shunk dissolved 1.0 g. KH2PO4, 0.5 g.
reported growth with all nitrogen sources
MgS04, 10.0 g. sucrose and 10.0 g. or
employed.
15.0 g. agar in tap water. This me-
Added nutrients: The author added one of
dium was employed for the isolation
the following nitrogen compounds as a
of nodule bacteria. He reported that
nitrogen source:
if mannitol be substituted for sucrose,
KNO3 the medium could be used to main-
(NH4)2S04
tain cultures.
sodium ammonium tartrate
References: Buchanan (1909 p. 381), Peklo
ammonium citrate
(1910 p. 470), Tanner (1919 p. 50), Shunk
ammonium succinate
(1921 p. 241).
ammonium acetate
ammonium lactate 1446. Lohnis' Congo Red Sucrose Agar
ammonium malate
asparagin Bonstituents:
Reference: Groenewege (1913 p. 25). 1. Water 00.0
2. Sucrose
1444. Owen's Sucrose Agar 3. K2HPO4
Constituents: 4. MgS04
1. Water 1000.0 cc. 5. Agar
2. Agar (2.0%) 20.0 g. 6. Congo red
3. Sucrose (Second 80)
(10.0%) 100.0 g.
Preparation: Dissolve 2 and 3 in 1.
(1)
Sterilization: Method not given.
Use: To determine bacterial count in cane
sugar products.
Reference: Owen (1914 p. 338).
2. Agar 10.0 g.
3. Sucrose 20.0 g.
4. KH2PO4 0.2 g.
5. MgS04 0.01 g.
Preparation
(1) Dissolve 2, 3, 4 and 5 in 1.
sour in peas.
Reference: Wyant and Tweed (1923 p. 12).
Constituents
426 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(a) Omitted the CaS04 and NaNO, and Sterilization: Not specified.
added 2.8589 g. NajCOs. Use: Isolation of Leptothrix ochracea.
(b) Omitted the NaCl. Inoculate the agar plates when the sur-
(c) Omitted the NaNO 3. face is still moist.
(d) Omitted the Na2S04. Reference: Lieske (1919 p. 418).
(e) Omitted the MgSOi.
1460. Sohngen's Petroleum Agar
(f) Omitted the K2SO4.
(g) Diluted any of the above media with Constituents:
2 or 3 volumes of water. 1. Distilled water 1000.0 cc.
Reference: Sackett (1912 p. 109). 2. Bipotassium phosphate 0.5 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 427
(2) Add a slight excess of copper car- of water instead of 1000.0 cc. as indi-
bonate. cated by Kellerman and McBeth.
(3) Shake vigorously. Tanner then mixed equal parts salt
(4) Allow to stand over night and then solution on cellulose solution.
siphon off the supernatant solution. (c) Tanner prepared starch agar by dis-
(5) Add 15.0 g. of unwashed sheet filter solving one-half the quantity of salts
paper. given in variant (a), step (3), in
(6) Shake occasionally until the paper 500.0 cc. tap water. Then Tanner
is dissolved. mixed equal parts of salt solution
(7) Dilute to 10 liters and add slowly a and starch water and proceeded as
one to five solution of hydrochloric in variant (a).
acid with vigorous shaking until the (d) Giltner prepared a starch agar as
precipitation of the cellulose is follows:
complete. (1) Dissolve 1.0 g. K2HPO4, 1.0 g.
(8) Dilute to 20 liters, allow the cellu- MgS04-7H20, 1.0 g. NaCl, 2.0 g.
lose to settle and decant the super- (NH4)2S04, and 2.0 g. CaCOa in
natant liquid. 1000.0 cc. of water.
(9) Wash by repeated changes of water, (2) Add 10.0 g. washed agar.
adding HCl each time until the (3) Weigh.
copper color disappears. Then wash (4) Boil over a free flame until the
with water alone until the solution solution of the agar is complete.
is free from chlorine. (5) Weigh and make up the loss in
(10) Allow to settle several days and weight with boiling water.
decant as much of the clear solution (6) Make a smooth paste of 10.0 g. of
as possible. potato starch or other starch in a
(11) If the cellulose percentage is too little cold water.
low centrifuge a portion of the fluid (7) Add 800.0 cc. of boiling water
to bring the cellulose content up to to (6).
one per cent. (8) Concentrate to 500.0 cc. by boiling.
(12) Dissolve 4, 5, 6, 7 and 8 in 1000.0 cc. Mix (5) and (8) and boil a few
(9)
of tap water. minutes.
(13) Mix 500.0 cc. of (11), 500.0 cc. of (12). (10) Strain thru two thicknesses of
(14) Dissolve 10.0 g. of agar in (13). cheese cloth.
Sterilization: Method not given. (11) Add 1.5% china blue rosolic acid
Use: Cultivation of organisms fermenting for indicator.
cellulose. (12) Tube, taking care to keep the
Variants CaCOs well mixed with the media.
(a) The author prepared a similar me- (13) Sterilize (method not given).
dium using potato starch instead of (e) Khouvine prepared a cellulose in the
cellulose as the polysaccharide. The same manner as Kellerman and
medium was prepared as follows McBeth and dissolved 20.0 g. agar in
(1) To 800.0 cc. of boiling water add 1000.0 cc. of water by heating. The
10.0 g. of potato starch suspended inorganic salts, as used by Keller-
in cold water. man and McBeth, were dissolved in
(2) Evaporate by boiling to 500.0 cc. the agar solution. Five hundred
(3) Dissolve 1.0 g. K2HPO4, 1.0 g. cubic centimeters of the cellulose
MgS04, 1.0 g. NaCl, 2.0 g. solution was then added to the agar
(NH4)2S04, and 2.0 g. CaCOj in solution, mi.xed well, tubed and steri-
1000.0 cc. of water. lized at 110 for 15 minutes.
(4) Mix 500.0 cc. (2) with 500.0 cc. (3). (f) Khouvine prepared a starch agar in
(5) Dissolve 10.0 g. of agar in (4). the following manner:
(6) Sterilization not specified. (1) Prepare a paste with 10.0 g. of
(b) Tanner used one-half the amounts of potato starch in a little cold water.
salts and dissolved them in 500.0 cc. (2) Add (1) to 800.0 cc. of boiling water.
430 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1. Distilled water 1000.0 cc. jar. Author reported that the filter paper
2. K2HPO4 10 g. showed no signs of being attacked.
3. MgS04 10 g. Growth occurred, however. The agar
4. NasCO, 1.0 g. became turbid. After incubation no
5. (NH4)2S04 2.0 g. clearing occurred around the colonies.
6. Cotton 30.0 g. Cellulose was not decomposed.
7. Agar 2.5 g. Reference: Vierling (1920 p. 206).
8. CR indicator (1.0%)
1474. Proskauer and Beck's Glycerol Am-
Preparation
monium Carbonate Agar (Klimmer)
(1) Dissolve 2, 3, 4 and 5 in 500.0 cc. of
distilled water. Constituents:
Prepare a 0.5% agar solution with 1. Water 1000.0 cc.
(2)
the remaining 500.0 cc. distilled 2. Glycerol 15.0 g.
Add 1.0% CR
indicator with constant Preparation: (1) Dissolve 2, 3, 4, 5 and 6
(5)
stirring. (CR indicator prepared by in 1.
3. Glycerol ^.0 g.
ticum liquefaciens
Reference: Tausz and Peters (1919 p. 49). 4. Ammonium phosphate 1.0 g.
5. Lactose 10-0 g.
1480. Simmons' Citrate Agar 6. Azolitmin 10 g.
Preparation
Constituents
1000.0 cc. (1) Purify agar
by cutting it in small
1. Distilled water
20.0 g. pieces and soaking it in distilled water
2. Agar
5.0 g. 24 hours.
3. NaCl
Dissolve (1), 2, 3 and 4 in 1.
4. MgS04 0.2 g. (2)
10 phthalein.
6. K2HPO4 g-
Dissolve 5 in (3).
7. Sodium citrate (2.77 g. of (4)
Dissolve l.C g. of Kahlbaum's C. P.
sodium citrate, 5^ H2O) .
. . . 2.0 g. (5)
azolitmin in 100.0 cc. distilled water
8. Brom-thymol blue (1.5%
alcoholic) 10-0 cc. and boil, 15 minutes. This gives a
blue solution.
Preparation
and 7 in 1.
6 (6) Add 10.0 cc. of (5) to (4).
(1) Dissolve 3, 4, 5,
Method not given.
(2) Add 20.0 g. clean washed agar and Sterilization:
p. 622).
5. Glucose 2.0 g.
6. Agar 20.0 g.
1483. Bengis' Ammonium Lactate Agar Preparation
Constituents: (1) Dissolve 2, 3, 4, 5 and 6 in 1.
Agar 15.0 g.
black colonies, typhoid organisms did not. 2.
Glucose or sucrose 10.0 g.
Reference Harrison and VanderLeck (1909
:
3.
p. 622).
4. KNO3 10 g-
5. CaCl.2 0.5 g.
1488. Fischer's Nitrate Tartrate Agar 6. MgS04 5.0 g.
3. MgS04 0.5
4. KCl
5. K2HPO4
6. FeS04
7. NaNOa
8. Sucrose
Preparation: (1) Dissolve 2,
7 and 8 in 1.
(NH4)2S04 3.0 g.
El. Only one type of organic
carbon added. 2.
3. Asparagin 1-5 g-
Fi. Carbohydrates added.
Groenewege's Starch Asparagin 4. K2HPO4 2.0 g.
CaCU 0-25 g.
Agar. 1507 5.
MgS04 0.25 g.
Noyes' Starch Asparaginate Agar 1508 . .
6.
Agar 150 g.
Stutzer & Hartleb's Glycerol As-
7.
1509 Preparation:
paragin Agar,
(1) Dissolve 2, 3, 4, 5, 6,
and 7 in 1.
Fj. Alcohols added.
(2) Dissolve one of
the sterile added
Sullivan's Glycerol Asparagin Agar. 1510
nutrients to sterile (1).
Peklo's Mannitol Asparagin Agar. 1511 .
Preparation Preparation
(1) Dissolve 2, 3, 4 and 5 in 1. (1) Dissolve 2, 5, 6, 7, 8 and 9 in 1.
(2) Adjust to 0.8 or 1.0% normal
Sterilization: Method not given. acid to
Use To produce chlamydospores by Sporo-
:
phenolphthalein.
thrix schenckii. Author reported that (3) May be clarified with egg
or by
the growth of the organism was slightly simply heating for 30 minutes at 15
accelerated if pure or impure sugars be pounds pressure in such a way as not
added. to disturb the sediment and then
Reference: Davis (1914 p. 485), (1915 p. decanting thru cotton filter.
179). (4) Add 1.0 g. glucose and 1.0 g. sodium
asparaginate to (3).
1503. Bengis' Phosphate Asparagin Agar Sterilization: Method not given.
Use: Bacterial counts in soils. Author
Constituents:
1. Distilled water 1000.0 cc. reported that there may be other satis-
2. Asparagin 10.0 g. factory combinations of the salts than the
NaCl 0.02 g.
instead of Na2HP04. 4.
reS04 trace
(b) Tanner described a similar medium 5.
Calcium phosphate trace
as Dolt's asparagin agar. One of 6.
Dextrose 10.0 g.
these media was composed of 100.0 7.
Asparagin 1-9 g-
cc. of distilled water, 350.0 cc. of a 8.
Agar (2.0%) 20.0 g.
3.0% purified agar solution in water, 9.
1506. Dolt's Lactose Asparagin Agar Use: General culture medium, primarily
Constituents: used to study organisms of the soil.
1. Distilled water Reference: Noyes (1916 pp. 93-94).
1000.0 ec.
2. Agar, purified 30.0 g. 1509. Stutzer and Hartleb's Glycerol
3. Asparagin 10.0 g. Asparagin Agar
4. NasHPO^ 2.0 g.
5. Lactose Constituents:
10.0 g.
6. Azolitmin (1.0% solution).. 10.0 cc.
1- Water 1000.0 cc.
Preparation 2. Agar 2O.O g.
(1) Purify agar by cutting it in small 3. Asparagin 10 g.
pieces and soaking it in distilled water 4. Glycerol 1.0 g.
Add 5.
Variants : Fraenkel used 15.0 g. agar instead
(4)
Prepare 6 by dissolving 1.0 g. of of 20.0 g.
(5)
Kahlbaum's C.P. azolitmin in 100.0 References: Stutzer and Hartleb (1897 p.
g. distilled water and boil for 15
403), Fraenkel (1898 p. 10).
(2) To 100.0 cc. of (1) add 400.0 cc. dis- 1514. Beijerinck's Malate Asparagin
tilled water and dissolve 2.0 g. as- Agar
paragin, 1.0 g. mannitol and a trace of
Constituents
Fe2Cl6 in the mixture.
1. Water.
(3) From sterile (2) prepare a 0.75% agar.
2. Agar.
(Method or final sterilization not
3. Sodium malate.
given.)
4. Asparagin.
Sterilization: times
Sterilize (2) several
5. Potassium phosphate.
(method not given). A turbidity forms
6. Mohr salt (NH4)2S04
but disappears when the medium cools.
Preparation
Use: Cultivation of plant actinomyces.
(1) Prepare a water solution of agar that
Author reported good growth.
has been washed free of soluble
Reference: Peklo (1910 p. 473).
material with distilled water.
(2) Pour distilled water over the solidified
1512. Thornton's Mannitol Asparagin Agar
mass and renew the water often.
Constituents Melt and boil.
(3)
1. Water 1000.0 cc.
(4) Add a small amount of nutrient solu-
2. K2HPO4 ,.
10 g.
tion consisting of sodium malate,
3. MgS04-7H20 0.2 g.
asparagin and potassium phosphate
4. CaCU 0.1 g.
(amounts not specified).
FeCls 0.002 g.
5.
(5) Boil again to remove all the air.
6. KNO3. 0.5 g.
(6) Add a drop of neutral solution of
Asparagin 0.5 g.
7. Mohr salt, (NH4)2S04, containing a
8. NaCl 0.1 g.
trace of Na^COs, there should be no
9. Agar., 15.0 g.
turbidity.
10. Mannitol 10 g.
Sterilization: Not specified.
Preparation Use: Cultivation of Spirillum desulfuri-
(1) Dissolve 2, 3, 4, 5, 6, 7, 8 and 9
in 1 by
cans, Spirillum tenue and other sulphate
steaming for 30 minutes. reducing organisms.
(2) Neutralize to turmeric paper. Reference: Beijerinck (1895 p. 108).
(3) Filter thru cotton-wool.
(4) Add 1.0 g. mannitol to (3). 1515. Fraenkel's Lactate Asparagin Agar
(5) Tube in 8.0 cc. quantities. (Klimmer)
Sterilization: Sterilize in the autoclave at
22.5 pounds pressure. Constituents
Use: Cultivation of soil forms. 1. Water 1000.0 cc.
References: Thornton (1922 p. 241) taken 2. K2HPO4 2.0 g.
from (1923 p. 277), Cunningham (1924 3. NaCl 5.0 g.
p. 136). 4. Ammonium lactate... 6.0 g.
5. Asparagin 4.0 g.
1513. Conn's Glycerol Asparaginate Agar 6. Agar 15.0 to 30.0 g.
Preparation
Constituents and 6 in 1.
(1) Dissolve 2, 3, 4, 5,
1. Water 1000.0 cc. (indicator
(2) Make distinctly alkaline
2. Agar 15.0 g.
not specified).
3. Glvcerol 10.0 cc.
Sterilization: Not specified.
4. K2HPO4 0.5 g.
Use: Synthetic culture medium.
5. Sodium asparaginate 1-0 g.
Reference: Klimmer (1923 p. 172).
Preparation
(1) Dissolve 2, 3, 4, and 5 in 1. 1516. Voges' Lactate Asparaginate Agar
Sterilization: Method not given.
Same as medium 4416, but containing 10.0
Use : Cultivation of actinomycetes.
g. of agar per liter of medium.
Reference: Conn (1921 p. 7).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 441
6. MgS04 2.0 g.
Sterilization: Method not given.
7. CaClj 0.2 g.
Use: Cultivation of B. Acaciae. Author
8- FeaCle trace reported that slime was produced.
9. Agar (1.5%) I5.0 g. Reference: Smith (1905 p. 383).
Preparation
1520. Higgins' Basic Lactose Asparagin
(1) Dissolve 2 and 3 in 250.0 cc. of 1.
(2) Dissolve 4, 5, 6, 7 and 8 in 500.0 Agar
cc.
of 1.
Solidify Basal Medium 386 by the addition
(3) Neutralize (2) by the addition of of agar.
KOH.
(4) Mix (1) and (3) and make up to a 1521. Sullivan's Glycerol Nitrate Asparagin
liter. Agar
(5) Dissolve 1.5% agar in (4).
Tube. Constituents:
(6)
Sterilization: Method not 1. Water 1000.0 cc.
given.
Use: To study denitrification. 2. Asparagin 10.0 g.
Reference: Lohnis (1913 p. 98). 3. MgS04 0.2 g.
4. K2HPO4 1.0 g.
1518. Conn's Asparaginate Agar 5. Ammonium lactate 0.5 g.
Constituents: 6. NaCl 5.0 g.
3. Na2HP04 3.0 g.
10. Basic fuchsin solution
4. KH2PO4 10 g.
Preparation
5. CaCl2 0.1 g.
(1) Dissolve 2, 3, 4 and 5 in 500.0 cc. of
6. MgS04-7H20 0.3 g.
distilled water.
7. NaCl 0.1 g.
Dissolve 6 in 100.0 cc. distilled
(2)
8. Fe2Cl6 0.01 g.
water. Add to (1).
9. Agar (2.0%) 20.0 g.
(3) Dissolve 7 in distilled water.
and and make up to Preparation
(4) Mix (2) (3)
(1) Dissolve 4, 5, 6, 7 and 8
in 100.0 cc. of
1000.0 cc.
agar and add to (4). water. (Meyers solution).
(5) Wash 8.0 g.
450.0 cc. of water.
pounds pressure for (2) Dissolve 2 and 3 in
(6) Sterilize at 15
(3) Mix 50.0 cc. of (1) and (2) and solidify
15 minutes.
a 10.0% NajSOs by the addition of 2.0% agar.
(7) Add 12.0 cc. of
(4) Distribute in 50.0 cc. lots in 200.0 cc.
solution.
Erlenmeyer flasks.
(8) Titrate to neutral to litmus using
Sterilization: Method not given.
N/10 HCl.
of saturated alcoholic Use: Isolation of uric acid splitting bac-
(9) Add 30 drops cobayae,
teria from feces and soil, Bac.
solution of basic fuchsin.
Bac. capri, Bac. guano, Bac. musculi,
(10) Mix thoroly and pour
into plates.
step Bac. hollandicus.
Sterilization: Sterilization given in
preparation.
Reference: Stapp (1920 p. 3).
(6) of
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 443
1529. Stapp's Hippurate Agar Levine's Boric Acid Peptone Agar.. 1547
I3. Containing salts of divalent cations.
Same as medium 507, but solidified by the
addition of 2.0% agar.
Bacto Lead Acetate Agar (Dehy-
drated) 1548
Lipman and Brown's Glucose Pep-
SUBGROUP II-C. SECTION 3
tone Agar 1549
Basal or complete media containing Waterman's Glucose Peptone Agar. 1550
agar and peptone (or other commercial Pfeiler and Lentz' Ringer Solution
digest); additional constituents, if any, of Agar 1551
known chemical composition. Harden's Glucose Peptone Agar 1552
Ai. Not containing additional materials. G2.* Disaccharides only added.
Jacobi's Peptone Agar 1530 Hi. Sucrose employed.
Molisch's Manganese Peptone Agar.. 1531 Greig-Smith's Sucrose Peptone
Roux and Rochaix' Peptone Agar 1532 . .
Agar 1553
Hesse and Niedner's Nahrstoff Hey- Owen's RawSugar Peptone Agar. . . 1554
den Agar 1533 Vierling's Sucrose Peptone Agar. . . . 1555
Lichtenstein's Yeast Extract Agar.. 1534 H2. Lactose employed.
A2. Containing additional materials. Levine's Eosine Methylene Blue
Bi. All additional materials inorganic. Agar (Dehydrated) 1556
Banning's Basal Salt Peptone Agar. 1535 Bacto Endo Agar (Dehydrated)
Matzuschita's Basal Sodium Chlo- Formula of Levine I557
ride Peptone Agar 1536 Levine's Eosine Methylene Blue
Glaessner's Nahrstoflf Heyden Agar. 1537 Agar 1558
Crendiropoulo and Panayotatou's Levine's Endo Agar 1559
Alkaline Peptone Agar 1538 Robin's Lactose Peptone Agar 1560
Stutzer and Hartleb's Phosphate Harvey's Lactose Peptone Agar 1561
Peptone Agar 1539 H3. Maltose employed.
Molisch's Salt Peptone Agar 1540 Sabouraud's Maltose Peptone Agar
B2. One or more of the added materials (Wurtz) 1562
organic. G3. Polysaccharides, only, added.
Ci.* Not containing nitrogen in addition to Molisch's Dextrin Peptone Agar. 1563 . . .
Di.* Only one type of additional organic Vierling's Starch Peptone Agar 1565
carbon supplied. Gibson's Starch Peptone Agar
El. Carbohydrates only added. (Harvey) 1566
Fi. One carbohydrate used. F2. Two or more carbohydrates used.
Gi. Monosaccharides only added. Bacto Eosine Methylene Blue Agar
Hi. Inorganic salts not added. (Dehydrated) 1567
Bacto Sabouraud's De.xtrose Agar Levine's Rosolic Acid China Blue
(Dehydrated) , 1541 Peptone Agar 1.568
Sabouraud's Glucose Peptone Agar E2.* Alcohols only added.
(Anderson) 1542 Sabouraud's Basal Glycerol Peptone
Hj. Inorganic salts added. Agar (Park, Williams & Krum-
Ii. Containing salts of monovalent cations wiede) 1569
only. Omelianski's Alcohol Peptone Agar. 1570
Matzuschita's Glucose Peptone Robinson and Rettger's Glycerol
Agar 1543 Opsine Agar 1571
Glaessner's Glucose Peptone Agar.. 1544 Hesse's Glycerol NahrstofT Heyden
Hucker and Wall's Glucose Peptone Agar.... 1572
Agar 1545 Molisch's Glycerol Peptone Agar. 1573
. .
Hesse and Niedner's Glucose Pep- Spengler's Glycerol Somatose Agar. 1574
tone Agar 1546 E3. Organic acids or their salts added.
Ij. Containing salts of trivalent cations. Omelianski's Formate Peptone Agar. 1575
Beijerinck's Glucose Peptone Agar. 1581 (5) Filter thru cotton, using compressed
Sarbouraud's Glucose Peptone Agar air to effect a fast filtration.
(Serena) 1582 (6) Distribute into smaller flasks.
Chantemesse's Phenol Peptone Agar (7) Distribute sterile (6) into sterile test
(Bezan5on) 1583 tubes that have been autoclaved in
Boekhout and de Vries' Maltose the autoclave for 2J hours. (The
Peptone Agar 1584 tubes are contained in an enamel
Cheyney's Maltose Peptone Agar... 1585 container in the autoclave. The
Sabouraud's Glycerol Peptone Agar 1586 inner temperature reaching about
Ea. Other combinations of organic carbon 150C.)
added. Sterilization: Heat (6) in streaming vapor
Robinson and Rettger's Glycerol for two hours (if the glassware used has
Opsine Citrate Agar 1587 been sterilized in the autoclave).
Botelho's Lacto-phenol Peptone Use: General culture medium.
Agar 1588
Reference: Jacobi (1888 p. 538).
Cj. Containing nitrogen in addition to
peptone.
1531. Molisch's Manganese Peptone Agar
Di. Inorganic nitrogen added.
Sullivan's Ammonium Lactate Pep- Constituents:
tone Agar 1589 1. Water (Moldau River) 1000.0 cc.
Lipman and Brown's Nitrate Pep- 2. Manganese peptone 0.5 g.
tone Agar 1590 3. Agar 10.0 g.
Beijerinck's Ferric Ammonium Ci- Preparation
Agar (Janke)
trate Peptone 1591
(1) Dissolve 2 and 3 in 1.
Vierling's Nitrate Peptone Agar 1592
(2) Pour in plates.
Heinemann's Asparagin Peptone Sterilization: Method not given.
Agar. 1593
Use: Cultivation of iron bacteria, Lepto-
Dj. Inorganic nitrogen not added; addi-
thrix ochracea.
tional organic nitrogen supplied.
Reference: Molisch (1910 p. 36).
MacConkey's Basal Bile Salt Pep-
tone Agar 1594
Harrison and van der Leek's Aescul- 1532. Roux and Rochaix' Peptone Agar
in Bile Salt Agar 1595
Constituents:
Harvey's Brilliant Green Bile Salt
1596 1. Distilled water 1000.0 cc.
Agar
2. Agar 25.0 g.
1530. Jacobi's Peptone Agar 3. Peptone 10.0 to 15.0 g.
Constituents Preparation
1. Water 1500.0 cc. (1) Soak 25.0 g. of chopped agar for 24
2. Meat peptone (Kem- hours in 500.0 cc. of water acidulated
merich's) 7.5 g. by the addition of 6.0% HCl. Stir
3. Peptone (siccum) 15.0 g. occasionally.
4. Agar-agar 15.0 to 22.5 g. (2) Wash thoroly with water.
CULTURE MEDIA FOR CULTIVATION OF MICRO ORGAXISMS 445
(3) Soak the agar for 24 hours in 500.0 Sterilization: Not specified.
cc. of a 5.0% ammonia solution. Use: The authors used the medium pri-
(4) Wash thoroly. marily for water analysis, but various
(5) Place in a liter of distilled water and investigators have used the same or
heat until the agar is dissolved. similar media for a large variety of
(6) Add 50.0 cc. of water containing 10.0 purposes.
to 15.0 g. peptone. Variants
(7) Neutralize by the addition of a (a) Wimmer used the medium with only
saturated solution of NaHCOa. 980.0 cc. water instead of 1000.0 cc.
(8) Pass thru flannel and then filter using to study nitrification.
a hot water funnel. (b) Klimmer used 8.0 g. Nahrstoff-
(9) Distribute into flasks or tubes. Heyden and 13.0 g. agar.
Sterilization: Sterilize at 115 to 120 for References: Hesse and Niedner (1898 pp.
30 minutes. 454-462), Wimmer (1904 p. 139), Smith
Use: General culture medium. (1905 p. 196), Tanner (1919 p. 50), Percival
Variants (1920 p. 121), Klimmer (1923 p. 194).
(a) Noyes dissolved 15.0 g. of the best
1534. Lichtenstein's Yeast Extract Agar
agar and 0.05 g. of Witte's peptone in
1000.0 cc. of water. Same as medium 518, but solidified by the
(b) Harvey dissolved 18.0 g. of agar and addition of agar.
peptone in 1000.0 cc. of water.
30.0 g.
1535. Banning's Basal Salt Peptone Agar
This medium was used to cultivate
hyphomycetes. Constituents
(c) Mortensen solidified medium 560 by 1. Water 1000.0 cc.
the addition of 2.0 or 2.5% agar. 2. KH.PO4 3.0 g.
(d) Moll used a 2.0% Witte peptone 3. MgS04 2.0 g.
solution solidified with 2.0% agar as 4. Peptone 10.0 g.
a basal medium and added a variety 5. Agar 10.0 g.
of inorganic salts to study the effect Preparation
of salts on the growth of molds. He (1) Dissolve 2, 3, 4 and 5 in 1.
reported that the toxicity of salts is (2) Do not adjust the reaction.
an additive characteristic of the (3) Dissolve one of the added nutrients
ion. The toxicity depends on the in (1).
solubility and the ionization of the Sterilization: Heat to 75C. to sterilize
salt. (method not given).
References: Roux and Rochaix (1911 p. Use: To study o.xalic acid formation by
115), Noyes (1916 pp. 93-94), Harvey Bad. xylinum, Bad. aceti oxalici and
(1921-22 p. 102), Mortensen (1909 p. 523), Bad. diabeticum. Author reported that
Moll (1920 p. 258). when nitrogenous materials were added
oxalic acid was not produced.
1533. Hesse and Niedner's Nahrstoff- Added nutrients: The author added one
Heyden Agar of the following materials:
Constituents: methyl alcohol, 10.0 g.
"
Preparation
intestinal tract. Different investigators
(1) Prepare ordinary nutrient agar using
used similar media to cultivate yeast,
water instead of bouillon. (Peptone
molds and other organisms.
and NaCl in amounts indicated were
Variants
assumed to be the constituents of
(a) BezanQon used agar instead of
18.0 g.
nutrient agar.)
20.0 g. The medium was used to
cultivate hyphomycetes, Sporotri- (2)Add 2.0% glucose to (1).
chum beurmanni Not specified.
Sterilization:
Use: Cultivation of mammalian and
(b) Harvey cultivated tineae, molds and
chicken tubercle bacilli. Author re-
eporothrix on a medium prepared as
ported that the mammalian types grew
follows:
poorly. Chicken types gave colonies of
(1) Prepare: Granulated peptone 10;
commercial glucose 40; agar 18;
1.5 mm. after six days.
Variants
water 1000.
(a) Truche and Cotoni solidified variant
(2) Raise slowly in the autoclave to a
(a) medium 596 by the addition of
temperature of 120C. and then
agar.
allow to fall to 100C.
(b) Harvey solidified medium 595 with
(3) Shake, to mix, at a temperature of
agar.
100 C.
References: Matzuschita (1899 p. 128),
(4) Have in readiness 2 one-liter flasks
Truche and Cotoni (1911 p. 480), Harvey
with funnels and thick, filter
(1921-22 p. 109).
paper.
(5) Place these flasks in the hot
1544. Glaessner's Glucose Peptone Agar
autoclave.
(6) Filter.
Same as medium 1537 but
contains 1.0%
(7) Keep the unfiltered medium hot. glucose. The author also described a
(8) Replace the funnels and filter medium the same as 1537, containing in
paper by new funnels with filter addition 10.0 g. peptone and 10.0 g. glucose.
paper as soon as filtration becomes
1545. Hucker and Wall's Glucose Peptone
slow.
Agar
Note: The filtration of this
medium is particularly difficult Constituents
and slow. 1. Water 1000.0 cc.
(9) Distribute the filtrate into test 2. Peptone 40.0 g.
tubes. 3. Glucose 2.0 g.
Note: The tubes should be 4. K2HPO4 5.0 g.
capped during incubation (which 5. Agar 15.0 g.
may be long) but may be placed Preparation
in a covered receptacle with its (1) Dissolve 2, 3, 4 and 5 in 1.
References : Anderson (1917 p. 343), Tanner organisms. Add 1.0 cc. of a 10.0%
(1919 p. 51), Bezangon (1920 p. 646), phenol and 1.0 cc. of a 1.0% (available
A blue
to the surface of the agar slant. Sterilization: Sterilize in the usual manner.
color developing within 30 minutes Use: Primarily used for differentiating
denotes ammonia production. Sal. paratyphi from Sal. schotmuelleri
Reference: Hucker and Wall (1922 p. 516), Used also in the study of other organisms
(1922 p. 485). of this group.
Reference: Digestive Ferments Co. (1925
1546. Hesse and Niedner's Glucose Peptone
p. 12).
Agar
Constituents
1549. Lipman and Brown's Glucose Peptone
Distilled water Agar
1. 1000.0 cc.
2. Agar 12.5 g. Constituents
3. Peptone (Gehe & Co.) 10.0 g. 1. Water (tap) 1000.0 cc.
4. De.xtrose 10 g. 2. Dextrose 10.0 g.
5. Sodium phosphate (ampho- 3. K2HPO4 0.5, 1.0 or 1.5 g.
teric) 0.5 g. 4. MgS04 0.2 g.
Preparation 5. Peptone 0.05 g.
(1) Dissolve 2, 3, 4 and 0.5 g. amphoteric 6. Agar 20.0 g.
reacting sodium phosphate in 1 by Preparation
boiling. (1) Dissolve 2, 4, 5 and 6 in 1.
(3) Cool to 50C. and clarify with egg Sterilization: Not specified.
albumin powder. Add albumin, boil Use: Bacterial count of cane sugar
and filter. products.
Sterilization: Method not given. Reference: Owen (1914 p. 338).
Use: General inexpensive culture medium.
1555. Vierling's Sucrose Peptone A^ar
Author reported that organisms retained
their characteristic growth, their patho- Same as 622 but solidified by the addition
genicity, their ability to produce pigment, of 2.0% agar.
their agglutinative ability and their
1556. Levine's Eosine Methylene Blue Agar
gram stain on this medium. (Messer-
(Dehydrated)
schmidt in Centr. f. Bakt., 68: 107-111
1913, compares this medium with meat Constituents:
extract agar and finds meat extract 1. Distilled water
medium far superior. This medium does 2. Peptone, Bacto 10.0 g
Preparation
3. Peptone (Collas) 20.0 g.
4. Sodium phosphate 0.4 g.
(1) Dissolve 2, 3 and 4 in 1.
5. Blue, soluble
Sterilization: Method not given.
6. Lactose
Use: Cultivation of purple bacteria.
40.0 g.
Preparation Reference: Molisch (1907 p. 11).
bacilli. Author reported that coli colo- 1565. Vierling's Starch Peptone Agar
nies colored the medium blue while ty-
phoid colonies were colorless. Constituents:
Reference: Robin (1897 p. 50). 1. Water 1000.0 cc.
2. K2HPO4 1.0 g.
1561. Harvey's Lactose Peptone Agar 3. CaClo 0.1 g.
4. MgS04 0.1 g.
Same as medium 607 but solidified by the 5. FeCls,. trace
addition of agar. 6. NaCl..,.., trace
7. Starch 5.0 g.
1562. Sabouraud's Maltose Peptone Agar
8. Peptone 10.0 g.
(Wurtz)
9. Agar
Constituents Preparation
1. Water..,., 1000.0 cc. (1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1.
2. Maltose ^, 38.0 g. (2) Solidify with agar (amount not
3. Peptone 5.0 to 8.0 g. specified).
4. Agar 14.0 g. Sterilization: Not given.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 453
(2) Filter, while hot, thru well-wetted sterile 5, 1.0 cc. of sterile 6, 1.0 cc. of
thick filter paper. 7 and 1.0 cc. of 8 per 100.0 cc.
(3) Make a suspension of 10.0 g. potato (6) Mix thoroly and pour in sterile
starch with a little of the hot filtrate plates.
and add to the bulk of the filtrate. Sterilization: Sterilize (4), method not
(4) Mix. given.
(5) Add sterile litmus solution to sterile Use: To isolate dysentery bacilli. The
(4) to give the desired color. author reported that the dyes such as
Sterilization: Sterilize (4) at 100C. on eosin, methylene blue, the fuchsin-
each of three successive days. sulphite indicator, and an excess of
Use: Detection of V. cholerae. Rosolic acid and china blue inhibited
Reference: Harvey (1921-22 p. 112). many dysentery types.
Variants: Harvey used 5.0 g. peptone
1567. Bacto Eosine Methylene Blue Agar
instead of 10.0 g.
(Dehydrated)
Reference: Levine (1920 p. 39), Harvey
Constituents: (1921-22 p. 88).
1. Distilled water
1569. Sabouraud's Basal Glycerol Peptone
2. Peptone, Bacto 10.0 g
3. Agar, Bacto 15.0
A^ar (Park, Williams & Krumwiede)
g
4. Lactose, Bacto 5.0 g Constituents
5. Sucrose, Bacto 5.0 g 1. Water 1000.0 cc.
6. K2HPO4 2.0 g 2. Peptone (1.0 to
7. Eosine 0.2 g 2.0%) 10.0 to 20.0 g.
8. Methylene Blue 0.05 g 3. Glycerol (0.5%) 5.0 g.
Preparation 4. Agar to solidify
(1) Dissolve 37.0 g. of Bacto Eosine Preparation
Methylene Blue Agar (dehydrated) (1) Dissolve 2, 3 and one of the added
in 1000.0 cc. water by boiling or nutrients in 1.
autoclaving. (2) Solidify by the addition of agar.
Sterilization: Sterilize in the usual manner. (3) Do not adjust the reaction.
Use: Isolation of Esch. coli, Esch. Ebert, Sterilization: Not specified.
Esch. typhi, etc. Use : Cultivation of molds.
454 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents
1571. Robinson and Rettger's Glycerol 1. Water, Moldau 1000.0 cc.
Opsine Agar 2. Agar 18.0 g.
1. Distilled water 1000.0 cc. (2) Add 0.0, 2.0 or 5.0 g. of lactic acid or
2. Peptone 30.0 g. 0.0, 1.0, 2.0 or 5.0 g. NaOH to obtain a
3. Lactose 20.0 g. neutral acid or alkaline reaction.
4. Agar 20.0 g. Sterilization: Not specified.
5. Phenol Use: Cultivation of Bacillus fuchsinus.
6. Litmus Author reported that the colonies were
Preparation first red and then a metallic sheen de-
sterile (1).
1585. Cheyney's Maltose Peptone Agar
Use: Differentiation of colon-typhoid
group. Bezanfon reported that the colon Constituents
colonies were red, typhoid colonies color- 1. Distilled water 1000.0 cc.
less. 2. Agar (2.0%) 20.0 g.
did Bezangon but added just before use, 4. Peptone (1.0%) 10.0 g.
solution was prepared as follows: (2) Filter thru a thin layer of cotton.
(1) Grind up litmus in a mortar. (3) Add 1.0% maltose and 1.0% peptone.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 457
While hot add 5.0 g. of sodium tal violet, per 100.0 cc. of the
(5)
taurocholate, 10.0 g. of lactose and filtrate.
10.0 cc. of a 5.0% watery solution (6) Tube in sterile tubes in 10.0 cc.
of neutral red. quantities.
460 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(7) Sterilize by heating 20 minutes on (4) Filter while hot thru cotton wool.
3 successive days. (5) Add 1.0% lactose dissolved in a
(h) Levine gave the following medium as little water and 1.0% Andrades
Bile Salt (Rebipel) Agar (After indicator.
Savage): Sodium taurocholate 5 (6) Sterilize intermittently.
grams, Witte's peptone 20 grams and References: MacConkey (1900 p. 20), (1901
distilled water 1 liter, are boiled up p. 740), Frost (1903 p. 342), MacConkey
together, 20 grams of agar are added (1905 pp. 334, 336), (1908 p. 325), Abel
and dissolved in the solution in the (1912 p. 227), Ball (1919 p. 81), Tanner
autoclave in the ordinary way. The (1919 p. 49), Percival (1920 p. 307),
medium is cleared with white of egg Harvey (1921-22 p. 89), Klimmer (1923
and filtered. After filtration, 10 p. 214), Levine (1921 p. 116), Cunningham
grams of lactose and 5 cc. of recently (1924 p. 98).
prepared per cent neutral red solu-
1
1595. Harrison and VanderLeck's Aesculin
tion are added. The medium is then
Agar
Bile Salt
tubed and sterilized for 15 minutes on
three successive days. Constituents:
(i) Harvey gave the following method 1. Water 1000.0 cc.
of preparation 2. Peptone (Witte (1.0 or
(1) Dissolve 20.0 g. peptone, 5.0 g. 2.0%) 10.0 or 20.0 g.
sodium taurocholate in 1000.0 cc. 3. Sodium taurocholate
tap water and make faintly alkaline (Commercial) (0.5%) 5.0 g.
to litmus. 4. Aesculin (0.1%) 1.0 g.
(2) Steam 45 minutes. 5. Iron citrate (0.05%) 0.5 g.
(3) Add 15.0 g. powdered agar, making 6. Agar 15.0 g.
it into a paste or suspension, Preparation
before addition, with alittle of the (1) Dissolve the agar in part of 1.
taurocholate peptone solution. (2) Add 2, 3 and 4 to the rest of 1.
(4) Steam gently 2^ hours to bring the (3) Mix (1)and (2).
agar thoroughly into solution. (4) Boil and filter (may be clarified with
(5) Bring the volume up to 1000.0 cc. egg white).
by the addition of water. (5) Tube.
(6) Cool to 60C., clarify by the addi- Sterilization: Sterilize by steaming on 3
tion of egg and filter. successive days or autoclaving at 15
(7) Dissolve 10.0 g. lactose (or other pounds for 15 minutes.
sugar if desired) in 15.0 cc. sterile Use: Presumptive test for B. coli in water
water and steam 15 minutes. analysis. Author reported that B. coli
(8) Add the lactose solution to the colonies were black, typhoid colonies
hot, clear, nutrient agar. produced no blackening.
(9) Add 5.0 cc. freshly prepared sterile Variants
1 per cent neutral red by means (a) The authors used 0.25% commercial
of a sterile pipette. bile salt, 1.0% Witte's peptone and
(10) Distribute the resulting agar, 0.1% iron citrate instead of amounts
which is deep red, into flasks or used above.
test tubes. (b) Levine gave Eyre's method of prep-
(11) Steam 25 minutes. aration as follows:
(j) Cunningham prepared the medium as (1) Measure out 400.0 cc. distilled water
follows: into a tared 2 liter flask.
(1) Steam 5.0 g. sodium taurocholate (2) Weigh out 15 grams agar, 10 grams
and 20.0 g. peptone in 1000.0 cc. peptone, 5 grams sodium taurocholate
water for one hour. and make into a thick paste with 150.0
(2) Filter. cc. distilled water.
(3) Add 1.5% agar and steam for one (3) Add this paste to the distilled water
hour. in the flask.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 461
7. 1-1000 Brilliant-green 20.0 cc. Marpmann's Flour Casein Agar .... 1610
8. Picric acid (1.0%) 20.0 cc. Williams and Povitzky's Flour Pep-
Preparation tone Agar 1611
<1) Dissolve 2, 3, 4, 5, 6, 7 and 8 in 1000.0 Otabe's Wheat Peptone Agar 1612
cc. of water. Dawson's Flour Peptone Agar 1613
(2) Reaction to be 1.5% acid to phenol- Plaisance and Hammer's Stover
phthalein. Infusion Peptone Agar 1614
Sterilization: Method not given. C2. Potato derivatives used.
Use: Enrichment medium for colon- De Gaetano's Potato Extract Agar . . 1615
typhoid group. Jochmann's Potato Bouillon Agar.. 1616
Reference: Harvey (1921-22 p. 91). Nicholle and Alilaire's Potato In-
fusion Agar 1617
SUBGROUP II-C. SECTION 4
Shiga et al. Potato Blood Agar 1618
Basal or complete media containing agar Gaehtgen's Potato Peptone Agar. . . 1619
and peptone (or other commercial digest) C3. Derivatives of legumes used.
together with additional constituents of Matzuschita's Pea Bile Agar 1620
unknown chemical composition of plant or Tanner's Pea Extract Tryp. Agar... 1621
soil origin. Behrens' Pea Blood Agar 1622
Ai. Containing yeast or yeast derivatives. De Rossi's V. Faba Infusion Pep-
Bi. All additional constituents of known tone Agar 1623
chemical composition. C4. Miscellaneous plant derivatives used.
Gassner's Yeast E.xtract Peptone Owens' Molasses Peptone Agar 1624
Agar 1597 Seiffert and Bamberger's Chloro-
Cohen and Clark's Yeast E.xtract phyll Bouillon Agar 1625
Peptone Agar 1598 Dawson's Edestin Peptone Agar. 1626 . . .
462 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
A4. Containing soil or its derivatives. 1600. Sherman's Yeast Extract Peptone
Temple's Soil Infusion Peptone Agar
Agar 1627
Constituents:
1. Water 1000.0 cc.
1597. Gassner's Yeast Extract Peptone
2. Peptone 20.0 g.
Agar Yeast
3. 10.0 g.
(1) Exact method of yeast extract water Sterilization: Method not given.
not given. Use: Isolation of organisms causing flavors
Mix 1 and in cheese.
(2) (1).
1602. Sturtevant's Egg Yolk Yeast Agar (16) Adjust the reaction of (13) so that
upon the addition of 1.0 cc. of (15)
Constituents:
to 10.0 cc. of the agar, the final pH
1. Water 1000.0 cc.
value will be about 6.8.
2. Yeast (dry) 10.0 g.
(17) Tube the agar (without the addition
3. Peptone 10.0 g.
of egg yolk suspension) in 10.0 cc.
4. Sodium glycero-phosphate
lots.
(buffer)....' 5.0 g.
(18) When desired for use add 1.0 to 2.0
5. Agar 15.0 g.
cc. (15) to each tube of melted
of
6. Egg yolk
agar under aseptic conditions, mix
Preparation
M'ell and slant. The agar should be
(1) Heat 10.0 g. dry yeast, 10.0 g. pep-
cooled to about 55C.
tone and 5.0 g. buffer (sodium glyc-
Sterilization: Sterilize (14); method not
ero-phosphate) in 500.0 cc. of water
given. Sterilize (17) by autoclaving at
with flowing steam for 30 minutes.
15 pounds pressure for 15 minutes.
(2) Adjust to pH = 7.6 to 7.8.
Use: Isolation of organism causing
(3) Boil for one minute over a free
American foul brood, Bacillus larvae. To
flame.
isolate the organism, drop some infected
(4) Filter thru paper on a perforated
material into the water of condensation
porcelain funnel, using siliceous
and smear over the surface of the agar.
earth to clarify.
Author reported that best growth was
(5) Place 15.0 shred agar in 1000.0
g. of
obtained when 0.5% glucose was added.
water and allow to stand
cc. distilled
To make counts, add only 10 or 15 drops
for 24 hours at room temperature.
of the egg yolk suspension, or 1.0 cc. of
(6) Pour off as much of the water as
the supernatant fluid of the suspension
possible by placing a piece of cheese
to 10.0 cc. yeast extract agar containing
cloth over the top of the flask.
varying amounts of glucose. Mix thoroly
(7) Add as much distilled water as was
and pour in plates. Add a dilute solution
poured off and soak for another 24
of fuchsin to the plates when counting.
hours.
This colors the colonies.
(8) Filter thru a cotton flannel cloth,
Variants: Author added from 0.5 to 10.0%
and wash the agar with 500.0 cc. of
glucose.
distilled water.
Reference: Sturtevant (1924 p. 136).
(9) Allow to drain, and squeeze the agar
as free from water as possible. and Savini-Gastano's
1603. Savini
(10) Add enough water to (9) so that the Hemoglobin Bacterial Extract
total weight will be 515.0 g.
Agar
(11) Dissolve the agar in the water by
heating. Same as medium 977 but agar employed
instead of bouillon.
(12) Filter.
(13) Mix equal parts of (4) and (12)
1604. Mankowski's Fungus Infusion
(double strength agar).
Peptone Agar
(14) Prepare a series of flasks containing
200.0 cc. of water. 0.5 to 1.0% Constituents:
neutral buffer salt may be added to 1. Fungus infusion 1000.0 cc.
keep the acidity down which 2. Agar 15 g.
Use: Differentiation of typhoid bacilli and 1606. Plaisance and Hammer's Corn Juice
Bacterium coli communis. Author re- Peptone Agar
ported that Bacterium coli grew in a
Constituents
silver white, solid and dry film. Typhoid
1. Corn juice 1000.0 cc.
bacilli grew slower, appeared as trans-
2. Agar 15.0 g.
parent, shining, damp strips. When 3. Peptone 10.0 g.
fuchsin indigo carmine be added typhoid
Preparation
bacilli colored the blue medium red,
(1) Obtain corn juice by pressing green
Bacterium coli communis gave a bluish
corn.
green color and finally completely de-
(2) Dissolve 2 and 3 in (1).
colorized the medium.
(3) Adjustment of reaction not specified.
Variants
Sterilization: Not specified.
(a) The author prepared a medium con-
Use: Study mannitol producing organisms
taining fuchsin and indigo carmine
from silage.
by continuing from step (4) above as
Reference: Plaisance and Hammer (1921 p.
follows:
432).
(5) Prepare a saturated solution of
acid fuchsin in a 1.0% solu- KOH 1607. Peglion's Grape Must Peptone Agar
tion (acid fuchsin may be added Constituents
to a 1.0% KOH solution until a 1. Grape must 500.0 cc.
dark black brown color is reached). 2. Peptone 10.0 g.
(6) Prepare a watery saturated solu- 3. Ammonium phosphate 2.0 g.
tion of indigo carmine. 4. Glycerol 10.0 cc.
(7) Add 2.0 cc. of (2) and 1.0 cc. of 5. Agar 10.0 g.
(3) to 22.0 cc. of distilled water. Preparation
This solution is dark blue and Dissolve 4 and 5 in 1.
(1) 2, 3,
reaction slightly alkaline.
(2) Pour into petri dishes or plates.
(8) Add (7) drop by drop to (4) until Sterilization: Not specified.
the agar is colored blue and then Use: Cultivation of Saccharomyces ellip-
violet blue.
soideus.
(9) Distribute into test tubes. Reference: Peglion (1898 p. 477).
(10) Add to each test tube a drop of
watery
saturated solution of
1608. Harvey's Banana Agar
indigo carmine. Constituents:
(b) Wiegert solidified medium 701 by the 1. Agar (nutrient) 1000.0 cc.
addition of agar. 2. Banana (10.0%) 100.0 g.
References: Mankowski (1900 p. 23), Preparation
Wiegert (1921-22 p. 110). (1) Use banana in the form of cut cylinder
or as 10.0% pulp incorporated with
1605. Owens' Cane Juice Peptone Agar
nutrient agar. (Medium 779 variant
Constituents: (bb) solidified by the addition of
1. Cane juice 1000.0 cc. agar.)
2. Agar (2.0%) 20.0 g. Sterilization: Not specified.
3. Peptone (1.0%) 10.0 g. Use: General culture medium.
Preparation Reference: Harvey (1921-22 p. 119).
(1) Heat fresh raw cane juice.
1609. Meacham et al. Malt Extract Agar
(2) Filter thru cotton.
(3) Add 1.0% peptone (peptone may be Constituents
omitted). 1. Malt extract (2.5%) agar. . . . 1000.0 cc.
(4) Solidify by the addition of 2.0% agar. 2. K2HPO4 M/50
Sterilization: Not specified. 3. Acetic acid N/50
(1) Preparation of
potato infusion not Preparation
given. (1) Extract 500.0 g. of
finely chopped beef
Potato 125.0 g.
to stand in a glass flask at about
2.
1622. Behrens' Pea Blood Agar (4) The reaction is slightly acid or
alkaline.
Constituents
Sterilization: Not specified.
1. Distilled water 1000.0 cc.
Use: Cultivation of bacteria found in the
2. Peas 10.0 g.
nodules of leguminous plants.
3. Peptone 20.0 g.
Variants: The author used 2.0% glucose
4. NaCl 5.0 g.
instead of sucrose.
5. CaClj 0.1 g.
Reference: de'Rossi (1907 p. 301).
6. N/1 NajCO, soln 10.0 cc.
7. Agar 20.0 g.
1624. Owen's Molasses Peptone Agar
8. Blood (rabbit) 2000.0 cc.
Preparation: Constituents
(1) Boil 10.0g. peas in 1000.0 cc. distilled 1. Water 1000.0 cc.
water to obtain extract. Length of 2. Molasses (Final 75 Brix) . . 160.0 g.
boiling not specified. 3. Agar 20.0 g.
(2) Strain, boil the extract and filter. 4. Peptone 10.0 g.
(3) Dialyze the filtrate in a large collo- Preparation
dium sac against running distilled (1) Dissolve 160.0 of final molasses,
g.
water for 24 to 48 hours. 75 Brix, 20.0 agar and 10.0 g. pep-
g.
(6) Distribute in test tubes in 1.0 cc. lots. Use : Bacterial counts of organisms found in
(7) Shortly before use the desired number corn juice and cane sugar products.
of sterile agar tubes are melted in Reference: Owen (1914 p. 338).
the water bath, cooled to 60C. and
1625. Seiffert and Bamberger's Chlorophyll
two volumes of defibrinated rabbit's
Bouillon Agar
blood are added.
(8) Mix well and solidify in slanting Constituents
position. 1. Neutral agar 1000.0 cc.
Sterilization: Sterilize by autoclaving at 2. Chlorophyll solution ("So-
105 to 108C. for 15 minutes. lutio spirituosa," Merck)... 25.0 cc.
(10) Add about 15.0 cc. of a 10.0% sterile Ai. Animal cells and tissues or their deriva-
Na2S0j solution to decolorize (9). tives added.
(11) Pour in sterile plates. Rivers and Kohn's Basal Blood Cell
(12) Dry the plates in the air. Peptone Agar 1628
Sterilization: Method of sterilization of Krumwiede, Pratt and Grund's Egg
(4), (5) or neutral agar not given. Peptone Agar 1629
Use: Selective medium for cholera. Au- Weiss' Nahrstoff Heyden Gelatin
thor reported that the cholera colonies Agar 1630
were red. Other organisms were inhibited. Deycke's Alkaline Albumin Gelatin
Reference: Seiffert and Bamberger (1916 p. Agar 1631
288). Deycke's Albuminate Glycerol
Agar :.... 1631a
1626. Dawson's Edestin Peptone Agar
Krumwiede and Pratt's Gelatin
Constituents: Serum Agar 1632
1. Water 1000.0 cc. Dunschmann's Bile Salt Peptone
2. Peptone 5.0 g. Agar (Bezanyon) 1633
3. Edestin 5.0 g. Capaldi's Peptone Gelatin Agar 1634
4. Agar 20.0 g. Smyth's Egg Trypsinized Peptone
Preparation: Agar 1635
(1) Dissolve 2, 3 and 4 in 1, Aj. Animal fluids added.
(2)Reaction to be alkaline. Bi. Blood employed.
Method not given.
Sterilization: Czaplewski's Blood Nahrstoff Hey-
Use: Culture medium to study variation of den Agar (Klimmer) 1636
B. coll. Norris et al. Blood Peptone Agar. 1637 . .
Use: Cultivation and preservation of stock (c) Weiss made bacterial counts of water
cultures of fusiform bacilli. in a medium prepared as follows:
Variants: The authors substituted ascitic (1) Dissolve 15.0% gelatin and 1.0%
that coli colonies were large milky colored solution and digest for 3 hours at
by reflected and brown by transmitted 40 to 45 C.
light. Typhoid colonies were small, (6) Dilute (5) to one liter with Ringer's
glistening and colorless. Abt used a solution (see medium 180) and boil
similar medium to produce anthrax for 20 minutes and replace loss with
spores. distilled water.
specified by Capaldi and omitted the (9) Dissolve 15.0 g. agar in one liter
KCl. Ringer's solution in the Arnold
(b) Tanner used either 10.0 g. glucose or steam sterilizer or autoclave
mannitol. (10) Clarify with egg.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 473
(5) Add chloroform to each bottle until and streptococci developed abnormally
0.5% chloroform has been added. large colonies on this medium.
(6) Add a drop of sterile oil to the stop- Reference: Fildes (1917 p. 492).
6. Agar to solidify
test sterility.
Prepare a 2.5 to 3.0% agar solution in 7. Blood (defibrinated ox)
(16)
water containing salt and Morson's 8. Serum (ox)
this mixture to 400.0 cc. of (16) and to 250.0 cc. of ox serum obtained
References: Tochtermann (1895 p. 965), blood cell extract was added. Otherwise
Kolle and Wassermann (1912 p. 406), Bes- no growth.
son (1920 p. 53), Harvey (1921-22 p. 79), Reference: Teague and Deibert (1922
Klimmer (1923 p. 221). p. 70).
1646. Browning's Telluric Acid Serum Agar 1648. Emlle-Weil's Pleuritic Serum
(Wood) Peptone Agar
Constituents Constituents:
1. Peptone water agar 1000.0 cc. 1. Water 1000.0 cc.
2. Serum, sheep 50.0 cc. 2. Peptone (Chapoteaut) 20.0 g.
3. Telluric acid (1.0%) 9.0 cc. 3. Glucose 8.0 g.
Preparation: (1) Add 50.0 cc. of sterile 4. Glycerol 20.0 g.
eheep serum, and 9.0 cc. of a 1.0% solution 5. Agar 24.0 g.
of telluric acid in distilled water to 6. Pleuritic serum (human) . . 250.0 cc.
1000.0 cc. of peptone water agar. Preparation
Sterilization : Sterilize the serum by heating (1) Dissolve 2, 3, 4 and 5 in 1.
(6) Centrifuge (5) and obtain the super- 5. Peptone, Bacto 20.0 g.
natant fluid from (6) and (7) and add Agar (Dehydrated) in 1000.0 cc. dis-
an agar solution containing 5.0 g. tilled water by boiling or autoclaving,
NaCl and 20.0 g. agar per 1000.0 cc. preferably the latter.
Sterilization: Not specified. (2) Restore the loss by the addition of
Sterilization: Sterilize in the autoclave at (2) Dissolve 10.0 g. lactose and 10.0 g.
15 pounds pressure for 20 minutes. peptone in 1000.0 cc. of (1).
Use: General culture medium, and in water (3) Dissolve 15.0 g. of agar in (2).
Variants
water (method not given).
(a) Ecker specified the use of Difco pep- (2) To 100.0 cc. of one add 0.5 cc. of a
tone, used 10.0 g. lactose as an added 1.0% aqueous solution of malachite
nutrient and used the medium to green and 0.5 cc. of bile.
study effect of bile on growth of (3) Add 0.75 cc. to 1.0 cc. of a 10.0%
(4) Dissolve 20.0 g. lactose in 400.0 cc. Sterilization: Sterilize at 15 pounds pres-
hot water. sure for 20 minutes.
(5) Add (4) to (3). Use: Culture medium, particularly for bac-
Add the contents of two ox sheep bile teria in dairy products.
(6)
(about 60.0 cc). The bile having Reference: Digestive Ferments Co. (1925
been removed under aseptic condi- p. 14).
tions.
1654. Sabouraud and Noire's Whey Peptone
(7) Add 1.0% of a standard solution of
Agar (Weil and Noire)
Merck's litmus (Brown,
purified
C. W., Litmus media, 47th Ann. Rep., Constituents
Michigan Board of Agriculture, pp. 1. Water 500.0 cc.
(7) Add (4) and (6) to the filtrate of (5). Use: Cultivation of gonococcus.
(8) Mix well. Variants Bezangon prepared the medium
:
(10) Autoclave for 10 minutes at (1) Boil 1000.0 cc. of milk for 5 minutes.
pounds pressure.
12 (2) Add 2.0 cc. of HCl.
References: Northrup (1912 p. 420), Rector (3) Filter thru linen.
(1913p. 154),Stitt(1923p. 47). (4) Add one-half the volume of water to
the whey.
1653. Bacto Whey Agar (Dehydrated) (5) Neutralize by the addition of a
Constituents 10.0% soda solution.
1. Distilled water 1000,0 cc. (6) Autoclave (5) for 10 minutes at
2. Whey (dry) 13,0 g. 120C.
Agar 1747
Wilson and Darling's Brilliant
Robinson and Rettger's Opsine In- Green Bile Salt Agar 1770
enous compound of known chemical com- E2. Organic nitrogen not added.
position. Fi. Containing carbohydrates and alcohols.
Lubenau's Lactose Caffeine Agar.. 1749 Bacto Saccharose-Mannitol Agar
Harvey's Caffeine Endo Agar 1750 (Dehydrated) 1772
Gathgen's Caffeine Fuchsin Sulphite Hesse's Lactose Glycerol Agar
Agar (Bezangon) 1751 (Stokes and Hachtel) 1773
C2.* Extracts specified. Schniirer's Saponin Glycerol Agar. 1774 .
Di. Only one additional source of carbon F2. Containing carbohydrates, without
added. alcohols.
El. Carbohydrates employed. Gi. Containing two carbohydrates.
Fi. Monosaccharides, only, added. Bacto Russell Double Sugar Agar
Viehoever's Basal Glucose Extract (Dehydrated)., 1775
Agar 1752 Nichols and Woods' Russell Double
Bacto Dextrose Agar (Dehydrated) . 1753 Sugar Agar 1776
Henneberg's Glucose Extract Agar. . 1754 Bailey and Lacey's Phenol Red Lead
Oldekop's Neutral Red Glucose Ex- Acetate Agar ^. 1777
tract Agar 1755 Holt-Harris and Teague's Eosine
F2. Disaccharides, only, added. Methylene Blue Agar 1778
Bacto Purple Lactose Agar (Dehy- Krumwiede, Pratt and McWilliams'
drated) 1756 Brilliant Green Agar 1778a
Bacto Litmus Lactose Agar (Dehy- Aronson's Fuchsin Sulphite Agar. 1779 . .
Agar 1782
* See C3, page 484. Amoss' Four Sugar Agar 1783
484 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Amoss' Sucrose Salicin Agar (Top- Penf old's Monochlorhydrin Agar. 1814 . .
Fildes' Blood Digest Agar (Kristen- Harvey's Telluric Acid Serum Agar. 1923
sen) 1891 Francis' Cystine Serum Agar (Stitt) 1924 .
Wolbach, Chapman and Stevens' Ci- F4. Animal than blood, serum,
fluids other
trated Blood Agar 1898 or ascitic fluid employed.
Noguchi's Serum Plasma Agar 1899 Steinschneider's Hydrocele Fluid
Soparkar's Citrated Blood Agar Agar 1932
(Liston) 1900 Heiman's Pleuritic Serum Agar 1933
Hirsch and McKinney's Chocolate Lentz and Tietz's Malachite Green
Agar 1901 Bile Agar (Klimmer) 1934
Harvey's Glucose Blood Agar 1902 E3. Containing animal secretions, excre-
H2. Blood neither citrated nor oxalated. tions or their derivatives.
Grassberger's Blood Agar 1903
Fi. Milk or its derivatives employed.
Ruediger's Blood Agar 1904
Gi. Nutrose used.
Dieudonne's Alkaline Blood Agar
Lentz and Tietz's Malachite Green
(Tanner) 1905
Nutrose Bile Agar (Klimmer) . . . 1935
Sherwood and Downs' Glucose
V. Drigalski and Conradi's Crystal
Blood Agar 1906
Violet Nutrose Agar 1936
Hall's Testicular Infusion Blood
Galli-Vallerio's Neutral Red Nu-
Agar (Stitt) 1907
trose Agar 1937
Cutler's Blood Clot Infusion Agar
Teague and Travis' Eosin Bismark
(Klimmer) 1908
Brown Nutrose Agar 1938
Harvey's Trypsinized Blood Agar. 1909 .
Kutscher's Serum Placenta Agar.. 1917 Costa and Boyer's Gum Infusion
BezanQon's Serum Placenta Agar. . . 1918 Agar 1948
Shamine's Nucleic Acid Serum Agar. 1919 Tulloch's Ox Heart Infusion Pea
Robey, et al., Glucose Serum Agar. . 1920 Flower Agar 1949a
Noguchi's Ringer Solution Serum D2. Not containing additional materials of
Agar 1921 unknown chemical composition of animal
Todd's Lactose Serum Agar 1922 origin.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 487
C3. Whether infusions or extracts were em- G2. At least one additional material or-
ployed not definitely specified. ganic.
Di. Containing tissues, cells or their Mandelbaum's Lactose Blood Agar. 1977
derivatives. Thompson's Glucose Plasma Agar. . 1978
El. Egg employed. Avery's Oleate Blood Agar (Stitt). 1979
Fi. Yolks only used. Esch's Ascitic Fluid Blood Agar 1980
Capaldi's Egg Yolk Agar 1949 Liston's Trypsinized Casein Blood
Nastiukoff's Egg Yolk Nutrient Agar 1981
Agar (Rechtsamer) 1950 F2.* Whole blood used.
Bezangon and Griffon's Glycerol Gi. Blood citrated or oxalated.
Egg Yolk Agar 1951 Bernstein's Basal Blood Agar 1982
Pergola's Tellurite Egg Yolk Agar. 1952 . Wordley's Oxalated Blood Agar 1983
F2. Whites of eggs only used. Wilson and Darling's Laked Blood
Lipschutz's Egg Albumin Agar 1953 Agar 1984
Krumwiede, Pratt and Grund's Egg Wilson and Darling's Lactose Blood
Albumin Agar 1954 Agar 1985
Oberstadt's Egg Albumin Agar 1955 Stitt's Glycerol Blood Agar (Choco-
F3. Whole egg (white and yolk) used. late Agar) 1986
Besredka and Jupille's Egg Agar Besson's Citrated Blood Agar 1987
(Besson) 1956 G2. Blood neither citrated nor oxalated.
Scales' Whole Egg Agar 1957 Hi. Additional constituents, if any, inor-
Stitt's Glycerol Egg Agar 1958 ganic, (exclusive of dyes).
Robertson's Alkaline Egg Agar Bezangon, Griffon and LeSourd's
(Park, Williams and Krumwiede) 1959 .
Blood Agar 1988
E2. Tissues or their derivatives other than Fleming's Brilliant Green Blood
eggs employed. Agar 1989
Fi. Tissue used. Hachla and Holobut's Alkaline
Cantani'juns' Sperm Agar 1960 Blood Agar 1990
Pettersson's Brain Ascitic Fluid Matthews' Trypsinized Blood Agar. 1991
Agar 1961 H2. At least one of the added constituents
Smith and Taylor's Fetus Agar 1962 organic.
Noguchi's Ascitic Fluid Tissue Agar . 1963 Bieling's Glucose Blood
Agar 1992
Gozony's Kidney Agar 1964 Mandelbaum and Heinemann's
Duval's Trypsinized Tissue Agar. . . 1965 Glycerol Blood Agar (Kolle and
F2. Tissue derivatives used. Wassermann) 1993
Gi. Containing gelatin.
Wassermann's Nutrose Blood Agar
Thoinot and Masselin's Gelatin Agar (Abel) 1994
. 1966
Fremlin's Phosphate Gelatin Agar. Savini and Savini-Castano's Bac-
. 1967
teriaBlood Agar 1995
G2. Not containing gelatin.
Bieling's Optochin Hydrochloride
Fat Agar
Vierling's 1968
Blood Agar 1996
Abe's Meat Water Infusion Agar. 1969 . .
Joas' Alkaline Serum Agar (Klim- (3) Heat over a free flame until the ma-
mer) 2004 terials are completely dissolved.
Muhlen and Hoffman's Glucose (4) Make up to the original volume by
Serum Agar (Stitt) 2005 the addition of water.
Cantani's Glycerol Serum Agar (5) Add Na2C03 or sodium phosphate
(Besson) 2006 until the reaction is slightly alkaline.
Kodama's Fuchsin Sulphite Serum (6) Pour into a flask and steam until
Agar 2007 the albuminous material still to be
Wassermann's Nutrose Serum Agar. 2008 removed is completely separated
Kliglers' Nasal Secretion Serum (usually 2 hours if sodium phosphate
Agar 2009 is used and longer if Na2C03).
Meyers' Tuberculin Agar 2010 (7) Filter thru cotton, using compressed
Douglas' Tellurite Trypsinized Se- air to effect a fast filtration.
rum Agar 2011 (8) Distribute into smaller flasks.
Czaplewski's Alkaline Serum Glu- (9) Heat in streaming steam for 2 hours
cose Agar 2012 (if the glassware has been sterilized
(10) Place in a steamer for 12-15 hours. (5) Dissolve 5.0 g. NaCl and 10.0 g.
(12) The steaming for 12-15 hours is (6) Boil 15 minutes, preferably over
sufficient for sterilization. a free flame.
(b) Schultz (1891). (7) Add 10.0 to 20.0 g. agar to (6) and
Place 3 to 4 liters of distilled boil until dissolved.
(1)
water in an enamel container and (8) Neutralize if necessary and add
add 20.0 g. finely cut up agar- glycerol if desired.
glass container fitted with a lid. lean meat to 1000.0 cc. of water
Store in a cool place until the (2) Strain thru a cheese cloth or a
(5)
ne.xt day. coarse towel and squeeze in a
(10) Neutralize (indicator not speci- (6) Cool the autoclave at 100C. be-
fied). fore opening.
(11) Filter. (7) Cool to 75C.
(12) Sterilization not specified. (8) Mix (7) and (3).
(f) Jensen (1898) studied denitrification (9) Add 10.0 g. peptone and 5.0 g.
using the following medium: NaCl to (8).
(1) Prepare a meat infusion from (10) Boil for 3 to 5 minutes.
500.0 g. of meat and 1000.0 cc. (11) Neutralize (indicator not speci-
of water. fied).
(g) Committee A. P. H. A. (1899). (1) Cut lean beef into small pieces in
(3) Add 1.0% peptone and 0.5% NaCl (4) Make up the loss of water and
to the filtrate. Heat until solu- filter thru a filtering cloth.
utes and boil for 5 minutes over a (8) Cool (in a closed container) and
free flame. filter.
(6) Filter while hot thru paper or cot- (9) Distribute in 300 to 500.0 cc. por-
ton and cloth, and add 1.0 to tions in clean flasks with pat-
2.0% agar to filtrate. Dissolve ented sealers.
by boiling or autoclaving. (10) Sterilize in streaming steam for
(7) Add N/1 HCl to the filtrate to ob- one hour.
tain the desired reaction (+1.5). (11) Add 1.5% agar to (10).
(8) U the medium is clear distribute (12) Bring to a boil on a concentrated
in tubes or flasks. If not clear, salt solution bath and boil for
one egg to the agar cooled to 50 (13) Take 30.0 cc. of (12) add a drop
of alcoholic phenolphthalein solu-
to 60C., and then boil vigor-
ously. Filter. tion and add N/1 sodium solution
until a red coloration is formed.
(9) Sterilize by the fractional
either
(14) Estimate the amount (12) and
or continuous method.
calculating from (13) add f the
(h) Ravenel (1899).
amount of sodium solution re-
(1) Mi.x 500.0 g. of freshly chopped
quired to give neutralization.
meat with 500.0 cc. of water.
(15) Keep in the autoclave or hot
(2) Allow to stand in a cool place
water for 15 minutes.
over night.
(16) Filter.
(3) Strain thru a towel. Tube.
(17)
(4) Chop agar into small pieces and (18) Sterilize (exact method not spec-
add to 500.0 cc. water. ified).
(2) Add 10.0 g. of finely cut agar to (10) Cool to 90C. and allow to remain
1000.0 cc. of (1). at that temperature for several
<3) Allow to stand until the agar is hours.
completely soaked up. The time (11) Decant the clear agar and filter
(9) Then add a 36B. soda solution (8) After the last washing allow the
drop by drop until the red litmus agar to drain for 10 minutes.
is turned blue. (9) Add 500.0 cc. of (4), 10.0 g. pep-
(10) Add 10.0 g. agar and dissolve by tone and 5.0 g. NaCl to the
heating. washed agar.
(11) Heat at 115 to 120 in the auto- (10) Autoclave at 115C. for 30 min-
clave. utes or in the steamer for 90 min-
(12) A precipitate forms which com- utes.
pletely clears the medium. (11) Make (10) up to a liter by the
(13) Filter and distribute in tubes. addition of (4).
(14) Sterilize at a temperature lower (12) Make slightly alkaline to litmus
than in (11). by the addition of KOH.
(q) Walbaum (1910). (13) Cool to 60C. and add the beaten
(1)Method of preparation of meat white of an egg.
infusion not given. (14) Heat in the autoclave for 45 min-
(2) Add 100.0 g. of finely cut agar to utes or in the steamer, for
1000.0 cc. of (1). 90 minutes.
(3) Place in the autoclave for 30 min- (15) Filter thru moistened filter paper,
utes at 0.5 atmosphere extra using a hot water funnel.
pressure. (16) Tube.
(4) Prepare a solution of 10.0 g. pep- (17) Steam for 30 minutes on each of
tone and 5.0 g. NaCl by heating 2 successive days.
at about 70C. (Material in (s) Abel (1912).
which the peptone and NaCl is (1) Chop 500.0 g. of fat free meat and
to be dissolved not specified, add to a water at 50C.
liter of
whether it be some of the infu- (2) Keep at 50C. for 30 minutes and
sion or water). then boil for 30 to 45 minutes.
(5) Place the agar, after autoclaving (3) Filter or strain the fluid from the
is completed, on a free flame, and meat.
add the distilled water to make (4) Make up the fluid to 1 liter.
up the loss in weight due to (5) Soak 1.5 or 2.0% agar in (4) for
evaporation. several hours.
(6) Add (4) to (5). (6) Add 1.0% Witte or Chapoteaut's
(7) Neutralize and boil again for a peptone and 0.5% NaCl.
short time. (7) Heat in the steamer until solution
(8) Filter thru a single or double is complete.
thickness of filter paper. (Re- Neutralize to litmus if necessary.
(8)
quires about 10 minutes.)
(9) Heat in the steamer for 15 to
(9) Sterilization not specified,
30 minutes.
(r) Abel (1912).
(10) Filter thru cotton-wool to clarify.
(1) Chop 500.0 g. of fat free meat and
The agar may be allowed to
add to a liter water at 50C.
of
solidify in the steamer in straight
(2) Keep at 50C. for 30 minutes and
walled vessels and cut away the
then boil for 30 to 45 minutes.
bottom opaque layer.
(3) Filter or strain the fluid from the
heat. (11) Sterilize on each of 3 successive
Make up the fluid to 1 liter.
days in the steamer or autoclave
(4)
Place 20 agar in a 3 liter flask at 120 C. for 15 minutes.
(5) g.
and add 500.0 cc. of tap water and (t) Wilcox (1916) produced toxin by
2.5 cc. of glacial acetic acid. growing B. tetani on the follow-
(6) Allow (5) to soak 15 minutes and ing medium:
drain carefully. (1) Dissolve 5.0 g. agar, 10.0 g.
(7) Wash the agar thoroly with 4 lots Witte's peptone and 5.0 g. NaCl
of water. in 1000.0 cc. veal infusion.
494 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Adjust reaction so that it is (16) Bring the volume up to 1000.0 cc.
neutral to phenolphthalein. by the addition of hot water.
(3) Tube in 8 to 10.0 cc. quantities. (17) Estimate and adjust the reaction
(4) Autoclave at 15 pounds for J hour. to a definite pH value, or to
(5) To transfer cultures, a semi-solid faintly alkaline to litmus or
agar culture is melted and 1.0 cc. 1.0% acid to phenolphthalein.
portions transferred to fresh (18) Steam 30 minutes.
semi-solid melted agar tube. (19) Filter, while thru well-
hot,
(u) Meier (1918). wetted, thick paper or two
filter
(1) Boil 500.0 g. of fat and tendon paper or two layers of absorbent
free beef in 1 liter of water. cotton wool, by placing filter
(2) Filter. funnel, stand, and receptacle for
(3) Dissolve 15.0 g. agar, 10.0 g. filtrate in the steam sterilizer
Witte's peptone and 5.0 g. NaCl and steaming till filtration is
(2) Add 500.0 to 1000.0 cc. distilled Note: Raw meat juice, 15.0 cc.
water or clear tap water. per liter of medium may be sub-
(3) Heat the mixture 20 minutes stituted for white of egg.
over a free flame at a temperature (b) Add to the medium little by
not exceeding 50C. littlebefore filtration and at a
(4) Skim off fat floating on the sur- temperature not exceeding 60 C.
face. (c) Stir to mix.
(5) Raise the temperature to boiling (d) Steam 30 minutes.
point. (e) Remove from steamer and shake
(6) Boil 10 minutes. up well to mix.
(7) Pour the mixture onto a wet, (f) Steam again 15 minutes.
thick, clean cloth. (g) Filter in the steamer thru thick
(8) Collect the fluid which drains filter paper, or thru two layers
thru the cloth together with that of absorbent cotton,
obtained by squeezing the meat (h) Refilter, if necessary, the first
(9) Add 10 g. peptone 5.0g XaCl. (20) Distribute into flasks on test
(10) Mix fiber agar 1 part, glacial tubes,
acetic acid 0.05 parts, and water (w) Harvey (1921-22).
2 parts. (1)Mince finely fat-free beef.
(11) Allow the agar to soak in the (2) Add 500.0 g. to 1000.0 cc. dis-
acidified water 15 minutes. tilled water or clear tap water.
(12) Remove the fiber agar and wash
(3) Heat the mi.xture 20 minutes over
thoroly with water imtil quite
a free flame at a temperature not
free from any trace of acid reac-
exceeding 50C.
tion to litmus paper.
(4) Skim off fat floating on the sur-
(13) Squeeze in a cloth to get rid of
face.
excess of water.
(5) Raise the temperature to boiling
(14) Add (13) to (9).
point.
(15) Steam gently 2^ hours to bring
the agar thoroly into solution. (6) Boil 10 minutes.
(8) Collect the fluid which drains (4) Raise the temperature to boiling
thru the cloth together with that point.
obtained by squeezing the meat (5) Boil 10 minutes.
in the cloth. (6) Pour the mixture on to a wet,
(9) Filter the fluid collected thru thick, clean cloth.
well-wetted, thick filter paper. (7) Add 2.5 g. XaCl to the filtrate.
(10) Add to the filtrate 10.0 g. pep- (8) Bring the volume up to 1000.0 cc.
tone, 5.0 g. sodium chloride. by the addition of water.
(11) Steam or boil 45 minutes. (9) Estimate and adjust the reaction.
(12) Bring the volume up to 1000.0 cc. (10) Steam 30 minutes.
by the addition of water. (11) Filter, while hot, thru well-
(13) Cut up 20.0 g. fiber agar into wetted, thick filter paper.
small pieces. (12) g. fiber agar in 500.0 cc.
Place 15.0
(14) Add to 1000.0 cc. hot bouillon tap water.
(12). (13) Wash the agar well by squeezing
(15) Bring the agar into solution with it thru the hands.
heat after having been allowed to (14) Decant and reject the dirty
soak 2 hours in the hot bouillon. water.
(16) Estimate and adjust the reaction (15) Replace with the same amount of
to a definite pH value or faintly clean tap water.
alkaline to litmus or 1.0% acid (16) Heat over a free flame with con-
to phenolphthalein. stant stirring to dissolve the agar.
(17) Steam 30 minutes. (17) Add peptone 5.0 g., sodium
(18) Clarify and filter. chloride 2.5 g.
(a) Beat up the white of one or two (18) Add to 500.0 cc. of meat extract
eggs along with the crushed (11), 10.0 g. egg albumin which
shells in about 20.0 cc. water. has been made into a paste or
Note: Raw meat juice, suspension with a little of the
15.0 cc. per liter of medium, meat infusion.
may be substituted for white of (19) Add the dissolved agar slowly to
egg. the meat extract thus prepared,
(b) Add to the medium little by with constant stirring.
before filtration and at a
little (20) Steam 60 minutes or heat 45 min-
temperature not exceeding 60C. utes at 120C.
(c) Stir to mi.x. (21) Bring the volume to 1000.0 cc.
(d) Steam 30 minutes. by the addition of water.
(e) Remove from steamer and shake (22) Estimate and adjust the reaction.
well to mix.
(23) Boil 15 minutes over a free flame.
(f) Steam again 15 minutes.
(24) Make up for any loss of volume
(g) Filter in the steamer thru thick
by addition of water.
filter paper, or thru two layers
(25) Filter, while hot, thru well-
of absorbent cotton wool.
necessary, the
wetted, thick filter paper by
(h) Refilter, if first
placing filter funnel, stand, and
portion of the filtrate.
receptacle for filtrate in the
(19) Distribute into test tubes.
steamer and steaming till filtra-
(20) Sterilize in the steamer or auto-
tionis completed.
clave.
Harvey (1921-22). (26) Distribute filtrate into flasks or
(x)
test tubes.
(1)Mince 250.0 g. fat-free beef with
500.0 cc. of water. (27) Sterilize in the steamer or auto-
(2) Heat the mixture 20 minutes over clave.
Broth (see variant (bb) medium Add 20.0 g. peptone and 5.0 g.
(6)
779) for 2 hours. NaCl to 1000.0 cc. of the filtrate
(2) Bring the agar into solution by and dissolve by shaking.
heating.
(7) Filter thru a wetted filter paper.
(3) Sterilization not specified,
(8) Make slightly alkaline to litmus
(z) Harvey (1921-22). by the addition of NaOH or
(1) Add 500.0 cc. of water to 500.0 cc. NaaCOs.
of the variant given by Harvey (9) Soak 20.0 g. of agar in (8) for
above, several hours.
(aa) Abbott (1921). (10) Boil until solution is complete,
(1) Add 500.0 g. chopped lean beef stirring constantly.
to 1 water and soak for
liter of
(11) Test, and readjust the reaction if
24 hours, kept at ice box tem- necessary.
perature. Cool to 60C.
(12)
(2) Strain thru a coarse towel and (13) Add the white of one egg beaten
press until a liter of fluid is up in 50 to 100.0 cc. of water.
obtained. Add
(14) (13) to (12).
(3) Dissolve 1.0% peptone, and 0.5% (15) Heat in the autoclave at 118"
NaCI in (2). for 15 minutes.
(4) Dissolve 1.0 to 1.5% agar in (3) by (16) Filter while hot thru paper.
boiling in a porcelain lined iron Tube.
(17)
vessel. Add 250.0 to 300.0 cc. (18) Sterilize at 115 for 20 minutes,
of water and boil until this vol- (cc) Pitfield (1922).
ume water has evaporated,
of
Cover 500.0
(1) g. of finely cut fat
leaving one liter volume.
free beef with 1000.0 cc. of water.
(5) Place the vessel in a large dish
(2) Shake well and place on ice over
of cold water.
night.
(6) Stir the agar constantly until
(3) Squeeze out the fluid by means
cooled to 68 to 70C.
of a cloth and make up the volume
(7) Add the white of one egg which to 1 liter.
has been beaten up in about
(4) Inoculate with a culture of the
50.0 cc. of water (a 10.0% dry colon bacillus.
albumin solution may be used).
(5) Allow to stand at room tempera-
(8) Mix (7) thoroly with (6).
ture over night.
(9) Allow to boil slowly for 30 min-
(6) Boil and add 10.0 g. Witte's pep-
utes. Do not allow the volume of tone and 5.0 g. NaCl.
the liquid to fall below the liter
(7) Weigh the saucepan and contents
mark.
and heat to 60C.
(10) Filter thru a heavy folded filter
(8) Make up the loss in weight by
paper.
the addition of water.
(11) Sterilize by steam (method not
(9) Neutralize to litmus,
given).
(bb) Dopter and Sacquepee (dd) Klimmer (1923).
(1921).
(1) Add 1000.0 cc. of water to 500.0 g. (1) Preparation of meat water not
of finely chopped fat and tendon given.
(6) Add 10.0 g. peptone and 5.0 g. (gg) Park, Williams and Krumwiede
NaCl to (5). (1924).
(7) Neutralize. (1) Prepare double strength infusion
(8) Boil. broth, using double the amount of
(9) Readjust the reaction if neces- beef or veal. (See infusion broth
sary. Alkaline agar may be pre- medium 779, variant (kk) or (11)
(2) Heat then to a boil stirring con- if one wishes to remove the sugar,
stantly. 12 hours at 37C.
(3) Filter thru a filtering cloth, wash- (3) Place in an enamelled pot and bring
ing the residue with one liter of slowly to a boil.
water and squeeze out the residue. (4) Boil for 10 minutes.
(4) Distribute into glass flasks and (5) Throw on a thick cloth and press
sterilize twice before use (This the meat free from juice.
constitutes a stock meat infusion.) (6) Filter the juice thru moistened
(5) For the preparation of one liter of paper.
agar medium add 11.5 g. fibre agar (7) Add 10.0 g. of Chapoteaut's or
to 500.0 g. water. Allow the agar Defresne peptone, 5.0 g. XaCl and
to grow and swell in the water. about 1.0 g. of sodium phosphate.
(6) Add 8.0 g. ovi albumin (siccum) to (8) Boil stirring constantly until solu-
about 40.0 cc. of water. It dissolves tion is complete,
slowly. (9) Neutralize or make slightly alkaline
(7) Place 1000.0 g. of (4) (meat infusion) to litmus by the addition of soda
in a weighed kettle. solution.
(8) Add 250-300.0 cc. water to (7) and (10) Soak 20.0 g. of chopped thread agar
1.0 g. peptone. in cold water for several hours.
(9) Heat nearly to boiling and neutralize (11) Heat (10) and (9) at 100C. until
with NaOH until litmus paper be- the agar is dissolved.
comes strongly blue. (12) Readjust the reaction if necessary.
(10) Add (5) (the soaked agar) to (9) and (13) Allow to cool to 55 or 60C.
boil over a free flame to dissolve the (14) Beat the white of an egg in 100.0 cc.
agar. Do not over heat. of water and add to (13).
(11) Add 1.0 g. (NH4)2S04 and 1.0 g. (15) Mix well.
KNO3 to (10). (16) Autoclave at 120C, for one hour.
(12) Weigh and add distilled water until (17) Filter thru a moistened Chardin
the contents of the kettle weigh filter using a hot water funnel.
1000.0 g. (18) Tube.
(13) Cool to 50-55C. Sterilization:Sterilize at 115 for 20
(14) Add (6) (albumin solution) or the minutes.
whites of two eggs and place in a Use: General culture medium.
steamer for 50-60 minutes. Variants
(15) Filter thru a hot water funnel. (a) The agar may be treated with a 6 to
(May use a folded felty, so-called 100 solution ofHCl (500.0 cc. water
coffee filter paper.) to 30.0 cc.HCl for 24 hours and then
Sterilization: Method not given. with a 5.0% ammonia solution. Then
Use Cultivation of Spirillum undula majus
: thoroly wash the agar with water.
and other spirilla. (b) Besson also used 20.0 g. peptone in-
Reference: Zettnow (1895 p. 394). stead of 10.0 g.
Reference: Besson (1920 p. 41).
1663. Besson's Phosphate Infusion Agar
1664. Smith's Infusion Agar
Constituents:
1. Water 1000.0 cc. Constituents:
2. Beef 500.0 g. 1. Distilled water 2000.0 cc.
3. Peptone 10.0 g. 2. Beef 1000.0 g.
4. Agar 20.0 g. 3. Peptone (Merck, brown) . . 20.0 g.
5. Sodium phosphate 1.0 g. 4. Agar 20.0 g.
6. NaCl 5.0 g. Preparation
Preparation (1) Soak finely chopped fat free beefwith
(1) Remove all fat and tendons from 2000.0 cc. distilled water for some
beef and chop into small pieces. hours and then slowly raise to 65C.
(2) Allow 500.0 g. of (1) to macerate with on the water bath.
1000. cc. cold water for 6 hours, or (2) Finally steam and filter.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 499
(3) Add 20.0 g. Merck's brown peptone (14) Boil 2 to 5 minutes over a free
and 20.0 g. agar. flame, stirring constantly.
(4) Steam (time not specified). (15) ]\Iake up
loss due to evaporation.
(5) Cool and add the whites of 10 eggs to (16) Filter thru absorbent cotton and
clarify. cotton flannel, passing the filtrate
(6) Resteam and filter. thru the filter until clear.
(7) Neutralize the egg albumin with HCI (17) Titrate and record the final
and then render the medium alkaline reaction.
with XaOH. (18) Tube in 7.0 cc. quantities.
Sterilization: Not specified. (19) Sterilize 15 minutes in the auto-
Use: General culture medium. Smith cul- clave at 110 or for 30 minutes in
tivated Pseudomonas campestris (Pam- streaming steam on 3 successive
mel) on the medium and reported that the days.
colonies developed moderately fast. (20) Store in the ice chest in a moist
They were circular or nearly so, thin and atmosphere, to prevent evapo-
well defined margin pale yellow wet and ration.
ing the temperature to rise above (a). Sterilize in the autoclave for
(e) Meier prepared a similar medium as (1) Remove spleen from a healthy rab-
follows: bit and cut into pieces 3 mm. in
(1) Boil 500.0 g. of fat and tendon free diameter under aseptic conditions.
beef in 1 liter of water. (2) Rub one piece of (1) on each agar
(2) Filter. slant of (a), (b) or (c) and allow
(3) Dissolve 15.0 g. agar, 10.0 g. pep- the spleen to remain on the surface
tone and 5.0, 20.0 or 40.0 g. of lac- of each slant just above the water
tose or glucose in the filtrate. of condensation.
(4) Neutralize by the addition of (e) Cystine agar. Add 0.02% cystine to
KOH. Add KOH until turmeric solid sterile 1.0% stock agar and
paper is turned quite weakly steam to melt agar and sterilize the
brownish red. cystine.
(5) Sterilization not specified. (f) Spleen agar. Rub pieces of spleen
References: Smith (1897 p. 480), Com- over 1.0% stock agar slants in the
mittee A. P. H. A. (1901 p. 384), (1905 manner as indicated under (d) (1)
p. 108), (1905 Sup. #1), (1909 p. 285), and (2) above.
Meier (1918 p. 436). (g) Serum glucose cystine agar.
(1) Add 0.1% cystine and 1.0% glucose
1665. Francis' Basal Infusion Agar
to solid sterile 1.0% stock agar.
Constituents: (2) Steam to melt the agar and to steri-
1. Infusion agar (1.5%) 1000.0 cc. lize the cystine and glucose.
(1) Prepare a stock beef infusion agar (h) Serum glucose cysteine hydrochloride
using fresh beef, 1.0% agar and 1.0% agar. Substitute cysteine hydro-
peptone. chloride for cystine in medium (g)
(5) Suspend the precipitate in distilled 1668. Krasnow's et al. Meat Infusion Agar
water and add concentrated soda Constituents
solution until part of the precipitate
1. Tap water 1000.0 cc.
is brought into solution. Dissolve
Veal, Bacto
2. 75.0 g.
the remainder of the precipitate by
3. Peptone 10.0 g.
prolonged heating in steam. NaCl 5.0 g.
4.
(6) Correct the reaction to a weak
5. Agar 20.0 g.
alkalinity.
Preparation
(7) Dry 100.0 cc. of the solution and Bacto Veal
(1) Infuse 75.0 g. of in
determine the per cent of dry ma-
500.0 cc. of tap water in the Arnold
terial.
sterilizer at 100C. for two hours.
(8) Dilute (6) so that there is 2 to 3.0% Allow the coagulum thus formed to
(2)
solid materials present.
settle to the bottom of the container.
(9) Dissolve 1.0% peptone, 1.0% NaCl
Allow to cool very slowly.
and agar (amount not given) to (8), Strain thru a wire sieve.
(3)
method not given. Dissolve 3 and 4 in
(4) (3).
(10) Neutralize by Dahmen's method
(5) Adjust to pH 7.9.
with 0.33% soda.
(6) Steam in the Arnold for 15 minutes.
Sterilization: Not specified.
(7) Filter.
Use: Isolation of cholera bacilli from Add an equal quantity
(8) of a 4.0% agar
stools. Author reported that this me- solution.
dium eliminated practically all other
Sterilization : Sterilize in the autoclave for
organisms from stools.
45 minutes at 15 pounds pressure.
Reference: Deycke (1893 p. 888).
Use: Cultivation of streptococci.
Reference: Krasnow, Rivkin and Rosen-
1667. Harvey's Basal Infusion Agar
berg (1926 p. 391).
Constituents
1. Infusion agar 1000.0 cc. 1669. Dopter's Liver Infusion Agar (White)
Preparation Constituents:
(1) Prepare infusion agar according to 1. Water 1000.0 cc.
variant (v) medium 1661. 2. Liver 500.0 g.
(2) Add one of the added nutrients to (1). 3. Agar (1.5%) 15.0 g.
Sterilization: Not specified. 4. Peptone (1.0%) 10.0 g.
Use Cultivation of B. acnes and anaerobes.
: (Witte, Chapoteau or Fair-
Also used as an enrichment medium for child)
the colon typhoid group. 5. NaCl (0.5%) 5.0 g.
Added nutrients: The author added one of Preparation
the following: (1) Mix 500.0 g. of well minced calf or
(a) Oleic acid 10.0 cc. beef liver with 1000.0 cc. of distilled
glycerol 20.0 cc. water.
(b) Glycerol 20.0 cc. (2) Stir well and infuse over night in the
oleic acid 1.0 cc. ice box.
(c) Glucose 20.0 g. (3) Boil 5 minutes.
sulphin digotate 10 g. (4) Filter thru paper.
(d) Glucose 20.0 g. (5) Make up to original volume.
(e) Sodium formate 4.0 g. (6) Add 1.0% Witte, Chapoteau or Fair-
Equal parts agar and 1.0% caffeine child's peptone, 0.5% NaCl and 1.5%
solution. agar.
The combinations (a) and (b) were (7) Boil 20 minutes and adjust to 4-0.2
used to cultivate B. acnes, (c) and (d) to phenolphthalein.
used for the cultivation of anaerobes. (8) Boil 5 minutes and filter.
Agar containing caffeine (e) was used Sterilization: Sterilize in the Arnold on
as enrichment medium for members three successive days.
of the colon typhoid group. Use: General culture medium. Also used
Reference: Harvey (1921-22 pp. 87, 92, 111). to cultivate and carry stock meningococci
502 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
cultures. Bronfenbrenner and Schles- (2) Cook (1) in flowing steam one
inger reported that the medium gave a hour.
large amount of water of condensation. (3) Strain thru cheese cloth and
To keep a pure culture on this medium cotton.
for several weeks, inoculate the water of (4) Add peptone (1.0%) and NaCl
condensation and tilt the tubes every day (0.5%).
or so. Other investigators cultivated a (5) Add 20.0 g. agar and heat in flow-
single layer of cheese cloth. Boil liver (or other organ, placenta,
and stir continually (Time not etc.)and 1000.0 cc. water in the
given). same manner as for ordinary infu-
Strain thru a single layer of cheese sion broth. (See variant (bb)
(2)
cloth and a thin layer of cotton. medium 779.)
(3) Do not adjust the reaction, and (2) Dissolve 3.0% agar, 2.0% peptone
sterilize at 10 pounds pressure for and 1.0% NaCl in water.
10 minutes. (3) Sterilize (2).
Prepare a 3.0% agar solution in (4) Mix equal parts sterile (1) and (3)
(4)
water (30.0 g. agar to 1000.0 cc. while hot under aseptic conditions.
water. (5) Tube in sterile tubes.
(8) Mix equal parts of sterile (3) and refrigerator over night.
sterile (7) while hot. (2) Bring to boil and strain thru cheese
(9) Tube in sterile tubes. cloth.
(b) Heller isolated B. Welchii, B. oede- (3) Add 15.0 g. Peptone (Difco), 5.0 g.
matiens and other soil anaerobes on salt and and dissolve
20.0 g. agar
(2) Weigh the kettle used in (1) and (9) Adjust to pH = 7.0.
its contents. (10) Add 1.0% dissolved egg albumin.
(3) Heat at 45C. for an hour. (11) Mix well and place in flowing
(4) Boil for 30 minutes. steam for 90 minutes.
(5) Make up the loss by weight by the (12) Decant the liquid from the clot.
addition of water. (13) Remove small clumps of albumin
(6) Strain thru cheese cloth. Squeeze by filtering thru a discarded pres-
by twisting the cloth or use the sure cooker. Place glass wool,
meat press. previously washed with dilute
(7) If used at once dissolve 1.0% pep- acid, in the barrel of the filter and
tone and NaCl in (6). a 30 mesh copper screen funnel for
(8) Dissolve 1.5% agar in (7) by heat- collecting large coagulated par-
ing in the autoclave at 10 to 15 ticles.
pounds pressure for 30 minutes or (14) Readjust to pH = 7.0 if necessary.
by boiling over the free flame. The reaction should fall to pH 6.6
(9) If boiled over a ree flame make up following sterilization.
the loss in weight by the addition (15) Sterilize at 15 pounds pressure for
of water. 30 minutes.
(10) Adjust the reaction to +0.2% to (16) For isolation work add sufficient
phenolphthalein quantity of a saturated aqueous
(11) Cool to 50C. and add one egg. solution of gentian violet so that
(12) Test the reaction and readjust if the concentration be 1:10,000.
necessary. References: White (1917 p. 49), Bronfen
(13) If more than 0.2% normal soda is brenner and Schlesinger (1918 p. 125)
required per liter, heat again for (1918-19 p. 219), Heller (1921 p. 460)
ten minutes. Goss, Barbarin and Haine (1921 p. 615)
(14) Filter thru cotton. Harvey (1921-22 p. 69), Heller (1922 p. 9)
(15) Distribute in tubes or flasks. Klimmer (1923 p. 201), Park, Williams
(16) Sterilize at 15 pounds pressure for and Krumwiede (1924 p. 131), Huddleson,
30 minutes. Hasley and Torrey (1927 p. 356).
(g) Stafseth (Huddleson, Hesley and
Torrey used the following medium 1670. Richardson's Mucosa Infusion Agar
for isolation and cultivation of Bac-
terium abortus (Bang): Constituents
(1) Grind fresh fat free beef liver in a 1. Distilled water 500.0 cc.
meat chopper to a plastic mass. 2. Mucosa of hog intestine 250.0 cc.
(2) Mix (1) with 500.0 cc. of tap water. 3. Peptone 5.0 g.
(7) Mix 500.0 cc. of (6) with 500.0 cc. of from one hog).
tap water, 20.0 g. washed agar, (3) To 250.0 cc. of the mucosa add
10.0 g. Bacto peptone and 5.0 g. 500.0 cc. of distilled water. )
"^
NaCl, and place in a covered (4) Boil for 30 minutes. Cool.
container. (5) Boil again then neutralize and boil
(8) Place in flowing steam for 30 once more.
minutes. (6) Filter.
504 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(7) Boil and add 5.0 g. peptone, 2.5 g. Sterilization: Not specified.
NaCl and neutralize again. Boil Use: Cultivation of saphrophytic organ-
for 20 minutes. isms. Author reported that diphtheria
(8) Filter. (The filtrate may be divided bacilli and streptococci grew on this me-
into two and using half of the
parts, dium better than on glycerol agar.
infusion as a liquid medium.) Saphrophytic organisms requiring sugar
(9) Add 1.5% agar to (8) and boil until in ordinary agar, grew luxuriantly on this
solution is complete. medium.
(10) Neutralize and cool to 68C. Reference: Lichtenstein (1915-16 p. 362).
(11) Add a well beaten egg to (10) and
boil until the egg is thoroly co- 1672. Stuart's Stomach-Liver Infusion Agar
agulated.
and tube. Constituents
(12) Filter
Sterilization: Sterilizein the Arnold on 1. Water 1000.0 cc.
(3) Allow to cool, skim and filter (10) Place (8) in a saturated salt solution
thru filter paper. bath and boil for 45 minutes, making
(4) Adjust the reaction so that the provision for loss of water by evap-
acidity is equal to 0.025 g. H2SO4 oration.
per liter (0.5%). (11) Add saturated Na2C03 solution until
(5) Add 5.0 g. of agar and 10.0 g. De- slightly alkaline to litmus (allow for
fresne peptone and heat for 15 min- a slight augmentation of the acid
utes at 118C. during the subsequent steps).
(6) Filter and tube in 6 to 8.0 cc. lots. (12) Cool to 55C. and add the white of
(7) Heat at 112 for 15 minutes. an egg and steam in Arnold sterilizer
(8) When ready for use add 0.1 cc. of a until clarification of the upper
1 to 10 sterile solution of lead sub- strata of the medium has occurred.
acetate in distilled water to each (13) Pipette off the clear supernatant
tube. fluid or filter.
(b) Tilley adjusted Jordan and Victor- (14) Transfer definite quantities of (13)
son's medium to pH = 7.2, sterilized to flasks.
the agar in the autoclave and added (15) To 100.0 cc. of hot sterilized distilled
3 drops of freshly prepared 10.0% lead water, add 10.0 g. of one of the added
acetate to each tube. nutrients, and expose the solution
(c) Harvey added 1.0 drop (0.05 cc. to to the action of live steam for 10 min-
each tube (4.0 cc.) of agar (see utes. (Use old Jena glassware to
variant (v) medium 1661) cooled to avoid alkaline production.)
45C. (16) Add to sterile (14) following the
References: Jordan and Victorson (1917 third sterilization, 1.5% of sterilized
p. 554), Emile-Weil (1917 p. 379), Tilley Kubel and Tiemann's litmus solu-
(1923 p. 115), Harvey (1921-22 p. 107). tion.
(17) Transfer to sterile tubes, slant and
1677. Elser and Huutoon's Basal Litmus
expose to incubator temperatures
Infusion Agar
for several days.
Constituents (18) The final reaction of the medium
1. Distilled water 1000.0 cc. should be very faintly alkaline to
2. Beef (lean) 500.0 g. litmus.
3. Peptone 10.0 g. Sterilization: Sterilize (14) by steaming for
4. NaCl 5.0 g. 15 minutes on each of three successve
5. Agar agar 16.0 g. days.
6. Litmus, Kubel and Tie- Use: Cultivation of meningococci, pseudo-
mann's 15.0 cc. meningococci and gonococci. The me-
Preparation dium is suited primarily for stock cul-
(1) Mix thoroughly 500.0 g. of chopped tures.
lean beef with 1000.0 cc. distilled Added nutrients: The author added 1.0%
water. of one of the following:
(2) Boil over a free flame for 15 minutes, glucose mannitol
stirring constantly. galactose dulcitol
(3) Filter. levulose inulin
(4) Add an emulsion of Bacterium coli. lactose dextrin
(5) Incubate at 37C. for 24 hours. maltose sucrose
(6) Sterilize for 10 minutes. Reference: Elser and Huntoon (1909
(7) Filter until clear. p. 406).
(8) Add 3, 4 and 5 that have previously
been soaked in water, to (7).
1678. Emile-Weil's Neutral Red Infusion
Agar
(9) A portion of the filtratereinocu- is
(1) Prepare beef infusion 1 pound in two (6) Add 1.0% Witte's peptone and 0.5%
liters water. NaCl to (5).
(2) Add 30.0 g. agar. (7) Boil in the steamer.
(3) Add 7.5 cc. N/1 HCl. (8) Cool in a cold water bath.
508 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(16) Add 1.4 to 1.5 cc. of (15) to each 6. N/1 NaOH 4.0 cc.
100.0 cc. of sterile melted and cooled 7. Crystal violet 1/70 g.
to 60-65C. (14). Mix well. Preparation:
(17) Pour into plates. (1) Pass one pound of beef heart thru a
Sterilization: Method of sterilization of meat chopping machine and soak in
(14) not given. one liter of distilled water over night
Use: Isolation of typhoid bacilli from inan ice box.
feces. Enrichment of typhoid bacilli. (2) Squeeze the fluid thru a cheese cloth,
Smear the plates with the stool or feces heat to boiling and filter thru filter
suspension. Incubate 20 hours at 37C. paper.
Wash the surface of the plates with 8 to (3) Add 1.0% peptone, 0.25% NaCl,
10.0 cc. physiological salt solution, and 1.5% agar-agar, 4.0 cc. of N/1 NaOH
smear 1 to 3 loops of the salt solution sus- and heat for 30 minutes in the auto-
pension on Drigalski-Conradi medium. clave at 15 pounds pressure.
Reference: Werbitzki (1909 p. 205). (4) Adjust the reaction to -fl (hot titra-
tion), clear with white of egg.
1682. league's Victoria Blue Infusion Agar Filter thru cotton.
(5)
Constituents (6) Distribute in flasks in 200.0 cc. lots.
1. Meat infusion 1000.0 cc. (7) Add to the melted sterile agar 1/700%
2. Peptone, Witte (1.0%) 10.0 g. of crystal violet.
(5) Add stock solutions (method of 3. Gentian violet (1 : 1000) 25.0 cc.
preparation not given) of Victoria Preparation
blue 4R in varying amounts so that (1) Prepare heart infusion agar using
there is present 1/20, 1/30, 1/40 or 1/50 Berna peptone, according to Hun-
of dye, to sterile (4). toon's method. (See medium 1863
Sterilization: Sterilize by heating in Ar- for Huntoon's method.)
nold for 3 successive days. (2) Add 0.25% of a freshly prepared 10.0%
Use: Enrichment of B. paratijphosus A and solution) NasSOa and 1/400% (2.5 cc.
B. enteritidis. Author reported that the of a 1:1000 solution per 100.0 cc.) of
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 509
Felix). This organism produces an in- Use: General culture medium. The me-
tense black color on this medium. dium conforms to "Standard Methods"
Reference: Friedberger and Joachimoglu formula.
(1918 p. 805). Reference: Digestive Ferments Co. (1925
p. 10).
1688. Ragit Agar
Constituents: 1690. Bacto Nutrient Phosphate Agar
1. Water 1000.0 cc.
(Dehydrated)
2. Ragit agar 42.0 g. Constituents
Preparation: (1) Dissolve 42.0 g. of Ragit 1. Distilled water
agar in 1000.0 cc. of water. Ragit agar is 2. Beef extract (Bacto) 3.0 g.
a trade name of a dried medium marketed 3. Peptone (Bacto) 10 g.
by Merck. It contains "Maggibouillon" 4. Agar (Bacto) 15.0 g.
Agar and Peptone in such amounts that 5. NaaHPOi 5.0 g.
42.0 g. of powder in 1 liter of water gives Preparation
a nutrient agar of the usual composition, (1) Dissolve 33.0 g. Bacto Nutrient
of
(i e. Beef infusion with 1.0% peptone Phosphate Agar (Dehydrated) in
and 2.0% agar.) 1000 cc. distilled water by boiling or
Sterilization: Not specified. autoclaving preferably by boiling.
Use: General culture medium. (2) If sterilized 20 minutes at 15 pounds
Variants pressure pH = 7.5 .
(a) Tausz and Peter cultivated Bac- (3) Cool to 50C. before adding albu-
terium aliphaticum, Bacterium ali- minous enrichment materials if these
phaiicum Uquefaciens and Paraffin materials are to be added.
bacteria, and prepared the medium Sterilization: Sterilize in the usual manner.
as follows: Use General culture medium.
:
(1) Boil 42.0 g. Ragit agar with 1 liter Reference: Digestive Ferments Co. (1925
of water for one hour. p. 11).
510 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1691. Bacto Nutrient Agar 1.5% Sterilization: Do not heat over 75 C. when
(Dehydrated) sterilizing(method not given).
Constituents :
Use: To study oxalic acid formation by
acetic acid bacteria. Meier used a sim-
1. Distilled water
ilar medium to study the bacterial count
2. Beef extract (Bacto) 3.0 g.
of milk and whey.
3. Peptone (Bacto) 5.0 g,
4. Agar (Bacto) 15.0 g.
Added nutrients The author added one of
:
the following:
5. NaCl 8.0 g.
Preparation Methyl alcohol 10.0 g.
claving.
Amyl alcohol 5.0 g.
Glycerol 10.0 g.
(2) Distribute as desired (Blood may be
added in suitable proportions for
Ethylene glycocol 10.0 g.
Variants: Meier studied the bacterial (Blood serum may be used instead
counts of milk and whey, using the fol- of egg white.)
lowing medium: (8) Boil for 3 to 5 minutes and filter
(1) Add 10.0 g. peptone, 5.0 g. NaCl g. NaCl in 500.0 cc. distilled water
and 5.0 g. Liebig's beef extract to by boiling.
500.0 cc. water. (2) Dissolve 5.0 g. agar in 500.0 cc. dis-
meat extract, 10.0 g. Witte's pep- (2) Neutralize to litmus and then add
tone and 5.0 g. agar in 1000.0 cc. 10.0 cc. normal soda solution pe?
water. liter of medium.
(2) Distribute in 10.0 cc. lots into test (3) Sterilization not specified,
tubes that will not give up alkali (i) Kligler and Defandorfer (1918).
by sterilization. (1) Dissolve 10.0 g. peptone, 3.0 g.
(5) Make up the loss in weight by the (j) Hesse (Ball) (1919).
addition of distilled water. (1) Digest 5.0 g. agar in 500.0 cc. of
(6) Filter.
water.
(7) Adjust the reaction to neutrality (2) Dissolve 10.0 g. peptone, 5.0 g.
(indicator not specified). beef extract and 8.5 g. NaCl in
(f) Viehoever (1913) cultivated urea (5) Adjust the reaction to 1.0%.
splitting organisms on the follow- (6) Tube.
ing medium: (7) Sterilize in the autoclave,
(1) Dissolve 6.0 g. Witte's peptone, (k) Hesse (Tanner) (1919).
meat extract (amount not speci- (1) Dissolve 5.0 g. agar in 500.0 cc, dis-
(3) Tube in 5.0 cc. quantities. NaCl in 500.0 cc. distilled water.
(4) Sterilize (method not given). (3) Mix (1) and (2).
(h) Meier (1918) made bacterial counts of agar in coldwater for several
milk and whey using the following hours. Squeeze the water thru
medium a cloth.
(1) Dissolve 15.0 g. agar, 10.0 g. (3) Heat (2) and (1) at 100C. until the
Liebig's meat extract, 10.0 g. agar is dissolved.
Witte's peptone and 5.0 g. NaCl (4) Readjust the reaction if necessary.
in 1000.0 cc. distilled water. (5) Allow to cool to 55 or 60C.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 513
NaOH sp. gr. 1.226 or approxi- (6) In the morning dump out the
mately 20.0%.) jellymass and cut off and discard
(6) the tubes thoroly and cool.
Mix the bottom portion containing the
Do
not sterilize. sediment.
(o) Giltner (1921). (7) Melt the remainder of the agar.
(1) Place 500.0 g. of water in an agate (8) Distribute in flasks (or nursing
water pail and add 15.0 g. of agar. bottles).
(2) Wash the agar well, separating the (9) Sterilize (method not specified),
shreds and squeezing thru the (10) Store until ready for use.
hands. Wash until clean. (q) Stitt (1923).
(3) Make up to the original volume (1) Dissolve 15.0 g. of agar in 500.0 cc.
the addition of tap water. of water in the inner compartment
(4) Heat over a free flame until the of a rice cooker.
agar is dissolved, stirring con- (2) Cool to 55C.
stantly. (3) Dissolve the whites of one or two
(5) Add 3.0 g. of standard meat ex- eggs in 500.0 cc. of water.
tract (or use 500.0 cc. meat infu- (4) Prepare a paste from 3.0 g. Liebig's
sion) to 500.0 cc. of tap water. meat extract, 10.0 g. peptone and
(6) Add and 0.5% NaCl.
1,0%, peptone 5.0 g. NaCl by adding (3) little
(8) Add 10.0 g. of albumin mixed with (5) Add the remainder of (3) to (4).
100.0 cc. water. (6) Heat (5) to 50 to 55C.
514 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(7) Add (6) to (2). required per liter, heat again for
(8) Adjust to a desired pH value by 10 minutes.
the addition of normal acid or thru cotton.
(11) Filter
alkali. (12) Distribute in
tubes or flasks.
(9) Bring the agar to a boil in the inner (13) Sterilize at 15 pounds pressure for
compartment of a rice cooker. 30 minutes.
(10) Filter in an autoclave or Arnold References: Heim (1895 p. 193), Ravenel
sterilizer thru filter paper wetted (1899 p. 605), (1899-1900 p. 89), Frost
with boiling water. It may be (1903 p. 16), Hesse (1908 p. 441), (1908
filtered thru cotton or gauze if p. 89),Stokes and Hachtel (1909 p. 40),
clearness of the medium is not an Viehoever (1913 p. 214), Bengis (1916
essential. p. 392), Meier (1918 p. 435), Kligler and
(11) Sterilization not specified. Defandorfer (1918 p. 438), Ball (1919
(r) Stitt (1923). p. 80), Tanner (1919 p. 51), Besson (1921
(1) Prepare a paste of 3.0 g. meat ex- p. 43), Giltner (1921 p. 386), Wolf and
tract, 10.0 g. peptone, 5.0 g. NaCl, Shunk (1921 p. 325), Giltner (1921 p. 40),
15.0 g. powdered agar and the Heinemann (1922 p. 35), Stitt (1923
white of one egg by mixing with a pp. 36, 37), Park, Williams and Krum-
little water in a mortar. wiede (1924 p. 117).
(1) Dissolve, 2.0 to 5.0 g. Beef extract the pan. Add 300.0 cc. of water to
(Liebig's or Armour's), 10.0 g. pep- allow for evaporation. Heat over
tone, and 5.0 g. NaCl in 1000.0 cc. the gas.
water by boiling over a fire. (3) Add 15.0 g. of shredded agar to (2)
(9) Test the reaction and adjust if oration of water, or boil until the
necessary. proper weight is obtained if the
(10) If more than 0.2% normal soda is mixture weighs too much.
515
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(4) Soak 1.5% market agar (1.2% if Tanner (1918 p. 48), Ball (1919 p. 77),
oven dried) in water, and wash Dawson (1919 p. 142), Committee
under tap in a sieve. A. P. H. A. (1920 p. 96), Levine (1921
Add to (4) 600.0 cc. of distilled p. 108), Cohen (1922 p. 189), Committee
(5)
A. P. H. A. (1923 p. 4), Committee S. A. B.
water minus the water absorbed
(1923 p. 9), Park, Williams and Krum-
during the washing. Weigh.
wiede (1924 pp. 131, 132), Committee
(6) Mix (5) and (3).
A. P. H.A. (1925 p. 98).
(7) Heat on the stove until the agar is
completely melted, stirring con- 1696. Glaessner's Nahrstoff Heyden Extract
stantly. Agar
(8) Boil and stir constantly for Constituents
20 minutes.
1. Water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 517
Dissolve 3, 4 and 5 in 500.0 cc. water. Use: Show effect of pancreas extract on
(2)
Melt sterile (1) and cool to 44C. growth of organisms. Author reported
(3)
Mix equal parts of sterile (3) and that the organisms were inhibited by the
(4)
sterile (2) at 40C.
pancreas extract.
Pour in sterile plates. Reference: Kotlar (1895 p. 153).
(5)
Sterilization: Sterilize (1) and (2) sepa-
1700. Bacto Andrade Maltose Agar
rately (method not given).
(Dehydrated)
Use: Cultivation of diphtheria bacilli.
Variants: The author used 1.0 g. meat Constituents
extract and 5.0 g. NaCl instead of 1. Water
amounts indicated. 2. Beef extract (Bacto) 3,0 g.
Same as medium 807, but solidified by the 6. Andrade Indicator (Difco) . . 0.0275 g.
addition of 30.0 g. of agar. 7. NaOH
Preparation
1698. Conn and Breed's Nitrate Extract Bacto Andrade
of
(1) Dissolve 30.0 g.
Agar
Maltose Agar (Dehydrated) in
Constituents: 1000.0 cc. of distilled water by boiling
1. Water 1000.0 cc. or autoclaving, preferably the latter.
2. Agar 150 g.
(2) Tube.
Peptone 10-0 S- 20 minutes at 15 pounds
3. (3) If sterilized
4. KNO3 10 g- pH = 7.7.
5. Beef extract 3.0 g. Sterilization: Sterilize in the usual manner,
Preparation: (1) Dissolve 2, 3, 4 and 5 in 1. avoiding excess heat.
Sterilization: Method not given. Use: General culture medium.
Use To determine the ability of bacteria to
: Reference: Digestive Ferments Co. (1925
reduce nitrates. p. 16).
Variants
(a) Committee S. A. B. prepared beef ex- 1701. Bacto Andrade Mannite Agar
tract agar according to the method of (Dehydrated)
Committee A. P. H. A. (1920 see Same as 1700, but substituting Bacto
variant (g) medium 1695 and adjusted Andrade Mannite Agar (Dehydrated) for
the reaction to pH = 6.6 to 7.4. Bacto Andrade Maltose Agar (Dehydrated)
(b) Percival sterilized the
medium on 3
successive days for 20 minutes each 1702. Bacto Andrade Dextrose Agar
day. (Dehydrated)
References: Conn and Breed (1919 p. 278),
Same as medium 1700, but using Bacto
Committee S. A. B. (1920 p. 128), Percival
S. A. B. (1923
Andrade Dextrose Agar (Dehydrated) in-
(1920 p. 163), Committee
stead of Bacto Maltose Agar (Dehydrated)
p. 10).
(6) Test the reaction and adjust if 1711. Warden's Salt Agar
necessary.
Constituents
(7) If more than 0.2% normal soda is
1. Distilled water 1000.0
required per liter, heat again for
2. Bouillon 200.0
10 minutes.
3. Agar 2.5 g
(8) Filter thru cotton.
Distribute in tubes or
4. Sodium bicarbonate 0.2 g
(9) flasks.
5. CaCla 0.25 g
(10) Sterilize at 15 pounds pressure for
30 minutes.
6. KCl 0.45 g
(k) Park,
7. NaCl 10.8
Williams and Krumwiede (1924) g,
Preparation
reported a very satisfactory medium
(1) Dissolve 3, 4, 5, 6 and 7 in 1 by aid of
can be made by simply using 0.5% or
less of agar instead of the usual 1.5%
heat until fluid shows a translucent
employed. This can be diluted by
ground glass appearance.
the addition of i of its bulk of an (2) Add bouillon (composition or method
of preparation not specified).
enrichment fluid. The finished
(3) Filter while hot, through gauze or
medium should just "set" suffi-
cotton into test tubes or flasks.
ciently for stab culture purposes.
Nutrose (1.0%) may also be added. (4) Adjustment of reaction not specified.
Sterilization: Sterilize once in autoclave
(1) Park, Williams and Krumwiede (1924)
(time not specified).
gave the following medium as Mus-
Use: Culture medium for gonococci.
grave and Clegg's Agar for the culti-
vation of amoeba:
Author reported that when cool medium
was semi-solid, and had a translucent
(1) Mix 90.0% tap water, 10.0% ordi-
silvery appearance. Nearly all strains
nary nutrient broth (preparation
of gonococci did not grow on this me-
not given) and 1.0% agar.
dium. If a few loopfuls of sterile human
(2) Dissolve.
blood be placed on this surface, all
(3) Reaction neutral to phenol-
strains grew.
phthalein.
Reference: Warden (1913 p. 94).
(4) Sterilize as usual (method not
given).
1712. Stroszner's Regenerated Agar
(m) Cunningham (1924).
(1) Add 1.5% agar to bouillon. Constituents
(2) Steam for 30 minutes to dissolve 1. Bouillon.
the agar. 2. Agar (used).
(3) Boil over an open flame for Preparation:
15 minutes, stirring constantly. (1) Place the used agar in an enamel con-
(4) Adjust the reaction to a slight tainer and melt in streaming steam
alkalinity using turmeric paper (add no water).
as an indicator (distinctly brown). (2) Measure the agar in a graduated
(5) Filter while hot thru a plug of cylinder.
cotton-wool in the bottom of an (3) To each liter of agar add 40.0 g. of
enamelled funnel. powdered charcoal and boil for 30 to
(6) Tube. 40 minutes in the steamer.
(7) Sterilize intermittently in steam. (4) Cool to 50C. and add 40.0 cc. defi-
References: Wurtz (1897 p. 29), Smith brinated blood per liter of agar.
(1902 p. 92), Roux and Rochaix (1911 (5) Boil in streaming steam once more
pp. 115, 116), Hoffmann (1912 p. 387), for 40 minutes.
Lohnis (1913 p. 16), Roddy (1917 p. 43), (6) Filter (method not given).
Bezan^on (1920 p. 112), Hilgermann and (7) Add meat infusion or
300.0 cc. 1 to
Weissenberg (1917-18 p. 470), Klimmer 1.5% Liebig's bouillon to each liter
(1923 p. 229), Stitt
(1923 p. 36), Park, of agar.
Williams and Krumwiede (1924 pp. 117, Sterilization: Method not given.
118, 134), Cunningham (1924 p. 15). Use: Regenerated agar.
MICROORGANISMS 523
CULTURE MEDIA FOR CULTIVATION OF
1. Nutrient agar
1000.0 cc 4. Kahlbaum's litmus solution.
2. Litmus Preparation
Preparation: (1) Soak used agar slants and plates in
(1) Dissolve
2.0 to 4.0% of one of the a 3.0% HCl solution for one hour.
added nutrients to plain agar. Stir constantly.
use, melt (1) and add a Wash the pieces of agar with water,
(2) Just before (2)
quantity of litmus to give
sufficient and soak in water for 24 hours.
4.0% of any desired carbon source. (5) Melt the agar and add to 1 liter,
Variants: Bezan^on gave the
following 8.0 to 10.0 cc. of a 10.0% soda solution,
Reference: Besson (1920 p. 60). beef and 1000.0 cc. water. (Exact
method not given.)
1720. Gasser's Fuchsin Agar NaCl and
(2) Add 10.0 g. peptone, 5.0 g.
20.0 g. Dextrose to (1).
Constituents
Nutrient agar. (3) Adjust to pH = 8.2.
1.
3. Peptone 40.0 g.
Constituents:
1. Agar. 4. KNO3 4.0 g.
5. Glucose 4.0 g.
2. Dahlia.
Agar 60.0 g.
Preparation: (1) dahlia in the ratio of
Add 6.
(3) Strain and heat in water bath to (5) After warming this mixture in a
boiling and strain again. double boiler and stirring it for a
(4) Dissolve 3 and 4 in (3). few minutes to dissolve ingredients,
(5) Adjust to pH = 7.5. titrate with N/20 sodium hydrate,
(6) Filter and autoclave at 15 pounds using phenolphthalein as an indicator,
pressure for 30 minutes. and neutralize with normal sodium
(7) Dry agar thoroly, weigh and wash in hydrate.
running water over night. (6) Boil vigorously for 30 minutes in a
(8) Dissolve 60.0 g. (7) in 1000.0 cc. of double boiler, and 5 minutes over a
water in the autoclave. free flame with constant stirring to
(9) Adjust to pH = 7.5 and clear by prevent the caramelization of the
straining through cotton and gauze. dextrose.
(10) Add glucose to (6) just before addi- (7) Make up any loss in weight by evapo-
tion of agar solution. ration and filter thru cotton flannel
(11) In making the mixtures the amount and filter paper.
of hot fluid, 6.0% agar, necessary to (8) Tube.
obtain the various percentages, is Sterilization: Sterilize in an autoclave for
diluted with hot distilled water to 15 minutes.
a volume equal to that of the double Use: Cultivation of B. sporogenes and
strength broth (9) also hot, and other bacteria. Medium also used for
mixed. presumptive test for B. coli in water
(12) Check reaction (pH = 7.5). analysis.
Sterilization: Sterilize in autoclave at Variants: Harvey solidified medium 833
15 pounds for 20 minutes. variant (a) by the addition of agar. He
Use: To study growth at different agar reported that when 1.0 acid, B. bifidus
concentrations. The author recom- grew on medium. When 4.0% acid
this
mended a 0.1% agar concentration for B. acidophilus grew on this medium.
growth of aerobic and anaerobic bacteria. References Jackson and Muer (1911 p. 290),
:
1724. Jackson and Muer's Liver Infusion 1725. Hall's Testicular Infusion Agar
Agar
Constituents
Constituents 1. Distilled water 1000.0 cc.
1. Water 1000.0 cc. 2. Testicles, beef 500.0 g.
2. Liver, beef 500.0 g. 3. Peptone (Witte or Difco) . . 20.0 g.
3. Agar 5.0 g. 4. Agar 30.O g.
4. Peptone (Witte's) 10.0 g, 5. Glucose 5.0 g.
5. Glucose 10 g 6. NaH2P04 3 0g.
6. K2HPO4 1.0 g. Preparation
Preparation Soak over night, 500.0
(1) g. of ground
(1) Chop 500.0 g. beef liver into small beef testicles from which the tunica
pieces, add 500.0 cc. distilled water vaginalis has been stripped, in
and boil slowly for two hours, stir- 1000.0 water
cc. distilled at room
ring occasionally. temperature.
(2) Add 3
(dried at 105C. for 30 minutes) (2) Heat to 50C. Keep warm in 37
to 500.0 cc. distilled water and digest incubator for one hour.
for 30 minutes in an autoclave at Boil strain, and
(3) restore to 1000.0 cc.
120C. (15 pounds). If in excessdo not boil to reduce
(3) After making up the loss by evapora-
volume, overheating is injurious.
tion, strain the liver infusion thru a Add 3, 4, 5 and 6 to (3).
(4)
wire strainer, add 500.0 cc. filtrate to (5) Soak at least an hour to soften the
the agar solution. agar.
(4) To the filtrate add 4 and 5, then (2). (6) Melt in autoclave at 10 pounds for
Weigh the infusion and container. 30 minutes.
527
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Mince finely.
(7) Titrate with N/1 NaOH to neutral (2)
(3) Allow to macerate in water over-
point with phenolphthalein.
(8) Check the titre by repetition using ^l^^^' . ^ , ,. .^,
Heat in a water bath with con-
N/20 NaOH-5 cc. should require (4)
stant stirring until the proteins
1 to^2 cc. to display color while
, have been coagulated.
,
(5) Filter thru coarse cloth.
^
autoclave at (6) Make up to volume, 750.0 cc, with
Sterilization: Sterilize in
hot distilled water^
10 pounds for 30 minutes.
Cultivation of gonococci. The (7) Add to the fluid, while hot,
Use:
20.0 g. peptone and 3i) g. di-
author reported that media with 1.0%,
tried hydrogen sodium phosphate,
2 0%, 3.0% or 4,0% agar were also
Allow the temperature to fall to
with and without glucose and varying (8)
u f(7)
agar to 60 and
.c^
(S)
T.T^
Add melted m Tto TfiT"
(6).
(4) Cool sterile melted
^^^ ^^^ ^^^^.j^ ^^^^^-^^ ^f gl^.^se
(9) Add 5.0 g- glucose- to the agar under
^ .^ .
^
mto and lead acetate
(10) Mix thoroly and distribute
conditions,
desired containers
^^^^^^
(11) Autoclave at lo pounds for
[gj ^^.^^ate to test sterility.
blood slowly and stir to avoid the used directly in the preparation of
formation of clumps. agar after neutralization and fil-
(5) Boil in the steamer for 40 minutes. tration by adding 2 or 3% peptone
(6) Filter (method not given). per liter.
(7) Sterilize (method not given). (14) To prepare plain agar from used
(The agar is light red. Endo agar take the slants and
(8) To a liter of agar add 300.0 cc. of plates and treat with a 0.5% NaOH
infusion broth or 1.0 to 1.5% solution after washing (5) until the
Liebig's extract broth, 4.0 g. lac- red color disappears. Wash in
tose, 4,0 cc. alcoholic concentrated water until the agar reacts neutral.
fuchsin solution and 2.0 cc. of a Add peptone and meat extract as
10% NazSOs solution. (Alkali in (9). To completely remove the
need not be added.) pink color add 1.0 cc. of a 1.0%
(c) Zipfel prepared regenerated Endo bismark brown solution per liter
agar as follows: of agar,
(1) Remove the Endo agar slants and (d) Levine gave the following method of
plates from tubes and petri dishes. preparation as Kendal's modification
(2) Add 3 0% HCl to used Endo agar (1) Prepare plain, sugar-free nutrient
slants or plates until the HCl agar, using 15 grams of agar per
covers the agar. Stir. liter.
(3) Allow the acid to react for one (2) Adjust the reaction to a point just
hour, stirring continuously. alkaline to litmus.
(4) Pour the red agar on a sieve. The (3) Flask the agar, 100.0 cc. to a flask,
HCl solution may be saved and and sterilize in the autoclave.
utilized (see end). (4) Prepare a 10.0% solution of basic
(5) Wash agar with a
the pieces of fuchsin in 96.0% alcohol. This
stream of water until the wash solution is fairly stable if kept
water is clear. away from light.
(6) Soak the agar in water for 24 hours, (5) Prepare a 10.0% aqueous solution
changing the water frequently. of chemically pure anhydrous
(7) Place the agar on a sieve at the sodium sulphite (1.0 g. in 10.0 cc.
end of this time and allow to drip water). This solution does not
free from water. keep.
(8) Liquify agar particles in
the (6) Add 1.0 cc. of (4) to 10.0 cc. of (5)
streaming steam. and heat in the Arnold sterilizer
(9) To a liter of (8) add 8 to 10.0 cc. for 20 minutes. The color of the
of a 10% soda solution, 4.0 cc. of fuchsin is nearly discharged if the
a filtered and sterilized solution of solutions are of proper strength.
20.0% peptone and 20.0% meat This solution must be prepared
extract or meat equivalent.
each day it does not keep.
(10) Sterilize for one hour in the (7) Add 1.0 g. of C. P. lactose (free
steamer. from glucose) to 100.0 cc. of agar
(11) To prepare Endo agar add 3.0 cc. and place in the autoclave until
of a concentrated fuchsin solution, melted and the lactose is thoroly
25.0 cc. of a 10.0% NajSOs solution dissolved.
freshly prepared and 8.0 g lactose (8) Add a sufficient volume of (6)
dissolved in a little water, per (about 1.0 cc.) to impart a faint
liter of (10). pink color to the medium.
(12) Pour into sterile plates. (9) Pour into sterile Petri dishes and
(13) To utilize the HCl washings from allow to harden in a dark place
(4), boil and then neutralize with with the covers partly removed.
soda, (charcoal may be
filter When cool the medium should be
added) and concentrate by evap- colorless when viewed from above
oration. The washings may be and a very faint pink when viewed
530 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
from the edge. The medium must p. 722), Abel (1912 p. 131), Stroszner
be kept in a dark place because the (1917-18 p. 224), Zipfel (1917-18 p. 477),
color is restored by the action of Levine (1921 p. 114), Harvey (1921-22
daylight. pp. 92, 93).
(e) Harvey.
(1) Add 3.7 cc. of a 10% anhydrous 1730. Wurtz's Litmus Lactose Agar
sodium carbonate solution to
Constituents:
1000.0 cc. of infusion agar (see
1. Infusion Agar 1000.0 cc.
variant (v) medium 1661).
2. Lactose (2.0%) 20.0 g.
(2) Dissolve 2.0 g. lactose in 25.0 cc.
3. Litmus
of distilled water.
Preparation
(3) Dissolve 0.5 g. anhydrous NaoSOs,
(1) Add 2.0% lactose to infusion agar
and saturated alcoholic
1.0 cc. of a
with a slightly alkaline (0.5% re-
basic fuchsin solution in 10.0 cc.
action).
distilled water.
(2) Tint sterile (1) by the addition of a
(4) Mix (2) and (3).
sufficient quantity of sterile litmus
(5) Add (4) to (1).
solution.
(6) Pour in plates.
and the litmus
Sterilization: Sterilize (1)
(7) Dry the surface of the medium
solution separately, method not given.
for 15 minutes in the incubator.
Use: Determine fermentation of lactose.
(f) Harvey.
Add 10.0 cc. of a 10.0% crystalline Variants
(1)
(a) Smith (1905) gave the following
sodium bicarbonate solution to
1000.0 cc. of infusion agar (see method of preparation.
(3) Add 5.0 cc. of alcoholic fuchsin (3) Make up lost weight.
(1), keeping the temperature below References: Smith (1902 p. 94), (1905,
60C. p. 196), Heineman (1905 p. 127), Com-
(8) Titrate and adjust the reaction to mittee A. P. H. A. (1913 p. 129), Giltner
neutral to phenolphthalein adding (1921 p. 379, 380).
normal HCl or NaOH.
1731. Bitter's China Blue Malachite Green
(9) Heat on a water bath for 40 min-
Agar
utes.
(10) Make up lost weight. Constituents
(11) Readjust to neutrality if neces- 1. Infusion agar (containing
sary and boil 5 minutes. 2.0 or 3.0% peptone and
(12) Restore lost weight. NaCl) 1000.0 cc.
(13) Filter thru absorbent cotton and 2. Lactose 20.0 g.
cotton flannel. 3. China blue (sat. aqueous
(14) Titrate and record final reaction. solution (Hochster)) 90 drops
(15) Add 1.0% lactose and sufficient 4. Malachite green (0.1%
azolitmin solution. solution, crystalline extra
(16) Tube in 10.0 cc. quantities. Hochst.) 25.0 cc.
(17) Sterilize minutes in the
for 15 Preparation
autoclave at 120C. or for 30 min- (1) Prepare meat infusion agar with
utes on each 3 successive days. 2.0 or3.0% peptone and NaCl.
(d) Giltner gave the following methods (2) Neutralize (1) with N/1 NaOH.
of preparation. The medium was (3) Add 2.0% lactose.
used in water analysis. (4) Boil a few minutes.
I. (1) Prepare infusion agar using (5) To each add 9 drops
100.0 cc. of (4)
equal parts meat infusion and of a saturated (about 10%) watery
water, with 1.0% peptone and china blue solution (Hochster Farb-
1.5% agar. werks 10 g. 2M) by means of a 1.0 cc.
(2) Adjust the reaction to -f-1.0. pipette.
(3) Add 1.0% lactose and 2.0% (6) Then add to each 100.0 cc. of (5)
azolitmin just before tubing. 2,5 cc. of a 0.1% malachite green
(4) Tube. (cryst extra Hochst, ) solution.
(5) Sterilize for 30 minutes on 3 (7) Pour sterile (6) in thin layers in
successive days. sterile plates.
II. (1) Preparation of meat infusion Sterilization: Sterilize 10 minutes in the
not given. autoclave.
(2) Strain (1) thru a piece of clean Use: Diagnosis of typhoid fever. Author
cheese cloth. reported that all acid forming organisms
(3) Place 2.0% washed agar in (coli, etc.) gave bright blue colonies.
distilled water. All non-lactose fermenting organisms
(4) Weigh (3). (typhoid) or alkali forming organisms
(5) Digest over a free flame. gave a colorless or yellowish colony.
(6) Add distilled water to make Reference: Bitter (1911 p. 474).
up the loss in weight.
Add lactose 2.0% and peptone 1732. Guth's Alizarine Lactose Agar
(7)
and mix (Klimmer)
2.0% to (6) until solu-
tion is complete. Constituents
(8) Add (2) to (7). 1. Meat infusion (beef or
(9) Adjust the reaction to 0. horse) 1000.0 cc.
(10) Add 2.0% azolitmin solution. 2. Agar 30.0 g.
(11) Boil over a free flame. 3. Peptone 10.0 g.
(12) Distribute in 100.0 cc. lots in 4. NaCl 5.0 g.
250.0 cc. Florence flasks. 5. Lactose 10.0 g.
(13) Sterilization not specified. 6. Alizarin 0.8 g.
532 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Add 50.0 to 70.0 cc. of tenth normal (3) Prepare a 2.0% solution of meta-
NaOH and 10.0 g. lactose dissolved in chrome yellow (II RD) in water.
20.0 or 30.0 cc. water. (4) Boil two minutes.
(6) Dissolve 0.6 g. NaOH and 0.8 g. ali- (5) Prepare a 1.0% water blue (6B extra
zarine in 100.0 cc. distilled water and P) in solution in water and dissolve
boil several minutes. 100.0 g. lactose in 175.0 cc. of the
(7) Add (6) to (5). solution.
(8) Thoroly mix sterile (7) and pour into (6) Boil (5) 10 minutes.
sterile plates. (7) Add 125.0 cc. of (4) and 175.0 cc. of
Sterilization: Sterilize by steaming for (6) to 2000.0 cc. (2).
30 minutes. Sterilization: Method not given.
Use Detection of typhoid and paratyphoid
: Use: Detection dysentery or typhoid
of
bacteria. Author reported that B. coli bacilli. The author reported that Bad.
lightened the medium coloring it yellow, coli gave a deep blue medium. Typhoid
typhoid bacilli, paratyphoid and enteri- and dysentery gave a yellowish medium.
tidis bacilli left the medium opaque and Medium was green. This medium inhib-
formed greyish blue colonies. The addi- ited neither the typhoid, dysentery nor
tion of malachite green completely in- coli organisms.
hibited the colon bacteria; however, 40 Reference: Gassner (1917-18 p. 221).
to 48 hours were required for the develop-
ment of the typhoid colonies. 1735. Torrey's Brom Cresol Purple Lactose
Variants: Klimmer added 1.7 cc. of a 0.1% Agar
malachite green solution to each 100.0 cc. Constituents
ofmedium. 1. Water 1000.0 cc.
Reference: Klimmer (1923 p. 215). 2. Beef heart 500.0 g.
3. Peptone 10.0 g.
1733. Harvey's Starch Agar 4. Agar 15.0 g.
5. Lactose 1.0 g.
Constituents:
6. Brom Cresol Purple (Sat.
1. Infusion agar 1000.0 cc.
ale. soln.) 0.75 cc.
2. Starch (water soluble) 2.0 g.
Preparation
Preparation
(1) Boil 500.0 g. finely chopped beef
(1) See medium 1661, Variant (v) for the
heart (other meat material may be
preparation of infusion agar.
used) in one liter of water for 15
(2) Add 2.0 g. water soluble starch to
minutes over a free flame
1000.0 cc. of (1).
(2) Strain through canton flannel and
Sterilization: Not specified.
absorbent cotton.
Use: General culture medium.
(3) Make up the loss in filtrate and add
Reference: Harvey (1921-22 p. 112).
10.0 g. peptone and 15.0 g. flaked
agar.
1734. Gassner's Metachrome Yellow Water
(4) Heat in the Arnold until solution is
Blue Infusion Agar
complete.
Constituents: (5) Adjust to pH = 7.4 and place in the
1. Infusion agar 2000.0 cc. Arnold for 30 minutes.
2. Metachrome Yellow (II RD) (6) Readjust the reaction if necessary.
(2.0%) 125.0 cc. (7) Filter thru cotton and add 1.0 g.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 533
lactose and a sufficient quantity of a infusion agar (see variant (v) medium
saturated alcoholic solution of brom 1661). The medium was used to
cresol purple (generally about 0.75 cultivate V. cholerae.
CO.) to give the desired depth of (b) Harvey added 50.0 cc. of glycerol to
color. 1000.0 cc. of infusion agar (see
(8)Flask in 200.0 cc. quantities. variant (v) medium 1661).
Sterilization: Autoclave at 15 pounds pres- Reference: Abbott (1921 p. 132), Harvey
sure for 10 minutes. (1921-22 p. 87).
Use: Analysis of fecal flora.
1737. Kowalski's Glycerol Lung Infusion
Reference: Torrey (1926 p. 353).
Agar
1736. Abbott's Glycerol Infusion Agar
Constituents
Constituents 1. Water 2500.0 cc.
1. Water 1000.0 cc. 2. Lung, calf 1000.0 g.
2. Beef 500.0 g. 3. NaCl 18.0 g.
3. Peptone (1.0%) 10.0 g. 4. Potassium phos-
4. NaCl (0.5%) 5.0 g. phate 9.0 g.
5. Agar (1.0 to 1.5%) . . 10.0 to 15.0 g. 5. (NH4)2S04 9.0g.
6. Glycerol (5.0 to 7.0%). 50.0 to 70.0 g. 6. Na2S04 25.0 g.
Preparation: 7. Sug.ar 90.0 g.
(1) Add 500.0 g. of chopped lean beef to 8. Peptone 25.0 g.
1 water and soak for 24 hours,
liter of 9. Agar 40.0 g.
kept at ice box temperature. 10. Glycerol 20.0 to 25.0 g.
(2) Strain thru a coarse towel and press Preparation
until a liter of fluid is obtained. (1) Remove the lung of a calf imme-
(3) Dissolve 10.0 g. (1.0%) peptone, and diately after death. Grind it in a
5.0 g. (0.5%) NaCl. meat grinding machine, keeping as
(4) Dissolve 1.0 to 1.5% agar in (3) by clean as possible during the
boiling in a porcelain lined iron procedure.
vessel. Add 250 to 300.0 cc. water (2) Add two liters of water to 1000.0 g.
and boil until this volume water
of ground (1 )
of finely Place in a glass
.
To the clear, straw colored filtrate, of water and alcohol. Dilute with 20
(11)
whose reaction should be neutral, or parts water.
at the most slightly alkaline, add 8 (2) Compositionof usual infusion broth
Use: General culture medium. with 1000.0 cc. of water, and allow to
Reference: Harvey (1921-22 p. 111). stand for 24 hours at room tempera-
ture.
1740. Omelianski's Formate Agar Boil for 15 minutes over a free flame.
(2)
1. Meat infusion 1000.0 cc. paper and press out the residue.
20.0 g. Add chopped agar,
12.0 g. of finely
2. Agar (4)
10.0 g. peptone and 5.0 g.
10.0 g. of Witte's
3. Sodium formate
4. Phenolphthalein solution . . 200.0 cc. of NaCl, and boil over a free flame,
egg albumin stirred up well in a little (5) Adjust to +1 with 2X, NaOH.
water. (6) Heat in Arnold sterilizer for ^ hour.
(6) Add soda solution until the reaction (7) Adjust again to -|-1.
is distinctly alkaline and heat in the (8) Cool to 55C., clear with egg white
autoclave at 120C. for 20 minutes. and filter thru cotton.
(7) When taken from the autoclave pour (9) Flask in 100.0 cc. or 200.0 cc. lots.
immediately upon a Chardin filter (10) When ready for use melt sterile (9),
paper. add 1.0% lactose and 1.0% saccha-
(8) Distribute in about 10-12 cm. layer in rose. (1.0 g. to 100.0 cc. medium of
mm. by 22 cm. test tubes.
a 20 to 22 each sugar.)
Sterilization: Sterilize at 112 to 115C. for (11) Prepare a 3.0% stock solution of
15 to 20 minutes. yellowish eosin in distilled water.
Use: Cultivation of anaerobes. Meier (12) Prepare a 1.0% stock solution of
made bacterial counts from milk and brilliant green in 50.0% alcohol, a
whey in a similar medium. g% solution in distilled water.
Variants : Meier prepared a similar medium (13) Add1.0 cc. of (11) and 1.0 cc. of (12)
as follows: to each 50.0 cc. of medium.
(1) Boil 500.0 g. of fat and tendon-free (14) Shake well.
beef in one liter of water. (15) Pour into sterile Petri dishes.
(2) Filter. Sterilization: Sterilize (9) by heating in the
(3) Dissolve 2.5 g. lactose, 2.5 g. glucose, autoclave at 120C. for 20 minutes.
5.0 g. Witte's peptone, 5.0 g. NaCl Use: Isolation of typhoid bacilli from
and 5.0 g. agar in 1. stools. Author reported that typhoid
(4) Neutralize by the addition of KOH. colonies after 18 hours were grayish in
AddKOH until turmeric paper is color by reflected light. B. coli colonies
turned quite weakly brownish red. were red. In transmitted light typhoid
(5) Sterilization not specified. were colorless and transparent. Liebig's
References: de Gasperi and Savini (1911 p. meat extract cannot be substituted for
248), Meier (1918 p. 454). beef infusion.
References: Teague and Clurman (1916 p.
1743. Teague and Clurman's Eosin Brilliant
651), Tanner (1919 p. 54).
Green Agar
Constituents:
1744. Kan-Ichiro Morishima's Lead
Acetate China Blue Agar
1. Distilled water 1000.0 cc.
2. Beef, chopped 500.0 g. Constituents
3. Peptone (Witte) 10.0 g. 1. Meat infusion agar (compo-
4. XaCl(C. P.) 5.0 g. sition not given) 2000.0 cc.
5. Agar 15.0 g. 2. Neutral 2.0% solution lead
6. Lactose 10.0 g. acetate in distilled water. . . 50.0 cc.
7. Saccharose 10.0 g. 3. 1.0% solution China blue
8. 3.0% 3'ellowish eosin 4. N sodium hydrate solution
solution 20.0 cc. 5. Lactose 10.0 g.
9. 1.0% brilliant green solu- 6. Glucose 1-0 g.
tion 20.0 cc. Preparation
Preparation (1) Preparation or composition of meat
(1) Soak beef in water in the ice chest infusion agar not given except that
over night. it is to be prepared in the usual way.
(2) Squeeze infusion thru cheese cloth, (2) Clear with egg white.
heat in Arnold sterilizer and pass (3) Titrate while hot to -0.2 to -0.4
thru filter paper. with phenolphthalein.
(3) Add 3, 4 and 5 to warm (2). Rub (4) Prepare 2.0% neutral lead acetate
peptone into paste before adding. in sterile distilled water. Heat for
(4) Heat in autoclave for 30 minutes at 5hour at 100C. in water bath.
120C. (5) Melt (3), and add 5.0 cc. of (4) to
536 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
every 100.0 cc. agar. Cool agar to (7) Steam 15 minutes. The mixture
60C. or precipitate will be formed. becomes dark brown and very
(6) Transfer to small test tubes, to a cloudy.
depth of 1.5 cc. (8) Add 50.0 cc. of sterile (2), 50.0 cc.
(7) Stock solution of 1.0% china blue in of sterile (3), 2.5 cc. sterile (4) and
distilled water is prepared. 25.0 cc. of sterile (5) to hot (7).
(8) Add 0.4 cc. N sodium hydrate to (9) Place the flasks in a sloping position
10.0 cc. of (7). Heat on water bath to allow the precipitate formed to
for 10 minutes at 100C. (Color settle.
changes from blue to brown.) (10) Pour plates carefully, avoiding as
(9) added to 100.0 cc. of
1.2 cc. of (8) is far as possible the transference of
(3) reaction -0.2 to -0.4. settled precipitate.
(10) Add 1.0% lactose and 0.1% glucose (11) Place the plates to dry in the in-
to (9) and heat in water bath at cubator with their agar surface
100C. for 10 minutes. downwards.
(11) Cool to 60C. (12) Preserve in the dark for three days
(12) Add to (6) making a second layer in before use.
the tubes. Sterilization: Sterilize (1), (2), (3) and (5)
(13) Incubate over night make stab at 100C.
inoculation. Use : Isolation of V. cholerae.
Sterilization: Not specified.
Reference: Harvey (1921-22 p. 93).
Use: Differentiation of B. paratyphosus
A, B. paratyphotus B, B. enteritidis and
1746. v. Szaboky's Glycerol Lung Agar
B. coll.
Variants: The author prepared a similar Constituents
medium but used 1.0% inositol and 0.1% 1. Water 2000.0 cc.
arabinose instead of 1.0% glucose. 2. Lung 1000.0 g.
Reference: Kan-Ichiro Morishima (1918 3. Agar 10.0 g.
p. 19). 4. Peptone (Witte) 20.0 g.
5. Glycerol 100.0 g.
1745. Aronson's Fuchsin Sulphite Agar
6. Glucose 10.0 g.
(Harvey)
Preparation
Constituents (1) Boil 1 kilogram of lung with 2 kilo-
1. Infusion agar 1000.0 cc. (Time not specified.)
grams of water.
2. NasCOa (10.0% anhydrous (2) Filter and dissolve 3, 4, 5 and 6 in the
soln.) 60.0 cc.
filtrate.
3. Sucrose (20.0% soln.) 50.0 cc.
(3) Neutralize to litmus.
4. De.xtrin (20.0% soln.) 50.0 cc.
(4) Heat again.
5. Fuchsin (sat. alcoholic Distribute in 20.0 cc. lots into suitable
(5)
soln.) 2.5 cc. wide sterile test tubes.
6. Na.SOa (10.0% soln.) 25.0 cc.
manner
Sterilization: Sterilize in the usual
Preparation
by short heating in streaming steam (time
(1) Prepare a 10.0% anhydrous NaaCOs
or number of days not specified).
solution.
Use: Cultivation of tubercle bacilli. The
(2) Prepare a 20.0% sucrose solution.
author reported that colonies developed
(3) Prepare a 20.0% dextrin solution.
in one day when reaction was 0.5% alka-
(4) Prepare a saturated alcoholic fuch-
line to 0.5% acid.
sin solution.
Variants Tubercular lung may be used as
(5) Prepare a 10.0% NaaSOs solution :
Mix 60.0 cc. sterile (1), heated to material must be sterilized on 5 days for
(6)
2 hours each.
45C. and 1000.0 cc. melted sterile
infusion agar (see variant (v) Reference: v. Szaboky (1907 p. 652), Kolle
1661 for preparation), cooled to 45 C. and Wassermann (1912 p. 413).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 537
obtained.
(7) Tube and slant after final steriliza- 1749. Lubenau's Lactose Caffeine Agar
tion. Constituents
Sterilization: Partial sterilization given in 1. Distilled water 1000.0 cc.
step (3) above. Sterilize (7) once in the 2. Beef 5OO.O g.
Arnold sterilizer as further heating tends 3. Agar (2.0 to 3.0%) . . . 20.0 to 30.0 g.
to break up the carbohydrates. Incubate 4. Peptone 3.0 g.
24 hours and discard tubes showing 5. NaCl (0.5%) 5.0 g.
growth. 6. Lactose (0.5%) 5.0 g.
Use To differentiate between typhoid and
:
7. Caffeine
paratyphoid A and B bacilli. Author 8. Litmus
reported that typhoid bacillus fermented Preparation
dextrin-inositol with acid formation. Boil 500.0 g. of finely chopped lean
(1)
Para-typhoid bacillus B fermented dex- beef with one liter distilled water for
trin-inositol with acid and gas formation. 30 minutes.
Para-typhoid bacillus A did not ferment Filter, and make up to one liter.
(2)
dextrin-inositol. Shiga-Kruse and Hiss- Add
(3) 3, 4, and 5 to (2) and dissolve by
Russel dysentery types did not ferment boiling in a salt water bath.
dextrin-inositol. Flexner Rosen dysen- (4) Neutralize to litmus.
tery types fermented dextrin-inositol.
(5) Boil and filter.
B. typhi murium and B. pullorum did not
(6) Add 60.0 cc. of sterile litmus solution
ferment dextrin-inositol. B. aerogenes (preparation not given) to hot ster-
fermented dextrin-inositol with gas ile (5).
formation. Mix thoroughly, and allow to cool.
(7)
Reference: Hulton-Frankel (1918 p. 380). Add 110.0 cc. of a sterile 6.0% caffeine
(8)
solution containing 6.0% peptone. The (5) Add 7.0 cc. of normal soda solution
lactose, litmus and caffeine were added to or 10.0 cc. of a 10.0% soda solution.
the hot sterile nutrient agar. (6) Boil.
References: Lubenau (1907 p. 249), (7) Filter.
Harvey (1921-22 p. 92). (8) Add 10.0 g. c.p. lactose, 5.0 cc. of a
concentrated filtered alcoholic
1750. Harvey's Caffeine Endo Agar fuchsin solution and 25.0 cc. of a
Add 33.0cc. of 10.0% caffeine to 1000.0 cc. 10.0% freshly prepared sodium
of Harvey's modification of Endo agar (see sulphite solution.
variant (e) medium 1729). (9) Add 0.33% crystalline caffeine to
(8).
1751. Gathgen's Caffeine Fuchsin Sulphite Klimmer reported that typhoid,
Agar (Bezanson) paratyphoid and cholera colonies
Constituents were blue; colon colonies were red.
1. Infusion agar (3.0%) 1000.0 cc. References: Bezangon (1920 p. 345),
2. Lactose (10.0% solution). . . 120.0 cc. Harvey (1921-22 pp. 92, 93), Klimmer
3. Fuchsin (1923 p. 210).
4. Na.SOa (10.0% soln.) 25.0 cc.
5. Caffeine 3.3 g. 1752. Viehoever's Basal Glucose Extract
Preparation: Agar
(1) Add 120.0 cc. of a 10.0% soda solution
Constituents
to a liter of neutral 3.0% infusion
1. Water 500.0 cc.
agar.
2. Peptone (Witte) 6.0 g.
(2) Add 120.0 cc. of a 10.0% lactose
3. Meat extract (Liebig's) 4.0 g.
solution to (1).
4. NaCl 1.0 g.
(3) Add 10.0 g. of crystalline fuchsin to
5. Glucose 50 g.
100.0 cc. of 96.0% alcohol and allow to
6. Agar 10.0 g.
stand for 2 hours.
Preparation
(4) Decant (3) and add 2.5 cc. of the
(1) Dissolve 2, 3, 4, 5 and 6 in 1.
supernatant fluid to sterile (2).
(2) Dissolve one of the added nutrients
(5) Prepare a 10.0% solution of NazSOa-
in (1) and adjust the reaction as
(6) Add 25.0 cc. of sterile (5) to (4).
indicated.
(7) Add 0.33 g. of pure caffeine to each
(3) Tube in 5.0 cc. lots.
100.0 cc. quantity.
Sterilization: Method not given.
(8) Pour in plates.
Use: To study spore production by urea
Sterilization: Sterilize (2) at 100C. for one
splitting organisms, Bac. probatus.
hour. Sterilize (5) by heating at 80C.
Added nutrients: The author prepared one
for one hour.
colon typhoid of the mediums as indicated:
Use: Differentiation of
group.
(a) Neutral basal medium + 0.25%
NasCOs.
Variants
(a) Harvey added 33.0 cc. of a 10.0% (b) Neutral basal medium + 1.0%
caffeine solution to 1000.0 cc. of CaCOa.
Harvey's modification of Endo agar (c) Neutral basal medium + 2.0% urea
(see variant (e) 1729).
+ 0.3% (NH4)2C03.
(b) Klimmer prepared a similar medium Variants: The author gave the following
as follows: variants:
Soak 30.0 to 40.0 g. agar in meat (a) Basal medium with 0.3, 0.2 or 0.1
(1)
water or a 1.0% meat extract solu- concentration of nutrients 0.1% +
tion for several hours. NasCOa.
(2) Boil for 3 hours. (b) Basal medium with 0.3 concentration
1753. Bacto Dextrose Agar (Dehydrated) (7) Allow to stand for 3 hours at 100C.
(8) Neutralize once more with Na2C03
Constituents: and heat for a short time.
1. Distilled water
(9) Filter.
2. Beef extract (Bacto) 3.0 g.
Add 10.0 g. glucose and
(10) sterilize
3. Peptone (Bacto) 5.0 g.
on three successive days.
4. Glucose (Bacto) 10.0 g.
(b) Bredeman cultivated Bac. amylo-
5. Agar (Bacto) 15.0 g.
bacter and prepared the medium as
Preparation follows:
(1) Dissolve 33.0 g. of Bacto Dextrose Wash 215.0 g. of agar in running
(1)
Agar (Dehydrated) in 1000.0 cc. tap water for three or four hours.
distilled water by Ijoiling or prefer-
(2) Add water to (1) until the total
ably autoclaving for 10 minutes at 15
weight is 10 kilograms. Dissolve
pounds. the agar in the water.
(2) If sterilized 10 minutes at 15 pounds Clarify with egg white and filter.
(3)
pH = 7.4. (4) Dissolve 7.2 g. Witte's peptone,
Sterilization: Sterilize in the usual manner.
6.0 g. dextrose, 4.8 g. Liebig's meat
Use: General culture medium. Add 8.0 g. extract and 1.2 g. NaCl in 200.0
NaCl per liter to prevent hemolysis when cc. of water.
using as a base for blood agar.
(5) Adjust the reaction so that it is
Reference: Digestive Ferments Co., (1925
slightly alkaline.
p. 11).
(6) Boil and filter.
(7) Remove from the container in a (3) Boil one hour and filter.
solid state and cut away the (4) Adjust the reaction once more and
bottom part containing the sedi- add 0.3% agar to (3).
ment. (5) Dissolve the agar by boiling in the
(8) Melt the clear top portion in a autoclave for one hour.
steamer and pour onto a filter. (6) Filter while hot.
(9) Distribute into small test tubes (7) Add 1.0 or 2.0 cc. of a concentrated
(10 mm. in diameter and 12 cm. neutral red solution (exact con-
high). centration not given).
(10) Sterilize two times (method not (8) Dissolve 0.15% glucose in (7).
specified). (9) Distribute into 5.0 cc. lots in test
(f) Robinson and Rettger used a medium tubes.
containing 2.0% opsine, 0.5% NaCl, Sterilization : Sterilize for 15 minutes to two
1.5% agar, 10.0 g. glucose, and 5.0% hours in the autoclave (pressure not
Liebig'smeat extract. specified.)
(g) Dawson studied the variation of B.
Use: Differentiation between coli and ty-
on a medium composed of
coll 2.5 g.
phoid organisms. Author reported that
peptone, 10.0 g. meat extract, 10.0 g. coli types produced decolorization and
glucose and 20.0 g. agar (10.0 g. fluorescens after 24 hours. Typhoid
glycerol may be added). colonies caused no change in color of the
(h) Stitt prepared the medium as follows: medium.
(1) Mix 3.0 g. meat extract, 10.0 g.
References: Oldekop (1904p. 123), Klimmer
peptone, 5.0 g. NaCl, 10.0 g. glu-
(1923 p. 210).
cose and 15.0 g. agar with 1000.0
cc. water containing the whites of
1756. Bacto Purple Lactose Agar
one or two eggs.
(Dehydrated)
(2) Boil in a rice cooker until solution
is complete. Constituents:
(3) Filter. 1. Distilled water
(4) Sterilization not specified. 2. Beef extract (Bacto) 3.0 g.
References: Henneberg (1898 p. 18), Gott- 3. Peptone (Bacto) 5.0 g.
heil (1901 p. 432), Bredeman (1909 p. 4. Agar (Bacto) 10.0 g.
409), Zikes (1911 p. 148), Bachmann 5. Lactose (Bacto) 10.0 g.
(1912-13 p. 7), Burckhardt and Enriquez 6. Dibromcresolsulphonephth-
(1917-18 p. 16), Robinson and Rettger alein 0.025 g.
(1918 p. 202), Dawson (1919 p. 142), Preparation
Stitt (1924 p. 38). Dissolve 29.0 g. of Bacto Purple
(1)
Lactose Agar (Dehydrated) in 1000.0
1755. Oldekop's Neutral Red Glucose
cc. of distilled water by boiling or
Extract Agar
autoclaving.
Constituents necessary.
(2) Restore the loss if
1. Distilled water 500.0 cc.
(3) If sterilized at 15 pounds pressure for
2. Meat extract (Lie-
20 minutes, pH = 6.5.
big's) 5.0 g.
Sterilization: Sterilize in the usual manner.
3. NaCl 2.5 g.
4. Peptone (Witte sic- Use: To determine lactose fermentation.
cum) 10.0 g. Acid colonies are yellow, alkali producers
5. Agar 1.5 g. are purple. Author reported that this
6. Neutral red, concen- medium is superior to 1757.
trated solution 1.0 or 2.0 cc. Reference: Digestive Ferments Co. (1925
7. Glucose (0.15%) 0.75 g. p. 12).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 541
1757. Bacto Litmus Lactose Agar Sterilization: Sterilize (6) in the autoclave
(Dehydrated) at 15 pounds pressure (120C.) for 15
minutes.
Constituents:
Use: To determine the fermentation of
1. Distilled water
lactose.
2. Beef extract (Bacto) 3.0 g.
Variants: The following investigators pre-
3. Peptone (Bacto) 5.0 g.
pared similar media as indicated below:
4. Agar (Bacto) 10.0 g.
(a) Meyer.
5. Lactose (Bacto) 10.0 g.
(1) Dissolve 10.0 g. peptone, 10.0 g.
6. Azolitmin 10 g.
meat extract and 30.0 g. agar (3.0%)
Preparation
in 1000.0 cc. of water.
(1) Dissolve 29.0 g. of Bacto Litmus
(2) Distribute in 400.0 cc. portions.
Lactose Agar (Dehydrated) in 1000 not given.
(3) Sterilization
cc. of distilled water by boiling or add 16.0 g.
(4) To each 400.0 cc. lot
autoclaving.
of a sterile 25.0% lactose solution
(2) Restore the loss if necessary.
and 12.0 cc. of a sterile 8.0% litmus
(3) If sterilized at 15 pounds for 20
solution.
minutes pH = 7.0.
(5) Pour into sterile plates and allow to
Sterilization : Sterilize in the usual manner.
harden in open air.
Use: To determine fermentation of lactose.
(b) Bendick (Tanner).
Reference: Digestive Ferments Co. (1925 Dissolve 15.0 g. agar, 5.0 g. beef
(1)
p. 12). extract, 10.0 g. NaCl and 10.0 g.
peptone in 1000.0 cc. of water.
1758. A.P.H.A, Litmus Lactose Extract
(2) Clarify with egg and filter.
Agar (1917) not
(3) Adjustment of reaction is
Constituents: necessary.
1. Distilled water 1000.0 cc. (4) Distribute in 250.0 cc. lots in 500.0
2. Beef extract 3.0 g. cc. flasks.
and 12.0 g. agar, dried for 30 minutes tube from the flask, keeping the
at 105C. before weighing, to 1000.0 contents mixed well.
cc. distilled water. (c) Stitt.
Prepare nutrient agar by dissolving
(2) Boil over a water bath until solution (1)
sterile agar just before it is poured tribute in 50.0 or 100.0 cc. Erlen-
pounds for 15 minutes, or in the (12) Distribute in 200-400 cc. lots and
Arnold, store in the dark.
(d) Conradi-Drigalski (Stitt). (13) Sterilization not specified.
(I) Add 1.0 cc. of a 1 to 100 solution of (b) KastleandElvove.
crystal violet in distilled water to (1) Dissolve 10.0 g. Liebig's extract,
100.0 cc. of the medium as prepared 10.0 g. peptone and 5.0 g. NaCl in
by Stitt above (variant (c)). 1000.0 cc. distilled water by heat.
References: Committee A.P.H.A. (1917 (2) Cool and add 40.0 g. powdered
p. 97), Meyer (1917 p. 238), Tanner (1919 agar. Allow agar to settle.
p. 48), Committee A.P.H.A. (1920 p. 97), (3) Place in Arnold and cook 3 hours.
Stitt (1923 p. 49). (4) Neutralize to litmus with NaoCOs.
(5) Filter thru cotton on a Buchner
1759. Endo's Fuchsin Sulphite Agar
filteror allow to settle, rejecting
(Heinemann)
turbid bottom part.
Constituents Add 10.0 of a sterile
(6) cc. 10.0%
1. Extract agar (3.0%) 1000.0 cc. Na2C03 solution to^(5) after
2. Lactose 10.0 g. filtering.
3. Fuchsin (ale. soln.) 5.0 cc. (7) This medium may be storedTin
4. NaaSOa (10.0% soln.) 25.0 cc. 100-200 or 400.0 cc. flasks until
5. NaOH (10.0% soln.) 10.0 cc. desired for use.
Preparation (8) When desired for use melt (7) and
(1) Prepare a medium from 2, 3 and 4 to each liter add c.p. 10 g. lactose
using 3.0% extract agar as a base. and 5.0 cc. of a 10.0% alcoholic
(2) Add 10.0 cc. of a 10.0% NaOH fuchsin solution (freshly | pre-
solution. pared).
Sterilization: Not specified. (9) Prepare a 10.0% alcoholic fuchsin
Use: Isolation of typhoid bacilli. Coli solutionby shaking 10.0 g fuchsin
colonies large and red, typhoid colonies (not acid fuchsin) with 100.0 cc.
small, colorless and bluish. of 96% alcohol, allow to stand 24
Variants: The following authors have pre- hours. Decant supernatant fluid
pared similar media as indicated: and filter this fluid each time
(a) Klinger. immediately before use.
(1) Add 20.0 g. Liebig's meat extract, (10) After fuchsin and sulphite have
20.0 g. Witte's peptone, 10.0 g. been added to (7), shake vigor-
NaCl and 80.0 g. agar to 1000.0 cc. ously and place unstoppered in
of water. sterilizer for 5 to 10 minutes to
(2) Heat for either 3 hours in stream- allow foam to settle.
ing steam, or 2 hours at 110, or (11) Add 25.0 cc. of a freshly prepared
one hour at 120C. sterile 5.0% anhydrous Na.SO,
(3) Filter thru a thick layer of cbtton. solution to (10) and mix by rotat-
(4) Neutralize to litmus. ing flask.
(5) Add 20.0 cc. of a sterile 10.0% soda (12) Sterilize in Arnold for a few minutes
solution to (4). and pour in Petri dishes while
(6) Add 20.0 g. of chemical pure lac- steaming hot. Solidified medium
tose to (5). nearly colorless to transmitted
(7) Add 10.0 g. of crystalline fuchsin to light and rose or flesh to reflected
100.0 cc. of 96% alcohol and allow light.
to stand for 20 hours. (c) Kendall and Day.
(8) Pour off the saturated solution. (1) "Dust" 15.0 g. powdered agar
(9) Add 10.0 cc. of (8) to (6). upon the surface of cold tap water.
(10) Add 50.0 cc. of a freshly prepared Allow to settle into the medium
10.0% NasSOs solution to (9). before heat is applied or other
(II) INIix well. ingredients added.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 543
Heat in Arnold sterilizer until the (14) Pour each tube into sterile petri
(11)
agar is melted and the lactose dish and allow to harden.
to each 100.0 cc. of the medium. other half of each plate with a
Mix thoroly and pour into sterile similar coli culture.
(13)
plates. (16) Incubate for 24 hours.
Allow to harden (with covers (17) The plate showing most typical
(14)
removed) in the incubator for 30 luxuriant growth of both the coli
minutes. The plates are now and typhoid is chosen as the
ready for inoculation. proper amount of acid or alkali
Kinyoun and Deiter. to add to the remainder of the
(d)
Mix 80.0 g. Witte's peptone, 40.0 medium.
(1)
powdered (18) Add the necessary amount of acid
g. NaCl and 160.0 g.
agar with sufficient water to make or alkali to the entire lot of agar
a smooth paste. (9).
Add suflBcient water to make 8000.0 (19) Distribute in 100.0 cc. lots m
(2)
cc.
(20) Sterilize in the Arnold for an hour
(3) Add 80.0 g. Liebig's meat extract
(not necessary to mix). on two successive days.
Place in the Arnold sterilizer and (21) Store until ready for use.
(4)
steam until solution is complete. (22) When ready for use add 1.0 g.
crystalline lactose to each flask of
Usually requires about an hour.
Cool to 55C. and titrate to +1 melted agar.
(5)
using phenolphthalein as an (23) When the lactose has dissolved
indicator. add 5.0 cc. of a 5.0% solution of
Place in tall beakers, steam an anhydrous NaaSOs, freshly pre-
(6)
hour and allow to solidify. (Allow pared and still hot to each flask.
Add 1.0 cc. of a half saturated
the beakers to stay in the steamer (24)
alcoholic solution of basic fuchsin
over night.)
Remove the solid agar from the to each flask.
(7)
beaker and cut away all the agar (25) Mix well.
(26) Pour into plates.
containing sediment.
544 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(e) Committee A.P.H.A. (1913). (4) Adjust the reaction. Best results
(1) Add 30.0 g. of agar to one liter of are obtained when the medium is
cold water by sifting successive just slightly alkaline to litmus.
small portions upon the surface (5) Add exactly 3.0 cc. of a saturated
and allowing them to settle. alcoholic fuchsin solution and 30.0
(2) Add 10.0 g. Witte's peptone, 5.0 cc. of a 10.0% solution of anhy-
g. meat extract to (1) and boil drous Na2S03 (or 3.0 cc. of a satu-
until solution is complete. rated aqueous solution of Na2S03
(3) Neutralize to litmus by adding may be added).
NasCOs. (6) Tube.
(4) Distribute in 100.0 cc. quantities. (7) Sterilize for 20 minutes on each of
(5) Prepare a 10.0% solution of 3 successive days in an Arnold
Na2S03 in water. sterilizer.
(6) Prepare a 10.0% basic fuchsin (h) Committee A.P.H.A. (1917-1920).
solution in 96% alcohol. (1) Add beef extract, 10.0 g.
5.0 g.
(7) Add 2.0 cc. of (6) to 10.0 cc. of (5) peptone and 30.0 g. agar, dried at
and steam for a few minutes in 105C. for one hour before weigh-
the Arnold steamer. ing to 1000.0 cc. of distilled water.
(8) Add 1.0 g. chemically pure lactose (2) Boil on a water bath until all the
to each 100.0 cc. of sterile (4). agar is dissolved and then make
(9) Melt (8) and add 0.5 cc. of (7). up the water lost by evaporation.
(10) Pour in plates. (3) Cool to 45C. in a cold bath, then
(11) Sterilize (4) for 2 hours in stream- warm to 65 C. in the same bath
ing steam. without stirring.
(f Robinson and Rettger. (4) Make up lost weight.
(1)Dissolve 25.0 g. powdered agar, (5) Titrate and if the reaction is not
10.0 g. Peptone (American brand), already between neutral and +1,
and 5.0 g. Liebig's meat extract in adjust to neutral.
1000.0 cc. water. (6) Filter thru cloth and cotton until
(2) Make neutral to litmus. clear.
(3) Steam in the autoclave at 12 to 15 (7) Distribute in 100.0 cc. (or larger)
pounds extra pressure for 35 to 40 quantities in flasks.
minutes. (8) Sterilize in the autoclave at 15
(4) Filter thru absorbent cotton and pounds pressure (120C.) for 15
cheese cloth. minutes after the pressure reaches
(5) Add NajCOs and heat for 10 15 pounds.
minutes on boiling water bath. (9) Prepare a 10.0% solution of basic
Reaction 0.1 to phenolphthalein. fuchsin in 95.0% alcohol, allow to
(6) Add 10.0 g. lactose and 5.0 cc. of a stand 20 hours, decant and filter
saturated alcoholic solution of the supernatant fluid. This is a
fuchsin to hot liquid. The me- stock solution.
dium is now brilliant red. (10) When ready for use prepare a
(7) Add 10.0 cc. of a 10.0% anhydrous 10.0% anhydrous sodium sulphite.
sodium bisulphite solution. (11) To each 10.0 cc. of (8) add 2.0 cc.
(8) Distribute into large test tubes in of (7) and steam for 5 minutes in
20.0 cc. lots. the Arnold or water bath.
5 to 7 minutes at 10
(9) Sterilize for (12) To each 100.0 cc. of agar, add 1.0
pounds extra pressure. g. of lactose, and dissolve in
(g) Roddy. streaming steam or on a water
(1) Prepare a 3.0% extract agar. bath and 0.5 cc. of (9).
(2) Liquify (1) in the Arnold steam (13) Pour into Petri dishes and allow
sterilizer. to harden thoroly in the incubator
(3) Dissolve 10.0 g. lactose in (2). before use.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 545
(i) Krumwiede, Kohn, Kuttner and (3) Distribute in 100.0 cc. quantities
Schumm. in flasks.
(1) Dissolve 25.0 g. agar, 10.0 g. pep- (4) Prepare a 10.0% basic fuchsin
tone and 5.0 g. meat extract in solution in 96% alcohol.
1000.0 cc. water by heating over a (5) Prepare a 10.0% solution of chem-
gas stove. ically pure anhydrous sodium
Adjust reaction neutral to litmus sulphite (1.0 g. in 10.0 cc. water).
(2)
using 10.0% Na2C03 solution. (6) Add 1.0 cc. of (4) to 10.0 cc. of (5),
(10) To melted (9) just before use add (2) Add 120.0 cc. of a 10.0% lactose
0.5 a saturated alcoholic
cc. of solution to (1).
solution of fuchsin and 1.0 cc. of a (3) Sterilize at 100 for one hour.
dium bisulphite (per 100.0 cc.?). to 100.0 cc. of 96% alcohol and
Tanner (Hygienic Laboratory). allow to stand for 2 hours.
(j)
Add 3.7 cc. of a 10.0% anhydrous (5) Decant (4) and add 2.5 cc. of the
(1)
Na2C03 solution to 1000.0 cc. of a supernatant fluid to (3).
lots. These are aproximate, (2) Sterilize (1) in 100.0 cc. quantities
however, and the proper balance (method not given).
can easily be attained by a little (3) When desired for use add 1.0 g. of
Constituents :
composition of 3.0% beef extract agar
1. Water 1000.0 cc. not given.
2. Agar 20.0 g. (2) Adjust (1) to pH of 7.2 to 7.4.
3. Meat extract, Liebig's 10.0 g. (3) Add 1.0% lactose from a sterile 20.0%
4. Peptone 10.0 g. solution to sterile (2).
0.1% 10.0 cc. (5) Pour into sterile Petri dishes, about
Preparation :
20.0 cc. to each plate.
(2) Filter and boil again for 30 minutes. agar or lactose solution not given.
(3) Add 15.0 g. lactose to (2) and boil Sterilize the 0.04% brom cresol purple
for 15 minutes in the autoclave.
(4) Add a sterile soda solution (any Use: Isolation of typhoid and dysentery
desired strength, but not too strong) bacilli from stools. Author reported that
until red litmus paper is turned blue. medium was clear and deep blue in color.
(5) Add 130-0 Kubel-Tiemann's
cc. of Lactose fermenters produced greenish
litmus solution, and 10.0 cc. of a yellow colonies with a yellow zone. Non-
crystal violet solution that has been lactose fermenters produced bluish colo-
prepared by dissolving 0.1 g. Hochst nies. Typhoid colonies have typical
crystal violet in hot sterile distilled woolly appearance. In thickly seeded
water to (4) plates, typhoid colonies were bluish
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 549
green, while colon colonies were brilliant (4) Readjust the reaction if necessary.
yellow. The addition of brilliant green (5) Allow to cool to 55 or 60C.
did not interfere with the color produc- (6) Beat the white of an egg in 100.0 cc.
tion of the non-lactose fermenters. of water and add to (5)
Variants: The author prepared a similar (7) Mix well.
medium as follows: (8) Autoclave at 120C. for one hour.
(1) Prepare a 3.0% beef extract agar and (9) Add 5.0 g. of sucrose to (8).
adjust to pH = 7.6 to 7.8. (10) Tube.
(2) Clear (1) with white of an egg. Sterilization: Sterilize at 115C. for 20
(3) Sterilization not mentioned. minutes.
(4) Add 1.0% lactose from a sterile 20.0% Use: General culture medium.
solution. Reference: Besson (1920 p. 43).
(5) Add for every 100.0 cc. of the medium,
10.0 cc. of an 0.04% aqueous solution 1763. Hesse's Starch Extract Agar (Stokes
of phenol red. This solution may be and Hachtel)
sterilized by autoclaving.
Constituents:
(6) Pour into sterile Petri dishes, about
1. Distilled water 1000.0 cc.
20.0 cc. of agar to the plate.
2. Agar 5.5 g.
The author reported that the medium was
3. Beef extract (Liebig's) 5.0 g.
originally salmon pink or old rose. Lac-
4. Peptone (Witte's) 10.0 g.
tose fermenters produced vivid greenish
5. Starch 10.0 g.
yellow colonies with a surrounding zone
6. NaCl 8.5 g.
of green. The typhoid bacillus, the
7. Azolitmin solution (Kahl-
paratyphoid bacillus (A and B) and the
baum's)
dysentery bacilli (Shiga, Flexner and
Hiss Y) all produced pink colonies. In Preparation
very thickly seeded plates the colon (1) Dry agar at 105C. for 30 minutes.
(2) Dissolve 5.5 g. of (1) in 500.0 cc.
colonies assumed a bright green or yellow
distilled water.
green color and are opaque, whereas the
typhoid colonies were more translucent (3) Add 5.0 g. beef extract to 500.0 cc.
1762. Salomonsen's Sucrose Extract Agar (6) Make up the loss due to evaporation
(Besson) by the addition of distilled water.
Constituents: (7) Mix (6) and (2) and boil for 30
minutes, making up the loss in
1. Water 1000.0 cc.
2. Meat extract (Liebig's) .... 5
weight by the addition of distilled
g.
water.
3. Peptone 30.0 g.
(8) Filter.
4. NaCl 5.0 g.
5. Agar 20.0 g. (9) Adjust the reaction to neutral.
(10) Color with Kahlbaum's azolitmin
6. Sucrose 5.0 g.
solution.
Preparation:
Dissolve 4 in 1.
(11) Distribute in 10.0 cc. lots.
(1) 2, 3,
chopped thread agar (12) Store in the ice box until ready for
(2) Soak 20.0 g. of
use.
in water for several hours.
cold
Squeeze water thru a cloth. Sterilization: Autoclave at 16 pounds pres-
(3) Heat (2) in (1) at 100C. until the sure for 20 minutes.
agar is dissolved. Use: Differentiation of typhoid bacillus.
550 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1765. Coplin and Bevan's Glycerol Extract 1767. Fawcus' Dye Bile Salt Agar
Agar (Bezanfon)
Constituents: Constituents
1. Water 1000.0 cc.
1. Water 900.0 cc.
2. Meat extract 5.0 g.
2. Agar 30.0 g.
3. Peptone (albumin) 10.0 g.
3. Meat extract, Liebig's 20.0 g.
4. Glycerol 62.0 g.
4. Peptone (10.0% soln.) 100.0 cc.
5. Agar (1.5%) 15.0 g.
5. Brilliant green (1 1000) : 10.0 cc.
Preparation 6. Picric acid (1:100) 10.0 cc.
(1) Dissolve 2, 3, 4 and 5 in 1. 7. NaCl 20.0 g.
(2) Clear and filter in the same way as 8. Sodium taurocholate 5.0 g.
other media. (Method not given) Preparation
Sterilization: Not specified.
(1) Dissolve 2, 3, 4, 7 and 8 in 1.
Use: Cultivation of Micrococcus pyogenes (2) Make alkaline (Indicator not speci-
aureus. Authors reported that 3 or 4 fied).
days after inoculation the medium To 1.5 liters of agar add 10.0 cc. of
(3)
became opaque, and after 5 or 6 days the 1-1000 brilliant green and 10.0 cc. of a
medium appeared as if tapioca was mixed 1-100 picric acid solution.
with it. Sterilization: Method not given.
Reference: Coplin and Bevan (1892 p. 70). Use: Cultivation of colon typhoid group.
1766. Piettre and de Souza's Citric Acid Reference: Bezangon (1920 p. 345).
Extract Agar
1768. Percival's Urea Extract Agar
Constituents:
1. Water 1000.0 cc. Constituents
2. Meat extract 0.7 g. 1. Water 1000.0 cc.
3. Peptone (Chapoteaut) 10.0 g. 2. Beef extract (Lemco's) 5.0 g.
4. NaCl 5.0 g. 3. Peptone (Witte's) 10.0 g.
3. Peptone (Witte siccum) 20.0 g. (4) Add 0.4 cc. of a 1.0% solution of
4. NaCl 10.0 g.
brilliant green to each 100.0 cc. of
5. Agar 80.0 g.
melted medium, cooled to 50C. when
6. Lactose 20.0 g.
ready to pour in plates.
7. Fuchsin (10.0% alcoholic Sterilization: Not specified.
1774. Schniirer's Saponin Glycerol Agar (3) Add 1.0% lactose 0.1% glucose and
5.0% of a 0.02% watery solution of
Constituents:
phenol red.
1. Beef extract agar
pH 7.2-7.4 hot.
(4) Correct the reaction to
(3.0%) 1000.0 cc.
Sterilization: Not specified.
2. Glycerol 30.0 cc.
Use: To study fermentation or respiration
3. Saponin (Merck's) 10.0 to 80.0 g.
by the typhoid bacillus.
. .
Preparation
Reference Nichols and Wood (1922 p. 322).
:
be substituted for Liebig's meat extract. (1) Dissolve 15.0 g. agar in 500.0 cc.
References: Holt-Harris and Teague (1916 of water by heating over gas
stove.
p. 597), Stitt (1923 p. 49), Park, Williams
and Krumwiede (1924 p. 128). (2) Dissolve 3.0 g. of Liebig's extract
peptone and 5.0 g.
of beef, 10.0 g.
1778a. Krumwiede, Pratt and McWilliams NaCl in 500.0 cc. of 1 by heating
Brilliant Green Agar over gas stove.
Constituents (3) After complete solution mix (1)
1. Water 1000.0 cc. and (2).
2. Agar 15.0 g. (4) Adjust to 0.6 or 0.7% acid to
3. Salt (NaCl) 5.0 g. phenolphthalein (neutral to And-
4. Peptone (Witte) 10.0 g. rade indicator).
5. Beef extract (Lie- (5) Prepare Andrade indicator by
big's) 3.0 g. adding 16.0 cc. of a N/1 NaOH
555
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
When adding the brilliant green (see Kuttner and Schumm (1918 p. 275),
Kligler (1918 p. 320), Giltner (1921 p.
step (7) above) add 0.25 cc. of a 1.0%
solution of neutral red to each 100.0 366), Park, Williams and Krumwiede
cc. of agar. (1924 p. 128).
(8) Prepare an alcoholic saturated solu- 1780, Hesse's Malachite Green Agar
by pulverizing com-
tion of fuchsin (Klimmer)
mercial fuchsin in a mortar and
Constituents
adding absolute alcohol and placing
1. Water 1000.0 cc.
in the incubator for a day shaking
2. Peptone 10.0 g.
occasionally,
3. NaCl 5.0 g.
(9) Prepare a 10.0% solution of sodium
4. Meat extract 10.0 g.
sulphite. Boil several times to
5. Agar 35.0 g.
sterilize.
6. NazCOa (10.0%)
(10) To 100.0 cc. of liquid and sterile (4)
soln.) 30.0 to 40.0 cc.
add 6.0 cc. of sterile (5).
7. Sucrose (20.0%)
(11) Heat in streaming steam for 15 min-
soln.) 50.0 cc,
utes. The agar becomes dark brown
8. Dextrin (20.0%
and turbid.
soln.) 50.0 cc.
(12) Add 5.0 cc. sterile (6), 5.0 cc. sterile
9. Malachite green
(7),0.4 cc. (8) and 2.0 cc. (9) to each
(chlorzinc salt c.p.
100.0 cc. lot immediately after it is
sat. ale. soln.) 4 cc.
removed from the steamer.
10. Na-^SOs (10.0%
(13) Slant the flasks so that the precipi-
soln.) 25.0 cc.
tate settles quickly to the bottom.
Preparation
(14) Pour into plates, but leaving the
(1) Soak 35.0 g. of agar in a liter of water
settled precipitate in the flasks.
over night.
(15) Dry in the incubator or at 50C. for
(2) Add 2, 3 and 4 in (1).
30 minutes.
(3) Boil for 4 or 5 hours in the steamer.
(16) The plates are transparent and
(4) Distribute in 100.0 cc. lots in 200.0
yellowish brown in color.
cc. flasks.
Sterilization: Sterilize (5), (6) and (7) by
(5) Add 3.0 to 4.0 cc. of a 10.0% water
heating in steam for 30 minutes.
free soda solution, 5.0 cc. of a 20.0%
Use: Diagnosis of Author re-
cholera.
sucrose solution, 5.0 cc. of a 20.0%
ported that cholera organisms gave large
dextrin solution, 0.4 cc. of a con-
red colonies. Other organisms were
centrated alcoholic solution of mala-
inhibited. The more alkali added, the
chite green (chlorzinc, double salt,
more coli colonies were inhibited.
c.p.) and 2.5 cc. of a
crystalline,
Variants : Klimmer prepared the medium as
10.0% NajSOg solution to each 100.0
follows
cc. of agar.
(1) Soak 35.0 g. agar in a liter of water Sterilization: Not specified.
over night.
Use: Cholera diagnosis. Author reported
(2) Add 10.0 g. peptone, 5.0 g. NaCl and that cholera colonies were green after 24
10.0 g. meat extract to (1).
to 48 hours.
(3) Boil for 4 or 5 hours in the steamer. Reference: Klimmer (1923 p. 219).
(4) Distribute in 100.0 cc. lots in 200.0
cc. flasks. 1781, Bacto Krumwiede Triple Sugar Agar
(5) Add 6.0 cc. of a 10.0% solution of (Dehydrated)
water free soda, 5.0 cc. of a 20.0%
Constituents:
sucrose solution, 5.0 cc. of a 20.0%
1. Distilled water
dextrin solution, 0.25 cc. of a satu-
2. Peptone (Bacto) 10.0 g.
rated alcoholic fuchsin solution and
3. Agar (Bacto) 15.0 g.
2.5 cc. of a 10.0% NasSOa solution to 4. Lactose (Bacto) 10.0 g,
each 100.0 cc. of agar.
5. Sucrose (Bacto) 10.0 g.
(6) Sterilization not specified.
6. Glucose (Bacto) 1.0 g.
References: Aronson (1915 p. 1028), Klim- 7. NaCl 5.0 g.
mer (1923 p. 218). 8. Andrade indicator 0.025 g.
557
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
7. Rafhnose 2.5 g.
Constituents
8. Sucrose 2.5 g.
1. Water 1000.0 cc.
9. Salicin 2.5 g.
2. Agar 15.0 g.
3.0 g. 10. Lead acetate (0.25%
3. Meat extract
lO'O S- soln.) 250.0 cc.
4. Peptone
NaCl (salt) 5.0 g. Preparation
5.
(1) Dissolve 2, 3, 4, 5, 6, 7, 8, 9 and 10 in 1.
6. Lactose 100 g-
(10) Mix sterile (9) with (8) and stir with (d) Giltner added 1.0% glucose and 1.0%
a sterile glass rod. CaCOs to hot agar, and sterilized
(11) Distribute into small flasks or tubes. by the discontinuous method.
(12) Place in the steamer for 10 minutes. (e) Harvey added sufficient sterilized
pH at this point should be near pH precipitated chalk to render glucose-
= 7.4. agar white and opaque.
(13) Prepare a 1.0% neutral lead acetate (f) Puder cultivated Rhabditis pellio, a
(lead diacetate) by rapidly raising nematode, on a medium prepared as
30.0 cc. of sterile distilled water to a follows:
boil, and adding 0.3 g. of neutral
(1) Prepare bouillon. Reaction to be
lead acetate (lead diacetate). alkaline.
(14) Add (13) to (11) using a sterile (2) Mix 10.0 cc. of with 90.0
(1) cc.
pipette in the proportion of 5.0 cc. water.
of (13) to each 100.0 cc. of (11).
(3) Dissolve 1.5 g. of agar in (2).
(15) Shake the flask quickly and Add 2.0 g. of glucose and
(4) drops of 8
vigorously. concentrated (strength not given)
(16) Add with a sterile pipette, 5,0 cc. of a alkaline to (3).
1.0% solution, Na2HP04 for each (5) Sterilize on three successive days
100.0 cc. of medium. for 30 minutes each day in a
(17) Shake again. steamer.
(18) Place in the water bath at 55C. Pour into sterile Petri dishes.
(6)
(19) Distribute into tubes. References: Frost (1903 p. 64), Heinemann
Sterilization Sterilize (9) by placing in the
:
(1905 p. 20), Lohnis (1913 p. 17), Lignieres
steamer for 20 minutes. See step (8) for (1919 Giltner
p. 1091), (1921 p. 365),
sterilization of agar. Final sterilization Harvey (1921-22 p. 89), Puder (1923 p.
oftubed medium not specified. 99),Stitt(1923p.38).
Use: Enumeration of B. aertryke in feces.
The authors reported that 1.0% glucose 1786. Mankowaki's Indigo Carmine Glucose
gave better results than lactose, sucrose Agar
and salicin, if B. aertryke outnumber the Constituents
other organisms in the feces. 1. 0.33 to 0.5% glucose agar.
Reference: Topley and Ayrton (1923-24 2. Fuchsin (acid,
p. sat. solution in 1.0%
230). KOH).
3. Indigo carmine (sat. aqueous solution).
1785. Frost's Glucose Agar
Preparation
Constituents: Prepare a 0.33 to 0.5% glucose agar.
(1)
1. Nutrient agar 1000.0 cc. Reaction to be neutral.
2. Glucose (1.0%) 10.0 g. (2) Prepare a saturated solution of acid
Preparation fuchsin in a 1.0% solution. KOH
(1) Add 1.0% glucose to nutrient agar. (Acid fuchsin may be added to a 1.0%
(2) Tube. KOH solution until a dark black
Sterilization: Sterilize in the steamer. brown color is reached.)
Use: General culture medium. (3) Prepare a watery saturated solution
Variants of indigo carmine.
(a) Heinemann added 1.5% glucose to (4) Add 2.0 cc. of (2) and 1 cc. of (3)
nutrient agar and sterilized the to 22 cc. of distilled This
water.
medium in the autoclave at 120 'C. for solution is dark blue and reaction
5 minutes. slightly alkaline.
559
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Preparation
under investigation.
Prepare glucose agar in sufficient (4) Pour into plates.
(1)
When the medium has solidified cover
quantity. (5)
be pres- the surface with a layer of (1) mixed
(2) Add gentian violet so that it
0.3% glucose and 1.0 cc. dye per 100.0 cc. 3. Litmus (soln.) 30.0 cc.
agar. 4. Nutrient agar (3.0%)
Variants Preparation
(a) Makgill added 1.0% of a saturated (1) Dissolve 3.0 g. of glucose and 30.0
solution of neutral red to glucose cc. of litmus in 200.0 cc. distilled
agar. water.
(b) Heinemann added sufficient 0.5% (2) Add five parts sterile (1) to six parts
neutral red to give a red color to 1.0% of sterile nutrient 3.0% agar in tubes.
glucose agar. The mixture should be a violet tint.
(c) Bezangon prepared a similar medium Sterilization: Sterilize (1) by filtering.
as follows: Method of sterilization of 3.0% agar not
(1) Dissolve 5.0 g. of agar in 1000.0 cc. given.
of bouillon. Use: Differentiation of diphtheria and
(2) To melted (1) nearly solid, add 2.0 pseudo diphtheria. True diphtheria
g. glucose and 0.7 cc. of a aqueous bacilli changed the color of the medium
saturated solution of neutral red. to red and grew only anaerobically.
(3) Tube. Pseudodiphtheria strains did not change
(4) Sterilization not specified. the color and grew only on the surface.
(d) Rothberg and Scheffler(Klimmer) Reference: Martin and Loiseau (1919 p.
prepared a medium as follows: 73). Taken from (1919 p. 185).
(1) Add 0.3% glucose to melted sterile
nutrient agar. 1792, Frost's Lactose Agar
(2) Add 1.0 cc. of a saturated (cold)
solution of neutral red sterilized in Constituents
the steamer to each 100.0 cc. of (1). 1. Nutrient agar 1000.0 cc.
(e) Cunningham prepared a medium 2. Lactose (1.0%) 10.0 g.
as
follows: Preparation
(1) Add 1.5% agar to bouillon. (1) Add 1.0% lactose to nutrient agar.
(3) Boil over an open flame for 15 Use: General culture medium.
minutes, stirring constantly. Reference: Frost (1903 p. 64).
(4) Adjust the reaction to a slight
alkalinity using turmeric paper as 1793. Wurtz's Litmus Lactose Agar
an indicator (distinctly brown).
Constituents
(5) Filter while hot thru a plug of clean
1. Infusion agar 1000.0 cc.
cotton-wool in the bottom of an 2. Lactose (2.0%) 20.0 g.
enamelled funnel.
3. Litmus
(6) Add 0.5% glucose dissolved in a Preparation
few cc. of water.
(1) Add 2.0% lactose to sterile infusion
(7) Stir to thoroly mi.x.
agar.
(8) Tube.
(2) Tube.
(9) Sterilize intermittently in steam.
(3) Melt sterile (2) and add suflicient
References: SchefHer (1900 p. 202), Magkill tincture of litmus to give a violet
(1901 p. 431), Heinemann (1905 p. 127), color.
Tanner (1919 p. 50), Bezangon (1920 p. (4) Pour sterile (3) into plates.
114), Klimmer (1923 p. 211), Cunningham Sterilization: Method of sterilization of
(1924 p. 16), Savage (1901 p. 437), Irons or not given.
(1) (2) Sterilize (3) at
(1902 p. 315).
100 C.
Use: Differentiation of Bad. coli and
1791. Martin and Loiseau's Glucose Litmus
Eberth's bacillus. Author reported that
Agar
Bad. coli fermented lactose with the
Constituents production of acid. Typhoid bacillus
1. Distilled water 200.0 cc. colonies were colorless, colon colonies
2. Glucose 3.0 g. were red.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 561
Add 2.0% lactose and 2.0% (2) Place 1.5 g. of lactose into a sterile
(7)
peptone to hot (6) and mix flask.
(10) Add 2.0% azolitmin solution. (4) Boil 100.0 g. of distilled water 15
(11) Boil over a free flame. minutes.
(12) Distribute in 100.00 cc. lots in (5) Dissolve 0.1 g. crystal violet in (4).
250.0 cc. Florence flasks. (6) Add a weak soda solution that has
(13) Sterilization not specified. been boiled several times to (3) until
References: Wurtz (1897 p. 43), Smith the desired degree of alkalinity is
(1905 p. 94), Heinemann (1905 p. 127), obtained.
Committee American Public Health (1913 (7) Dissolve 0.04 g. azolitmin in a little
142), Giltner (1921 pp. 379, 380). (8) Add 1.0 cc. of (5) and the azolitmin
solution to (6) (the melted lactose
1794. Gassner's Metachrome Yellow Water agar) that has been cooled to 45C.
Blue Lactose Agar (Klimmer) (9) Mix well and after about ten minutes,
pour into sterile Petri dishes. Allow
Constituents the agar to solidify with the covers
1. Nutrient agar 2000.0 cc.
removed.
2. Metachrome yellow (2.0% Sterilization: Method not given.
125.0 cc.
soln.) Use: Diagnosis of cholera. Authors re-
3. Water blue (6B extra ported that cholera colonies were deep
"Afga" 1.0% soln.) 175.0 cc.
blue.
4. Lactose 100.0 g. Hirschbruch and Schwer
Reference:
Preparation (1904 p. 150).
(1) Make two liters of agar prepared from
yeast or meat and peptone, slightly 1796. Ramond's Rubine Acid Lactose Agar
alkaline. Constituents
(2) Add 125.0 cc. of a 2.0% solution of 1. Nutrient agar 1000.0 cc.
metachrome yellow (boiled two 2. Lactose 40.0 g.
minutes). 3. Eubine acid
(3) Dissolve 100.0 g. lactose in 175.0 cc. Preparation
of a 1.0% solution of water blue (6B Add 4 parts per 100 of lactose to
(1)
extra "Afga") and boil 10 minutes. nutrient agar.
(4) Add (3) to (2).
(2) Add several grains of rubine
acid
Sterilization: Not specified. until the medium is colored red.
Use: Detection of typhoid bacilli. Author add a satu-
(3) Heat to 70 or 80C. and
reported that B. were deep
coli colonies
rated solution of Na2C03 until the
blue. Typhoid and dysentery colonies color disappears.
lightened up the medium, coloring it a
(4) Filterthru paper.
greyish yellow. (5)Distribute in tubes.
Reference: Klimmer (1923 p. 216). Sterilization: Sterilize at 115C. for five
minutes.
1795. Hirschbruch and Schwer's Azolitmin Use Detection of typhoid bacilli. Author
:
Crystal Violet Lactose Agar reported that Bad. coli colonies colored
Constituents: the medium red. Typhoid colonies did
Nutrient agar (1.0 to 1.5%). 1000.0 cc.
1. not change the color.
15.0 g. References: Ramond (1896 p. 884), Wurtz
Lactose
2.
3. Crystal violet (0.1%, soln.) 10.0 cc. (1897 p. 43).
4. Azolitmin 0.4 g.
1797. Delta's Fuchsin Lactose Agar
Preparation
(1) Prepare nutrient agar containing 1.0
Constituents
Distilled water. .0 cc.
1.
to 1.5% agar.
CULTURE MEDIA FOR CULTIVATIOX OF MICROORGANISMS 563
2. Agar (3.0%) 100.0 cc. Use: Detection of typhoid fever and dys-
3. Lactose 1-5 g. entery. Author reported that typhoid,
4. Fuchsin acid (0.5%) 10.0 cc. paratyphoid and dysentery bacilli gave
5. NaaCOs light colorless colonies. Coli forms gave
Preparation small red colonies. They were inhibited.
(1) Prepare a 3.0% nutrient agar with a Cholera colonies were colorless but the
reaction slightly alkaline to litmus. medium was not selective for this
(2) Dissolve 1.5 g. lactose in 4.0 cc. of organism.
distilled boiling water. Boil for 30 Reference: Kindborg (1915-16 p. 445).
seconds.
(3) Prepare a 0.5% solution of acid 1799. Bitters' China Blue Malachite-green
fuchsin. Agar (Klinimer)
(4) Boil (3) and decolorize by adding four
Constituents
drops of a normal NaaCOs solution.
Boil again until it assumes a port wine
1. Agar (2.0 or 3.0%) 1000.0 cc.
(5)
2. Lactose (2.0%) 20.0 g.
color.
Add and 3. China blue (Hochst. sat.
(6) (2) (5) to 100.0 cc. of (1).
solution)
Sterilization: Not specified.
Use: Examination of feces. 4. Malachite green (Hochst.
Variants: The author reported that any 0.1% soln.) 25.0 cc.
Preparation
sugar may replace lactose. The medium
may be made more differentiating by the (1) Prepare a 2.0 or 3.0% nutrient agar.
addition of "nutrose or caffeine or mala- (2) Neutralize to litmus by the addition
chite green (in tested solution as for Lentz ofNaOH.
and Tietz's medium) or crystal violet
(3) Add 2.0% lactose.
(4) Boil several minutes.
(10 drops of a 1 to 1000 solution). In the
lasttwo combinations the background is (5) Add 9 drops of a saturated solution
green (or blue) and the coli colonies are of China blue (Hochst.) to each 100.0
cc. of agar.
violet (or red)."
Reference: Delta (1915 p. 1053).
(6) Add 2.5 cc. of a 0.1% solution of
Hochst. crystalline extra malachite
1798. Kindborg's Fuchsin Malachite Green green to each 100.0 cc. (The mala-
Agar chite green may be omitted.)
Constituents
KI solution in dilute HCl was added. If
1000.0 cc. a yellowish halo was produced around a
1. Nutrient agar
50.0 g.
colony when a dilute solution of Nessler's
2. Starch (rice) (5.0%)
reagent be added, ammonia was formed.
Preparation
(1) Add 40.0 cc. of a 10.0%, soda solution
1805. Khouvine's Cellulose Agar
to of agar neutral to litmus.
1 liter
acteristic growth after from 14 to 20 Variants: The author prepared the agar
hours. Also used to determine the as indicated:
production of diastase by bacteria. (1) Place a layer of sterile agar in a sterile
1806. Scales' Salt Cellulose Agar 1807. Cantani's Basal Glycerol Agar
Constituents Constituents
1. Water. 1. Nutrient agar.
2. Filter paper 2. Glycerol.
3. Nutrient agar. Preparation
4. Salts. (1) Mi.x equal amounts of glycerol and
Preparation one of the added nutrients. Place in
(1) Dilute 100.0 cc. of concentrated an Erlenmeyer flask.
H2SO4 with 60.0 cc. distilled water in (2) Melt tubes of sterile nutrient agar and
a two liter Erlenmeyer flask. add 0.5 to 0.75 cc. of sterile (1) to
(2) Cool to about 60 or 65C. each tube.
(3) Moisten 5.0 g. of filter paper with (3) Incubate 24 hours to test sterility.
water. Sterilization: Storing the fluid in glycerol
(4) Add (3) to (2) and shake until the tends to sterilize After a time test the
it.
paper is dissolved. sterility of mixture. Method of
the
(5) When solution is complete fill the sterilization of agar not given.
flask as quickly as possible with cold Use: Cultivation of parasitic and patho-
tap water. The task of dissolving genic forms.
the filter paper and filling the flask Added nutrients: The author added one
requires about one minute. of the following: Urine, pus, sperm, milk,
(6) The rapid addition of cold water egg white and other albuminous materials.
precipitates the cellulose in small Reference: Cantani (1910 p. 471).
flocks.
Throw the 1808. Wurtz's Glycerol Agar
(7) precipitate on a filter
paper and wash with distilled water Constituents
until free from H2SO4. This re- 1. Nutrient agar 1000.0 cc.
quires about 3 hours time and only 5 2. Glycerol (6.0%) 60.0 g.
liters of distilled water. A 12 inch Preparation
funnel with a folded filter is the best (1) Add 6.0% sterile glycerol to sterile
apparatus to filter with. nutrient agar under aseptic con-
(8) During the washing do not allow the ditions.
volume of water in the funnel to Sterilization: Method not specified.
get below 100.0 cc. Use: Cultivation of tubercle bacilli. Simi-
(9) When the wash water is free from lar media have been used to cultivate a
H2SO4 as shown by the addition of large variety of other pathogenic forms.
BaCl2 solution, allow the volume in Variants
the funnel to drain to about 200.0 cc. (a) Gllicksmann (1897) added 7.7% gly-
(10) Brush any cellulose particles cling- cerol to 1.5% nutrient agar and
ing on the dry filter paper into the cultivated diphtheria bacilli.
suspension. (b) Thoinot and Masselin added 4.0 to
(11) Punch a hole in the bottom of the 6.0% glycerol to nutrient agar,
filter, collecting the cellulose sus- (c) Dalton and Eyre added 5.0% glycerol
pension. to nutrient agar and adjusted the
(12) Wash the filter with a stream of reaction to -}-10 on Eyre's scale.
water from a wash bottle. They cultivated Micrococcus meli-
(13) Make the suspension to 500.0 cc. tensis.
(14) Prepare 500.0 cc. of nutrient agar (d) Smith cultivated plant parasites in a
with salts (composition not spe- medium prepared by the addition of
cified). 50.0 cc. of Schering's c.p. twice dis-
(15) Mix (13) and (14). tilled glycerol to nutrient agar.
Sterilization: Not specified. (e) Roux and Rochaix added 1.0 to 5.0%
Use: Cultivation of organisms capable of glycerol to agar. They suggested
utilizing cellulose. the addition of several drops of a
Reference: Scales (1916 p. 662). saturated gum arable solution. This
566 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
favored the adherence of the agar to 1811. Heinemann's Litmus Mannitol Agar
the walls of the containing vessels. Constituents
Reference: Wurtz (1897, p. 46), Glucks- 1. Sugar free agar 1000.0 cc.
mann (1897 p. 436), Thoinot and Masselin 2. Mannitol (1.0%) 10.0 g.
(1902 p. 35), Smith (1902 p. 92), Dalton 3. Litmus
and Eyre (1904 p. 159), Heinemann (1905 Preparation
p. 128), Smith (1905 p. 196),
Roux and (1) Add 1.0% mannite to sugar-free agar.
Rochaix (1911 p. 117), Roddy (1917 p. 43), (2) Tube in 8.0 cc. quantities.
Ball (1919 p. 74), Besson (1920 p. 43), (3) Add 1.0 cc. of 1.0% sterile litmus
Dopter and Sacquepee (1921 p. 128), solution to each tube before use.
Bitfield (1922 p. 117), Stitt (1923 p. 38), Sterilization: Not specified.
Cunningham (1924 p. 165) Use: General culture medium.
Reference: Heinemann (1905 p. 127).
red, a slight greenish coloration was (4) Pour ascending quantities of the
formed, in 48 hours a distinct green solution into Petri dishes and 15.0 cc.
fluorescence. At the end of 24 hours with of melted agar added to each plate.
saffarin, the under surface was lighter Not given.
Sterilization:
and after 48 hours decolorization occurred. Use To show inhibition of bacterial growth
:
colonies.
1810. Mandelbaum's Rosolic Acid Glycerol Reference: Benfold (1913-14 p. 38).
Agar
1813. Wurtz's Phenol Agar (Copeland)
Constituents Constituents
1. Glycerol agar 6.0%.
1. Wurtz agar.
2. Rosolic acid 1.0% alcoholic soln. Phenol 2.0% solution.
2.
Preparation Preparation:
(1) Brepare a 6.0% glycerol
agar. or composition
(1) Method of preparation
(2) Brepare a 1.0% alcoholic
solution of
ofWurtz agar not given.
rosolic acid. Add 0.2 cc. of a 2.0% solution of car-
(2)
(3) Melt sterile (1), cool to 50C. and to
bolic acid to the agar. Amount of
each tube add 0.3 cc. of (2). agar not specified.
Sterilization: Not specified. Sterilization: Not specified.
Use: Differentiation of typhoid and meta- Use: Isolation of Bacillus coli communis
typhoid bacilli. Author reported that from water.
metatyphoid bacilli gave red colonies. Variants: Behmer's (Klimmer) added 2.0
cc. of a 5.0% phenol solution to
each
The typhoid colonies were yellow.
Reference: Mandelbaum (1912 p. 48). 100.0 cc. agar.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 567
Reference: Copeland (1901 p. 493), Klim- Use: CultivatioQ of typhoid and inter-
mer (1923 p. 227). mediate group.
Reference: Hurler (1912 p. 357).
1814. Penfold's Monochlorhydrin Agar
p , , 1817. Finger, Ghon and Schlagenhaufer's
, , " Urea Agar
^
1. Agar.
2. Monochlorhydrin 20.0% solution. Constituents:
Preparation: 1. Nutrient agar (2.0% agar,
(1) Prepare a 20.0% solution of mono- 1.0% peptone) 1000.0 cc.
chlorhydrin. 2. Urea (2.0%) 20.0 g.
(2) Place ascending quantities of sterile Preparation:
(1) in Petri dishes. (1) Prepare nutrient agar containing
(3) Add 15.0 cc. of sterile agar to each 2.0% agar with 1.0% peptone,
plate. Mi.xwell. (2) Add 2.0% urea to (1).
Sterilization: Filter (1) to sterilize. Sterilization: Not specified.
Method of sterilization of agar not given. Use: Cultivation of gonococci. Author
Use To show inhibition of bacterial growth
: reported that growth was not as luxuriant
using members of colon aerogenes group. as in serum agar. Also used by other in-
Author reported that B. coli produced vestigators to study urea decomposition.
various sized colonies and papillae may Variants:
be formed. (a) The author used 3.0 or 5.0% urea
Reference: Penfold (1913-14 p. 39). instead of 2.0%.
(b) Lohnis studied urea decomposition
1815. Jacobson's Ethylcinnamic Ether Agar
^^ ^^^.^^ ^ ^^ ^^ ^ J5 0^^ ^q^^^^^
Constituents: solution of urea to each tube of
1. Agar. nutrient agar. The medium was then
2. Ethylcinnamic ether. heated in the steam sterilizer.
Preparation: (c) Cunningham added 2.0, 5.0 or 10.0%
(1) Liquify 10.0 cc. tubes of nutrient agar urea to nutrient agar, and sterilized
by heating on the salt water bath. by heating intermittently in the
(2) Add one small drop of ethyl cinnamic steamer. The medium was used to
ether to each tube by means of a fine study urea decomposition,
pipette. This is equivalent to 0.025 g. Reference: Finger, Ghon and Schlagen-
Sterilization: Not specified. haufer (1894 p. 14), Lohnis (1913 p. 95),
Use: Differentiation of dysentery bacilli. Cunningham (1924 p. 143).
Author reported that the Hiss type of -^^ ,,. -^ , , r^
, r -1 J .
J 4.U 4. 1818. Russell's Double Sugar
^ Agar
to develop in the pres-
1
dysentery failed ^
ence of ethyl cinnamic ether while the Constituents:
Flexner showed good development. 1. Nutrient agar (2.0 or
Reference Jacobson (1919 p. 726).
: 3.0%) 1000.0 cc.
2. Litmus solution
1816. Hurler's Caffeine Agar
^^ 0^^ ^q^^^^^ ^^1^.
Constituents: tion) 30.0 to 50.0 cc.
1. Nutrient agar 1000.0 cc. 3. Lactose 10.0 g.
2. Caffeine (0.3%) 3.0 g. 4. Glucose 1.0 g.
Preparation Preparation
(1) Prepare nutrient agar. (1) Prepare2.0or 3.0% nutrient agar with
(2) Adjust the reaction of (1) to slightly a reaction of about 0.8% acid to
alkaline. phenolphthalein.
(3) Add 0.3% caffeine to (2). The (2) Add enough of a 5.0% aqueous solu-
caffeine is dissolved in 5.0 cc. of tion of litmus to (1) to give a distinct
water by heating over the
distilled purple violet, the amount required
water bath before adding to the agar. depending on the original color of the
Sterilization: Method not given. agar.
568 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(3) Adjust the reaction by adding sodium and Bacillus murium pro-
enteritidis
hydrate until the mixture is neutral to duced browning while A type did not.
litmus. (b) Giltner prepared a similar medium
(4) Add 1.0% lactose and0.1% glucose as follows:
(dissolved in a small amount of hot (1) Prepare a 1.5% nutrient agar.
water) to (3). (2) Add 1.0% glucose and 1.0%o lactose
(5) Slant the tubes following sterilization to (1).
and store in small quantities in a (3) Make up a 0.5% solution of basic
dark place. lead acetate.
Sterilization: Sterilize in the Arnold. (4) Sterilize (3). (Method not given.)
Pack the tubes loosely in the sterilizer (5) Tube.
basket to allow good circulation of the (6) Add the necessary amount of (4)
and gas was produced. Bacillus bouillon with agar, and added 5.0%
typhosus reddened butt, colorless glycerol and 2.0% glucose.
slant and produced browning particu- References: Thoinot and Masselin (1902 p.
larly near the surface of the stab. 35), Besson (1920 p. 43), Dopter and
The dysentery bacillus reddened the Sacquepee (1921 p. 128).
butt but did not produce browning.
1820. Thoinot and Masselin's Sucrose
Paratyphosus bacilli produced gas
Glycerol Agar
while Bacillus hjphosus and Bacillus
dysenteriae did not. Bacillus para- Same as medium 1819 but substituting
typhosus B. and allied bacilli, Bacilli 5.0 to 10.0 g. sucrose for glucose.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 569
same constituents but substituting 0.5% 1824. Kitasato's Glucose Formate Agar
mannitol for 1.0% lactose. The authors
(Tanner)
reported they used the medium for the
differentiation of B. typhosus from dys-
Constituents
entery bacilli. Nutrient agar
1. 1000.0 cc.
Reference: Kligler and Defandorfer (1918 2.Glucose 20.0 g.
3. Sodium formate 4.0 g.
p. 439).
Preparation
(1) Prepare nutrient agar.
1822. Kendall and Ryan's Sucrose Mannitol
(2) Dissolve 2 and 3 in 1.
Agar
(3) Adjustment of reaction not given.
Constituents (4) Tube.
1. Nutrient agar 2.5% 1000.0 cc. Sterilization: Method not given.
2. Mannitol 1.0 g. Use: General culture medium.
3. Sucrose, 10.0 g. Reference: Tanner (1919 p. 49).
4. Andrade indicator 10.0 cc.
Preparation :
1825. Sohngen and Fol's Glucose Butyrate
(1) Prepare nutrient 2.5% agar.
Agar
(2) Adjust to a reaction so that when Constituents
Andrade indicator is added that the 1. Water agar 50.0 cc.
medium be a faint pink while hot. 2. Nutrient agar 50.0 cc.
(3) Add 2 and 3 to (2). 3. Calcium butyrate 1.0 g.
(4) Tube. 4. Glucose 0.25 g.
(5) Slant so that slanted surface begins Preparation
1.0 cm. from the bottom. Medium is (1) Prepare tap water agar and nutrient
colorless when cold. agar.
Sterilization: Not specified. (2) Mix 50.0 cc. of tap water and 50.0 cc.
Use: Detection of intestinal and other of nutrient agar.
bacteria. (3) Dissolve 3 and 4 in (2).
Reference : Kendall and Ryan (1919 p. 403). Sterilization: Not specified.
Use: Cultivation of actinomyces, Acti-
1823. Hulton-Frankel and MacDonald's nomyces elastica and Actinomyces fuscus
Inositol-Dextrin Agar Reference: Sohngen and Fol (1914 p. 95).
each tube undiluted so that 0.1 cc. Variants: The author used one of the
equals 1.0% of acid. following instead of 10.0 cc. of fuchsin:
Safranin 10.0 cc.
(5) Add glucose.
Sterilization: Sterilize in autoclave at 15
Bismark brown 50.0 cc.
G. orange 60.0 cc.
pounds pressure one hour.
Neutral red 60.0 cc.
Use To show effect of lactic acid on growth
:
Preparation
10.0 cc. fuchsin solution.
Distribute as desired in sterile con- (1) Prepare nutrient agar.
(8)
tainers. (2) Add 2 and 3 to (1).
(5) Filter.
may be sterilized at 100C. in water and
(6) Add 2.0% lactose.
then added to the sterile agar if desired.
Tube.
Sterilize (3) by heating at 100 for 30
(7)
Sterilization: Method not given.
minutes. Sterilize (4) by heating at
Use: General culture medium.
100 C. for 30 minutes.
typhoid Reference: Heinemann (1905 p. 128).
Use: Differentiation colon
of
bacilli. The author gave the following 1830. Fleming's Oleic Acid Glycerol Agar
reactions:
Constituents
With fuchsin, typhoid blue; coli red. 100.0 cc.
Nutrient agar
With safranin, typhoid blue; coli orange.
1.
(2) Add 2.0% glycerol and 0.1% oleic 1832. Rosenow's Glucose Brain Agar
acid to (1).
(Haden)
(3) Tube. Constituents
Sterilization: Sterilize in the autoclave in 1. Water 1000.0 cc.
the usual way. (Details not given.) 2. Bacto nutrient broth 8.0 g.
Use: Cultivation of the bacillus of acne 3. NaCl 8.0 g.
vulgaris (Bacillus acne). 4. Glucose (c.p.) 2.0 g.
(3) Add and dissolve 1.0% glucose and 1833. Goldberger's Glucose Alkaline Egg
2.5% agar in (2). Agar (Abbott)
(4) Add 10.0% sterile ascitic fluid to Constituents
sterile (3). 1. Distilled water 1000.0 cc.
(5) Distribute in tall test tubes (column 2. Egg 1.0
of medium measuring 0.8 cc. in
3. Meat extract (Liebig's) 3.0 g.
diameter and 13.0 cc. in height. 4. Peptone (Witte's) 10.0 g.
(6) Add a piece of fresh sterile rabbit 5. NaCl (c.p.) 5.0 g.
kidney. 6. Glucose 1.0 g.
(7) Add a layer of sterile mineral oil.
7. Agar 30.0 g.
Sterilization: Method of sterilization of Preparation
(3) not given. (1) Mix a whole egg with an equal
Use: Cultivation pleomorphic strepto-
of volume of distilled water.
cocci causing poliomyelitis. The author (2) Mix one volume of (1) with an
reported that initial growth could best equal volume of 6.5% solution of
be obtained without the addition of the anhydrous NajCOa.
kidney. (3) Steam for 30 to 60 minutes.
Variants: The authors prepared the (4) Mix 3, 4, 5, 6 and 7 in 1000.0 cc. of
(4) Add 2.5 cc. of 5.3% anhydrous NaoCOa (3) Distribute in flasks or Petri dishes
solution for each 100.0 cc. of infusion. and add 10.0% sensitive litmus
(5) Filter sterile (4). (strength of solution not specified).
(6) Prepare a 3.0% meat extract agar. Sterilization: Not specified.
(7) Mix one volume of (5) with 3 volumes Use: Enrichment medium for typhoid
of hot melted sterile (6). bacilli. Author reported that mineral
(8) Pour plates. salts may be used as a basis for this
(9) Cover the plates with a piece of filter medium instead of extract or infusion
paper and place in the incubator for broth.
30 minutes until they are dry. Reference: Dunschmann (1909 p. 64).
CULTUEE MEDIA FOR CULTIVATION OF MICROORGANISMS 573
1837. Paneth's Glucose Ascitic Fluid Agar 1838. Plotz, Olitsky and Baehr's Ascitic
Fluid Agar
Constituents
1. Distilled water 3000.0 cc. Constituents
(13) Remove blood aseptically with a cal changes did not appear except near
syringe from the median basilic or the limiting pH values.
median cephalic veins of patient Reference: Reed and Orr (1923 p. 104).
(15.0 cc).
(14) Divide the 15.0 cc. blood among eight 1840. Becker's Defibrinated Blood Agar
sterile melted tubes of (11), 2.0 cc.
per tube. Constituents
(15) Add ascitic fluid to each tube of 1. Extract agar 1000.0 cc.
specific gravity of more than 1.015. Standard Methods, see medium 1695,
Must not be filtered, sterilized or and distribute in 40.0 cc. lots in 100.0
(16) Mix the content of each tube thoroly (2) Melt agar and cool to 45-50 C.
by pouring once or twice into (3) Add 1.0 cc. of defibrinated human
another sterile tube. Avoid air blood per 6.0 cc. agar.
(17) After thoro solidification cover each Sterilization: Method not specified.
(1) Prepare beef extract peptone agar cc. of N/1 caustic soda solution and
(4) Add whole rabbits blood to sterile steamer, or for 15 minutes in an auto-
(3) cooled to 80C. to make finished clave at one atmosphere pressure.
medium 1.0% blood. (5) Prepare "Ragit agar."
(5) Maintain at 80C. for 10 minutes. (6) Melt sterile (5) and add (4) to 85.0
range of 6.8 to 7.4 gave very nearly tion of alkaline hemoglobin. Method of
Use: Enrichment medium for cholera parent and light green. A great many
vibrio. of the intestinal forms were entirely
Reference: Galli-Valerio (1912 p. 550). inhibited.
Reference: Padlewsky (1908 p. 543).
1842. Padlewsky's Malachite Green Bile
Agar 1843. Fildes' Body Fluid Agar
Constituents Constituents
1. Meat extract agar 1. Extract agar.
(3.0%) 1000.0 cc. 2. Ascitic fluid or other body fiuid.
(2) Treat the ox bile with steam in a 0.5% chloroform has been added.
Koch's apparatus. (Time not (6) .Add a drop of sterile oil to the stop-
specified). per and fasten a dust cover tightly
(3) Filter the bile thru cotton. over the stopper.
(4) Add 2.0% peptone, 3.0%, (3) and (7) Place the bottles in the water bath
1.0% c.p. lactose to (1). for one hour at 45C. Shake
Adjust the reaction to slightly occasionally.
(5)
alkaline to litmus. (8) When bottles have cooled, remove a
(6) It is not necessary to filter the agar sample under aseptic conditions and
after the addition of the filtered bile. mix with agar. Incubate the mix-
(7) Distribute into flasks in 100 to 200 cc. ture at 37C. for 5 days to test
lots. sterility.
(8) When ready for use melt the sterile (9) Prepare ordinary lemco (or meat
agar, cool to 60 to 65 C. infusion) agar containing 2.5 to 3.0%
Mix the ascitic fluid and agar at as not sterilize with the method
low a temperature as possible. This employed.)
aids the production of a transparent (5) Add chloroform to each bottle
medium. until 0.5% chloroform has been
Sterilization: Sterilize (10) in the auto- added.
clave. (6) Add a drop of sterile oil to the
Use: Cultivation of meningococci and stopper and fasten a dust cover
other pathogenic cocci. tightly over the stopper.
Variants (7) Place in the air incubator at 37 C.
(a) Fildes substituted serum for ascitic for 24 hours, shaking constantly.
fluid. The serum was prepared as (8) After this time, remove a sample
follows: under aseptic conditions and test
(1) Collect horse or ox blood in tall its sterility by mixing it with agar
sterile jars from the slaughter and incubating. (Time not speci-
house. fied.) If sterile do not heat longer
(2) Allow to stand until all the cor- for heating tends to darken the
puscles have deposited. It may medium.
require 14 days for this to take (9) Prepare ordinary lemco (or meat
place. After 5 days examine the infusion) agar containing 2.5 to
jars and pipette or siphon off the 3.0% agar.
clear serum. Do not disturb the (10) Measure (9) into 200.0 cc. flasks in
sediment. 150.0 cc. lots.
(3) Distribute the clear serum in 200.0 (11) Autoclave (time and pressure not
cc. lots in perfectly fitting glass given).
stoppered flasks. (12) Add 25.0% of sterile laked blood
(4) Add chloroform each bottle
to (8) to (11). (Method of distribu-
until 0.5% chloroform has been tion not specified.)
added. Reference: Fildes (1917 p. 492).
(5) Add a drop of sterile oil to the
stopper and fasten a dust cover
1844. Haner and Frost's Milk Body Fluid
tightly over the stopper.
Agar
(6) Place the bottles ip the water
bath for one hour at 45C. Shake Constituents
occasionally. 1. Nutrient agar 1.0%
(7) When the have cooled,
bottles (dehydratedDif co) 1000.0 . cc.
remove a sample under aseptic 2. Milk, sterile 1000.0 cc.
conditions and mix with agar. 3. Serum (horse or
Incubate the mixture at 37C. for rabbit 6,0 to 12.0%). 120.0 to 240.0 cc.
five days to test sterility. Preparation
(b) Fildes also prepared the medium as (1) Prepare 1.0% agar from Difco de-
follows: hydrated product.
(1) Collect ox or horse blood from a (2) Add equal part of sterile milk to
slaughter house. sterile (1).
(2) Defibrinate the blood by strirring (3) Add 6.0% to 12.0% of sterile horse or
with a sterilized large wooden rabbit serum to (2).
stick wrapped in gauze. Sterilization: Method of sterilization not
(3) Lake the blood by the addition of given.
an equal volume of distilled water. Use: Cultivation of pneumococci and
(4) Distribute in 200.0 cc. lots into streptococci.
perfectly fitting glass stoppered Variants: The authors used whole blood
bottles. (The
author mentions instead of serun.
that the medium should contain Reference: Haner and Frost (1921
no suspended material, or it will p. 270).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 577
1845. Bacto Conradi-Drigalski Agar (3) Add 1.0% peptone, 5.0% beef extract
(Dehydrated) and 1.2% agar to the filtrate.
(4) Heat to boiling point until all the
Constituents:
agar is dissolved.
1. Distilled water.
(5) Correct the reaction to 1.2 using
2. Peptone, Bacto 10.0 g.
phenolphthalein as an indicator.
3. Sodium caseinate, Bacto. . 10.0 g.
(6) Cool to 45C. and add the whites of an
4. Lactose, Bacto 10.0 g.
egg.
5. Agar, Bacto 15.0 g.
(7) Heat to lOO^C. for 45 minutes and
6. NaCl 5.0 g.
filterthru cotton.
7. Dibromcresolsulphoneph-
(8) Correct the reaction if necessary and
thalein 0.03 g.
tube.
8. Crystal violet 0.02 g.
Sterilization : Sterilize three successive days
Preparation
in the Arnold sterilizer.
(1) Dissolve 50.0 g. of Bacto
Conradi-
Use: Cultivation of bacteria found in
Drigalski Agar (dehydrated) in 1000.0
cheese. Author reported that bulgaricus
cc. of distilled water by boiling or
types grew well on this medium.
autoclaving.
Reference: Eldredge and Rogers (1914
(2) Restore any lost weight.
p. 8).
(3) If sterilized at 15 pounds pressure for
20 minutes pH = 6.8. 1848. Ayres and Mudge's Milk Powder
Sterilization: Sterilize at 15 pounds pres- Agar
sure for 20 minutes.
Use: The author reported that acid produc- Constituents:
ing colonies were yellow. 1. Water 500.0 cc.
p. 12).
3. Na.HPOi + 2HoO
(Sorensen's phosphate). 1.0 g.
1846. Haner and Frost's Milk Agar 4. Peptone (Difco) 2.0 or 5.0 g.
(2) Add an equal part of sterile milk to phosphate in 5.0 cc. distilled water.
4. Beef extract (5.0%) 50.0 g. thru the filter. Then wash precipi-
5. Agar (1.2%) 12.0 g. tate with a little distilled water.
Preparation The filtrate is cloudy.
(1) Heat one liter of skim milk to boiling (7) Make up (6) to 250.0 cc. with dis-
sodium oleate solution. (In another for 10 minutes on each of 2 successive days
part of the article the author states holding the medium at 30C. between
that best growth was obtained with heating.
about sodium
1.0% oleate. The Use: Qualitative milk counts.
percentage used above is only 0.1%. Reference: Zoller (1923 p. 385).
(7) Filter. Agar
1853. Smillie's Tissue Infusion
(8) Tube.
Sterilization: Sterilize in the autoclave. Constituents
Use: Cultivation of Lactobacillus hulgari- 1. 2.0% veal infusion agar 100.0 cc.
1. Distilled water 5000.0 cc. (3) When ready for use, melt sterile (2),
2. Bacto Nutrient Agar (1.0%) 120.0 g. boil and cool rapidly to 50C.
(1.0%) into 4 liters of distilled water. (7) Add 15.0 cc. of (6) to a sterile tube
Add 2.0 g. sodium citrate and set in containing sterile rabbit tissue
(2)
streaming steam in the autoclave (kidney) (and the poliomyelitis
for about 5 minutes. materials).
(3) Stir 25.0 g. of any good grade of Sterilization: Method not specified.
(6) Remove the agar from the autoclave erobic organisms found in influenza
and stir. on a medium prepared as follows:
(7) Add 50.0 cc. distilled water to (5) (1) Prepare a 2.0% beef infusion agar.
following steaming. (2) Adjust (1) to pH = 7.4.
(8) Replace (6) and (7) in the autoclave (3) Mix one part (2) with two parts
and heat for 5 minutes at 5 pounds sterile human ascitic fluid in a
pressure. flask at 40C.
580 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(4) Pipette (3) into sterile tall test (8) Add sterile chalk in excess (about 5
tubes containing pieces of sterile spoonfulls) and distribute into sterile
rabbit kidney and the inoculum. tubes.
(5) Seal with sterile melted vaseline, (9) Add a sterile piece of liver to each
(b) Harvey cultivated anaerobic spiro- tube.
chaetes on the following medium: Sterilization: Sterilize at 100C. for one
(1) Transfer small portions of organs hour and slant.
aseptically removed from a rabbit, Use: Cultivation of Bad. butyricus and
specially killed for the purpose other anaerobes.
(2514 variant (a), to each sterile Reference: van Riemsdyk (1922 p. 248).
test tube 20 x 2 cm.
Mix one part infusion agar, 1855. Williams' Tissue Infusion Agar
(2)
1.0% acid to phenolphthalein at Constituents
45C. (see medium 1661 variant (v) 1. Water, tap 90.0 cc.
for preparation) with one part 2. Agar (1.0%) 1.0 g.
sterile ascitic fluid heated to 45C. 3. Infusion broth (10.0%) 10.0 cc.
(3) Fill, with sterile precautions, about 4. Brain
20.0 cc. of (2) into each test tube Preparation
(1). (1) Mix 90.0 cc. tap water, 10.0% ordinary
(4) Cover the medium to a depth of infusion broth and 1.0% agar.
3 cm. with sterile liquid paraffin. (2) Dissolve.
(5) Test sterility by incubation 48 (3) Reaction neutral to phenolphthalein.
hours. (4) Cut fresh sterile brain in small pieces
References: Smillie (1918 p. 324), Olitsky and place on the surface of sterile (3).
and Gates (1921 p. 715), Harvey (1921-22 Sterilization: Sterilize as usual (method
p. 97). not given).
Use: Cultivation of amoeba.
1854. van Riemsdyk's Liver Infusion Agar
Variants: Park, Williams and Krumwiede
Constituents substituted liver or kidney for brain.
1. Water 500.0 cc. Reference: Park, Williams and Krumwiede
2. Beef liver 0.5 lb. (1924 p. 134).
3. Peptone (Witte) 5.0 g.
4. NaCl 2.5 g.
1856. Olitsky and Gates' Ascitic Fluid
5. Agar 10.0 g.
Kidney Agar
6. 2.0% glucose agar 500.0 cc. Constituents
7. Liver tissue 1. Beef infusion broth.
8. Glucose 10.0 g. 2. Beef infusion (2.0%) agar.
9. Chalk 3. Human ascitic fiuid.
Preparation 4. Rabbit kidney.
(1) Pass raw beef liver thru a fine meat Preparation
grinding machine. (1) Prepare beef infusion broth and 2.0%
(2) Add 500.0 cc. of water to pound of beef infusion agar.
(1). (2) Adjust both the broth and agar to
(3) Boil for 30 minutes with occasional pH = 7.4.
stirring. Allow to settle. (3) Place in each 50.0 cc. Florence flask
(4) Pour off the liquid and force the liver i of a moderate size rabbit kidney.
thru a very fine metallic sieve. Sections are cut across the entire
(5) Add the pulverized liver and 3, 4 and kidney and placed with the cut sur-
5 to the liquid. face parallel to the base of the flask.
(6) Heat for one hour at 110C. and make (4) Inoculate the kidney.
alkaline to phenolphthalein using (5) Mix one part (2.0%) infusion agar,
N/10 NaOH. (2), with two parts sterile human
(7) Add 10,0 g. of glucose and mix with ascitic fluid of pH = 7.8 to 8.0, in a
an equal volume of 2.0% glucose agar. flask at 40C.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 581
wide meshed gauze into 500.0 cc. (3) Make up the original volume of (1)
distilled water and place in the ice by the addition of sterile infusion
box for 24 hours. broth.
(2) Filter thru cotton of varying degrees (4) Prepare a 2.0% solution of neutral
of compactness. The filtrate will sodium oleate. (Kahlbaum's pre-
not be clear. ferred.)
(3) Add 0.5%, KH2PO4 and 1.0% peptone. (5) Sterilize (4) in the autoclave.
(4) Prepare standard 2.5% agar from veal (6) To 94.0 cc. of hot 2.0%, nutrient agar
infusion with the addition of 0.5% (vitamin agar, see variant (e) medium
NaCl and 1.0% peptone. 1863 is recommended) add 5.0 cc. of
(5) Mix one part (2) with three parts (4). (5) and add 1.0 cc. of (3) under
(6) Adjust to pH 7.8. aseptic conditions.
(7) Tube. References: Harvey (1921-22 p. 87), Park,
(8) Following sterilization and solidify- Williams and Krumwiede (1924 p. 130).
ing replace the cotton plug with a
1859. Brown and Orcutt's Blood Cell Agar
sterile rubber cork.
Sterilization: Sterilize in the autoclave. Constituents:
Use: Cultivation of gonococci. 1. Veal infusion agar.
scanty growth. The author does not (3) Add some of the washed blood cor-
Use: To determine food requirements for (8) Dilute the red blood cells, freed
Bacillus pyogenes. Authors reported from serum, with 0.5% NaCl solution
that this medium supported the growth to the original blood volume.
of Bacillus pyogenes nearly as well as did (9) Shortly before use melt the desired
blood agar. tubes of sterile agar, cool to 60C.
Variants: The authors prepared a similar and add two volumes of (8) to each
medium as follows: tube.
(1) Prepare veal infusion agar. (10) Mix well and solidify in slanting
(2) Defibrinate blood and wash repeat- position.
edly with sterile physiological salt Sterilization: Sterilize (6) in autoclave by
solution. heating to 105-108 for 15 minutes.
(3) Lake thewashed corpuscles with Use: Special culture medium for Trypano-
distilled water and remove the cor- soma Brucei.
puscle stroma by centrifugation. Reference: Behrens (1914 p. 29).
(Centrifuge the laked blood cor-
1861. Sacquepee and Delater's Egg Albumin
puscles until the supernatant hemo-
Infusion Agar
globin solution no longer gives a
clouding reaction with salt.) Constituents
(4) Wash the corpuscle stroma repeatedly 1. Distilled water
in sterile distilled water until no 2. Egg white
visible trace of hemoglobin remains. 3. Meat 500.0 g.
(5) Add the stroma suspension to melted 4. Peptone 10.0 g.
agar (amount not given) and pour into 5. NaCl 5.0 g.
plates. 6. Agar 30.0 g.
This medium was inferior to blood cell Preparation
medium above. (1) Place the whites of two eggs in a
Reference: Brown and Orcutt (1920 pp. graduated glass cylinder.
222-223). (2) Shake constantly and add little by
littlethree times the volume of
1860. Behren's Blood Cell Agar
distilled water.
Constituents (3) Make alkaline by the addition of
1. Water 1000.0 cc. about 0.5 cc. of a 10.0% soda solution
2. Chopped beef 125.0 g. for each 100.0 cc. of (2).
3. Peptone 20.0 g. (4) Shake well.
4. NaCl 5.0 g. (5) Heat in the autoclave at 115 for 15
5. CaCl, 0.1 g. minutes.
6. N/1 NaaCOs solution 10.0 cc. (6) Macerate 500.0 g. of meat in a liter of
7. Agar 20.0 g. water.
8. Red blood cells from 2000.0 (7) Allow to stand in the cold for 24
cc. Rabbit blood hours and then bring slowly to boil.
Preparation (8) Filter.
(1) Digest chopped beef in 250.0 cc. (9) Dissolve 10.0 g. peptone, 5.0 g.
water over night in the cold, or for NaCl in (8).
one hour at 55C. (10) Dissolve 30.0 g. agar in (9).
(2) Strain (1), boil the infusion and (11) Exactly neutralize to litmus and
filter then add 9.0 cc. of a 10.0% soda
(3) Dialyze the filtrate in a large col- solution.
lodium sac against running distilled (12) Clarify by the addition of an egg
water for 24 to 48 hours. white to the agar at 60C. Heat in
(4) Dilute (3) to 1000.0 cc. the autoclave and filter thru paper.
(5) Dissolve 3, 4, 5, 6 and 7 in (4). (13) Mix (5) and (12). Approximately
(6) Distribute in test tubes in 1.0 cc. one part (5) to five parts (12).
lots. (14) Distribute as desired.
(7) Centrifuge defibrinated rabbit blood Sterilization: Sterilize for 15 minutes at
and draw off serum. 112C.
583
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Beef, fresh heart or steak. 500.0 g. mone agar as indicated above but
2. .
NaOH. hour.
Add 500 .p cc. of water. (5) Separate the clot from the sides of
(2)
Heat very slowly to 95C. the container and place in the steri-
(3)
Keep at 95C. for 60 minutes. lizer for another hour.
(4)
Filter thru cotton wool. (6) Clarify by straining thru glass wool.
(5)
(6) Distribute into flasks. (7) Adjust to pH = 7.2.
(7) When ready for use mix 10 parts (8) Tube or flask.
melted infusion agar, cooled to Sterilization: Sterilize the fractional
sterile
45''C. (see variant (v) medium 1661 method at 100C. in flowing steam.
for preparation) with one part sterile Use: Cultivation of Cyclostoma elegans
and other organisms living in symbiosis
(6) heated at 45C.
Sterilization: Sterilize (6) in the autoclave. with moUusks.
Use: General culture medium. Added nutrients:
Variants (a) The author used the basal medium
(a) Harvey reported that a clearer without additional material.
medium was obtained if the whites (b) The author added 10% defibrinated
of two eggs were mixed with 4.0 cc. rabbit blood to the melted agar just
of normal NaOH and 330.0 cc. of before use.
water instead of the quantities given (c) The author added 1.0 to 2.0% of
form and preserve in the ice chest (3) Adjust to slightly alkaline to litmus
for four weeks or sterilize in flowing with N/1 NaOH and then add 1.0
steam at 100C. for 30 minutes. cc. per liter of medium.
Reference: Meyer (1925 p. 47). (4) Cover the vessel and place in an
Arnold sterilizer or in a water bath
1866. Farcy and Chavaillon's Egg Albumin
at 100 for one hour
Serum Agar
(5) Remove the vessel from the sterilizer
Constituents and separate with a glass rod the
1. Infusion agar firm clot, which has formed, from the
2. Serum 100.0 cc. side of the vessel.
3. Egg white 20.0 cc. (6) Return to the Arnold sterilizer and
Preparation: heat at 100 for 1 hours.
(1) Add 100.0 cc. of horse serum obtained (7) Remove the vessel and allow to
under aseptic conditions to a medium stand at room temperature for about
sized sterile flask containing glass ten minutes in a slightly inclined
beads. position.
(2) Add 20.0 cc. of egg white to (1). (8) Pipette off the fluid portion or
Remove the egg white by means of a decant. If it is poured thru a fine
sterile pipette, and add to (1) under wire sieve, many of the fine pieces of
aseptic conditions. meat clot may be
caught. (Avoid
(3) Shake the flask vigorously for 5 to 10 filteringthru cheese cloth, cotton or
minutes. other adsorptive materials.)
(4) Prepare a 2.5% infusion agar. (9) Allow (8) to stand in tall cylinders
(5) Exactly neutralize (4) and add 0.2 for 15 to 20 minutes until the fat
cc. of a 10.0% soda solution per liter present has risen to the surface and
of agar. removed. The medium be may
(6) Mix one part (3) with 3 parts (5), further cleared by thru
filtering
melted and cooled to 50C. glass wool, asbestos wool, sedimenta-
(7) Distribute as desired. tion or centrifugation.
Sterilization: Not specified. (10) Tube in 10.0 cc. lots.
Use: Cultivation of meningococci. (11) Add a small amount (1.0 to 100.0
Variants: Authors suggested that sugars cc.) of blood (just
defibrinated
maj' be added if desired. enough to give a pinkish tinge to the
Reference: Faroy and Chavaillon (1915 p. poured plate) to each tube of sterile
455). (10).
SteriUzation : Sterilize (10) intermittently
1867. Huntoon's Hormone Blood Agar
in the steamer.
Constituents: Use: To cultivate highly parasitic organ-
1. Water 1000.0 cc. isms. Author reported the medium es-
2. Beef heart or steak (fresh). 500.0 g. pecially adapted to the cultivation of the
3. Peptone (Bacto) 10.0 g. meningococci. The medium is superior
4. Agar 16.0 g. to glucose ascitic agar.
5. Salt 5.0 g. Variants: Huntoon reported better results
6. Whole egg (one) were obtained using laked blood instead
7. Blood, defibrinated of defibrinated.
Preparation Reference: Huntoon (1918 p. 171).
(1) Chop 2 and then mix with 1, 3, 4,
5 and 6. Place in an enamel ware 1868. Torrey and Buckell's Ascitic Fluid
vessel or a large coffee pot. Egg Agar
(2) Heat over a free flame with constant Constituents
stirring until the red color of the 1. Distilled water 1000.0 cc.
meat infusion changes to brown at a 2. Beef heart 500.0 g.
temperature of about 68C. Do not 3. Whole egg 1
-
Preparation: ^'"*."!mI'; .
(preferably directly 1 Distilled water
(1) Place beef heart
.
Eggs
from slaughter house) and one whole 2.
3. Glycerol
egg in water.
Veal ^^^"^ S-
(2) Place (1) in a double boiler over a 4. ...
Gentian violet (1.0% soln.) 1.0 cc.
free flame and maintain at 60C. for
5.
. , ,
(1) Dilute egg whites
obtained under
aseptic conditions with 10 parts
(4) ld]usi\o slightly alkaline to litmus
distilled water. Shake thoroly
using 10.0% solution of Na.COa-
To clear, pass the Auid thru a thm
(5) Place (4) in flask, or preferably in a (2)
layer of cotton and then heat to
coffee pot and keat at 100C. in
100C.
Arnold steam sterilizer for one hour.
(3) Filter thru paper.
(6) Separate clot from sides of the _
Reference: Torrey and Buckell (1922 p. (17) Pour from one flask to another to
insure thoro mixing.
j2g)
588 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Sterilization: Sterilize (7) by heating in the (1) Extract one pound of lean chopped
autoclave at 120C. for 20 minutes. beef or veal in 500.0 cc. of water
for 18 hours.
Use: Cultivation of gonococci.
Reference: Leboeuf (1924 p. 768). (2) Add and dissolve in this infusion,
made up to 1 liter with water,
Agar 10.0 g. agar, 20.0 g. gelatin, 20.0 g.
1873. Besson's Gelatin Infusion
Witte peptone and 5.0 g. salt.
Constituents (3) Adjust to the neutral point using
1. Infusion broth 1000.0 cc. phenolphthalein as an indicator.
2. Gelatin 80.0 g. (4) Tube.
3. Agar 5.0 g. (b) Dopter and Sacqu6p^e prepared the
Preparation : medium as follows:
(1) Dissolve 80.0 g. of gelatin in 1000.0 (1) Add 1000.0 cc. of water to 500.0
cc. of infusion broth. g. of finely chopped fat and tendon
(2) Neutralize. free beef.
(3) Dissolve 5.0 g. of agar in (2). (2) Allow to stand in the ice box for
(2) Dissolve 50.0 g. of gelatin in (1). (4) Mix (3) and (1).
(5) Adjust reaction as desired (for B. (5) Dissolve 3, 4, 5 and 6 in (4) by heating
bifidus, +1 to phenolphthalein). over a free flame stirring constantly.
(6) Clear with eggs if necessary. (6) Titrate using phenol red as an indi-
(7) Add glucose and K2HPO4 to clear cator to pH = 7.4 to 7.8 or a reaction
filtrate. of 0.6 to phenolphthalein.
(8) To each 10.0 cc. of sterile medium (7) Tube and autoclave at 15 pounds for
add about 1.0 cc. of sterile defibri- 20 minutes.
nated rabbit blood just before pouring (8) Check titration.
the plate. (9) While (8) is still liquid, add human
Sterilization: Method not given. blood in proportion of 0.5 to 2.5%.
Use: Isolation of B. bifidus. To isolate Sterilization: See step (7) above.
B. bifidus pour medium in petri dish. Use: Isolation and cultivation of gono-
Prepare nutrient agar seeded with B. cocci. Author reported that using phenol
cereus and pour in cover of the Petri dish. red as an indicator primary acidity and
Allow the liver agar to solidify and secondary alkalinity may be determined.
nutrient only partially. When
agar In a case of mixed infection, methyl violet
nutrient agar semi solid, invert the
is in proportion of 1:200,000 to 500,000
inoculated liver agar plate and place it inhibited the growth of staphylococci.
in the semi solid agar. The solidifying Variants: The authors substituted 1.0 to
agar forms a seal. This gives partial 5.0% defibrinated rabbit blood for human
anaerobiosis. Author reported colonies blood.
were raised, more or less globular, opaque, Reference: Erickson and Albert (1922 p.
1 to 3 mm. in diameter, buff to reddish 277).
brown in color. B. acidophilus gave
serrated edge colony To isolate
flat.
1884. Harvey's Lactose Blood Agar
B. acidophilus use this medium omitting Constituents:
blood and adjusting to +4 acid. Grow 1. Infusion agar 1000.0 cc.
aerobically. 2. Lactose 30.0 g.
Variants: Harvey solidified medium 833 3. Blood, sterile defibrinated
variant (a) by the addition of agar. human
References: Torrey (1922 p. 435), Harvey 4. Rosolic acid (1.0%)
(1921-22 p. 110). Preparation
(1) See variant (v) Medium 1661 for
1883. Erickson and Albert's Testicular
preparation of infusion agar.
Blood Agar
(2) Mix 0.3 g. lactose with each 10.0 cc.
Constituents: of (1).
1. Distilled water 1000.0 cc. (3) Raise slowly to boiling water
2. Beef testicle 500.0 g. temperature.
3. Peptone 20.0 g. (4) Cool to 45 C.
694 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Add 1.0 cc. defibrinated sterile human (5) Add to each sterile tube of (4) 0.5 cc.
blood at 45C., and 3.0 cc. 1.0% alco- of defibrinated rabbit blood.
(2) Steam for 60 minutes. (13) Add 2.0% agar and dissolve.
(3) Desiccate in vacuo over sulphuric (14) Filter thru cotton wool and distrib-
acid or calcium chloride. ute in 600.0 cc. flasks.
(4) Grind the residue to powder. (15) The pH of sterile agar should be
(5) Make for use a 10.0% solution of about 7.2.
blood powder. (16) Add 30.0 cc. of (4) to each 600.0 cc.
(6) When ready for use add 3 parts of melted (15) cooled to 40 to 50C.
(5) at 45C. to 7 parts infusion Sterilization: Sterilize (10) by heating in
agar at 45 C. the steamer on each of 3 consecutive days.
(b) (1) Mix equal parts 12.0% crystalline Use: Cultivation of influenza bacilli,
Na2C03 solution and defibrinated Haemoglobinophilic bacteria.
blood. Reference: Kristensen (1922 p. 229).
(2) Mix 3 parts (1) with 7 parts of
1892. MacNeal's Blood Infusion Agar
4.0% infusion agar.
Reference: Harvey (1921-22 pp. 73, 75). Constituents:
1. Water 1000.0 cc.
1891. Filde's Blood Digest Agar 2. Chopped beef 125.0 g.
(Kristensen)
3. Agar 20.0 g.
Constituents: 4. Peptone 20.0 g.
1. Water 25,000.0 cc. 5. NaCl 5.0 g.
2. Beef 25,000.0 g. 6. N/1 NaoCOa soln 10.0 cc.
3. Peptone 1.0% 7. Blood, naturally sterile de-
4. NaCl 0.5% fibrinated rabbit 2000.0 cc.
5. Blood, defibrinated sheep Preparation
or horse (1) Extract 125.0 g. beef in 1000.0 cc.
Preparation distilled water.
(1) Add 6.0 cc. of pure concentrated (2) Dissolve 3, 4, 5 and 6 in (1).
HCl to 150.0 cc. of a 0.9% NaCl (3) Tube.
solution, and then 50.0 cc. defibri- (4) Add to sterile (3), cooled to about
nated horse or sheep blood and 2.0 g. 60 two volumes of naturally sterile
Langebek's concentrated pepsin. defibrinated rabbit's blood. Mix
(2) Pour into a sterile flask and incubate thoroly.
at 56C. for 5 or 6 hours. (5) Slant and solidify.
(3) Add NaOH solution until a sample Sterilization: Sterilize (3) in the autoclave.
diluted with distilled water to a Use: Isolation of Nagana parasite, Tr.
light brown color turns red on the Brucei and other trypanosomes. In-
addition of phenol red, without oculate Nagana blood in the small amount
giving the alkaline color change of liquid which collects on the surface
with a-naphtholphthalein. of the blood agar. Thompson cultivated
(4) Add 0.25% chloroform and stopper trypanosomes found in gold fish (T.
the flask with a sterile rubber and danilewskyi)
keep in a cold room. Variants
(5) Mince 25,000.0 g. beef. (a) The author used 4 times as much meat
(6) Add 25 liters of water and allow to (500.0 g. per liter) for succeeding
stand in the cold until the next day. cultures.
(7) Boil for a short time. (b) Tobey used the same medium and
(8) Press the fluid from the meat. added two volumes of defibrinated
(9)Add about 16 liters of water to the rabbit's blood. He inoculated the
meat, heat to boiling once more and water of condensation with a drop of
again press the juice from the meat. blood taken from the heart of a bird
(10) Mix the fluid from (7) and (8). by means of a drawn-out tube pipette.
(11) Add 1.0% peptone and 0.5% NaCl. The cotton plug is then cut short,
(12) Adjust to pH about 7.8 to 7.9. moistened with mercuric chloride
596 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
and the tube covered with a rubber (6) Cool sterile (3) to 45C. and add an
cap, after which it is placed at 25C. equal volume of (5).
for about one week, (7) Mix and slant.
250.0 cc. water over night in the and other trypanosomata. Other in-
cold, or for one hour at 55 C. vestigators cultivated a number of differ-
(2) Strain (1), boil the extract and ent organisms on similar media.
filter. Variants
(3) Dialyze the filtrate in a large col- (a) Duval (Gurd) prepared a medium as
lodium sac against running distilled follows:
water for 24 to 48 hours. (1) Prepare a beef infusion.
(4) Dilute (3) to 1000.0 cc. (2) Dissolve 10.0 g. peptone, 20.0 g.
(5) Dissolve 20.0 g. peptone, 5.0 g. agar and 5.0 g. NaCl in (1).
NaCl, 0.1 g. CaCh, 10.0 cc. N/1 (3) Adjust reaction to 0.6% acid to
NajCOa solution and 20.0 g. agar phenolphthalein (hot titration).
in (4). (4) Tube.
Distribute in test tubes in 10.0 cc. (5) Sterilize in the autoclave.
(6)
lots. (6) Cool to 52C. and add a small
(8) Shortly before use, the desired of blood are added to each 6 to
number of agar tubes are melted in 10.0 cc. of agar.
p. 265), Thompson (1908 p. 77), Behrens Gave a bright crimson almost trans-
(1914 p. 27). parent medium. If agar was above
60C. when mixed with blood, the
hemoglobin was destroyed and me-
1893. Smedley's Blood Infusion Agar
dium assumed a dirty brown color.
Constituents (b) Duval cultivated B. leprae on a
1. Bullock heart infusion 1000.0 cc. medium prepared as indicated. He
2. Peptone 10.0 g. reported that cultures without the
3. NaCl 10.0 g. blood and using B. typhosus and
4. Agar 20.0 g. Entameba coli gave growth of B.
5. Blood, defibrinated rabbit.. 1000.0 cc. leprae.
(c) Hagemeister cultivated pathogenic (3) Cool the flask to 50C. and thoroly
trypanosomes in the following mix 5.0 cc. of defibrinated human
medium: blood with the fluid agar.
(1) Infuse one pound of finely chopped (4) Pour this mixture into petri dishes
beef or horse meat with one liter for blood agar plates on the surface
of water for 24 hours. of previously prepared agar.
(2) Boil for one hour. (5) The agar for (4) is prepared by
(3) Filter thru a towel. dissolving 1.5% agar in normal
(4) Make up to 1 liter by the addition saline (0.85% NaCl), filtered,
of water. tubed and sterilized.
(5) Add 10.0 g. peptone (Sice), 20.0 g. (6) Pour the blood agar mixture (3)
agar and 5.0 g. NaCl to (4). on the plain agar (5) to a depth of
(6) Heat to boiling, stirring con- about 2 millimeters.
stantly. He reported that it was advisable to
(7) Neutralize by the addition of N/1 add the serum with the red blood cells
NaOH using litmus as an in- as the serum is an important aid in
dicator. hemolysin production.
(8) Add 5.0 cc. of N/1 NaOH per liter. (f) Kohman studied the oxygen tension
(3) Mix thoroly and pour in plates. infusion agar at 70C. (See
(i) Bell cultivated influenza bacilli in a variant (v) medium 1661.)
medium containing 4.0% defibrinated (2) Raise the temperature of the
rabbit blood to a 2.0% infusion agar. mixture to boiling point over a
(j) Yoshida cultivated Entamoeba tetra- free flame.
gena and Entamoeba coli from their (3) Shake to mix.
cysts in the water of condensation of (4) Raise to boiling point twice again.
an agar containing one part defibri- (5) Allow to deposit.
nated horse blood to two parts agar. (6) Decant the clear supernatant fluid
The blood and agar were mixed at into test tubes while the agar is
(1) Harvey mixed one part of sterile at 70C. with 20 parts of infusion
defibrinated blood at 45C. with two agar at 70C. (See variant (v)
di-sodium phosphate. The substitu- (3) Add 0.5 cc. of defibrinated horse
at 57C. Serum may be used instead (4) Remove a small piece of lung
of blood. tissue from an infected rat under
(o) Harvey cultivated B. influenza, etc., aseptic conditions and push the
on a medium prepared as follows: down into the tubes.
tissue
(1) Boil 1.0 cc. blood with 9.0 cc. (5) Seal the tubes with sealing wax.
water. (s) Pitfield added 1 part sterile defibri-
Allow to deposit. nated blood to 5 parts sterile melted
(2)
(3) Add 0.5 cc. clear colorless super- agar.
natant fluid to 5.0 cc. melted (t) Stitt prepared a N. N. N. medium
infusion. (See variant (v) me- for the cultivation of trypanosomes,
(1) Prepare an agar base using 500.0 g. (3) Heat the juice to coagulate the
veal, 5.0 g. NaCl, 15.0 g. agar and proteins. Filter.
10.0 g. peptone in the same manner (4) Dissolve 1.0% Witte's peptone and
as for the preparation of ordinary 0.5% Kahlbaum's NaCl in the
nutrient agar. filtered infusion.
(3) Tube in 12.0 cc. amounts. (7) Dissolve 2.0%, agar in (6).
(4) To prepare the blood agar melt the (8) Tube or distribute in flasks.
tubes of agar and place in a water (9) Cool melted tubes of (8)
sterile
bath at 45 C. for 15 minutes. to 50C. and add an equal volume
(5) Add 0.6 cc. defibrinated blood to of sterile defibrinated rabbit
each tube and mix thoroly. blood.
(6) Inoculate. (10)Mix well and slant,
(7) Pour into plates. ai) Autoclave (8) at 110C. for 20
(v) Beilin (Klimmer) cultivated influenza minutes. Blood collected and de-
bacilli on a medium prepared as fibrinated under aseptic con-
follows: ditions.
(1) Heat3.0%infusionagar with 1.0% References: Smedley (1905 p. 28), Gurd
peptone to boiling. (1908 p. 303), Duval (1910 p. 653), Hage-
(2) Add 15.0% of sterile fresh defibri- meister (1914 p. 228), Warden (1915 p.
nated horse blood to boiling (1). 428), Holman (1916 p. Kohlman
381),
(3) Remove from the flame. (1919 p. 574), Bernstein and Lowe (1919
(4) Cool to 50 to 60C. and mi.x p. 78), Wahl, White and Lyall (1919 p.
thoroughly. 420), Yoshida (1920 p. 361), Bell (1920 p.
(5) Pour in plates. 464), Anderson and Schultz (1921 p.
(w) Schottmuller (Klimmer) cultivated 656), Harvey (1921-22 pp. 73, 74, 76),
gonococci, meningococci and strepto- Jones (1922 p. 363), Pitfield (1922 p. 119),
cocci on a medium prepared by mix- Stitt (1923 pp. 43, 44, 52), Klimmer (1923
ing five parts liquified agar at 45C. p. 226), Park, Williams and Krumwiede
with two parts sterile defibrinated (1924 p. 133), Soule (1925 p. 248).
human blood.
1894. Harvey's Peptic Blood Digest Agar
(.\) Novy and MacNeal (Park, Williams
and Krumwiede) cultivated flagellate, Constituents:
trypanosomes and leishmania on a 1. Infusion agar
(3) Heat in the water bath at 55C. for Use: General culture medium.
2 to 24 hours with occasional shak- Reference: Harvey (1921-22 p. 84).
ing. (Note: The exact time is im-
1897. Harvey's Oxalated Blood Agar
material.)
(4) Add 12.0 cc. of 20.0% sodium Constituents
hydroxide. 1. Infusion agar
(5) Adjust the reaction by addition of 2. Blood, ox or sheep 400.0 cc.
20.0% sodium hydroxide until a 3. NaCl (0.85% solution)
sample gives with cresol red indi- 4. Ammonium oxalate (1.0%
cator solution (0.02%) the color of solution) 30.0 cc.
permanganate (corresponding to Preparation
pH = 7.6). (1) Collect 400.0 cc. ox or sheep blood
(6) Add pure Hydrochloric acid drop by at the slaughter house in a sterile
drop until cresol red indicator solu- flask containing 30.0 cc. of a 1.0%
tion gives practically no change of solution of ammonium oxalate and
color but phenol red gives red. 0.5 cc. formalin.
Note: Corresponding to pH = 7.0 (2) Mix well.
to 7.2. (3) Allow to stand 30 minutes.
(7) Add chloroform to 0.25%. (4) Dilute i with 0.85% sterile salt
1895. Bailey's Hormone Blood Agar 1898. Wolbach, Chapman and Steven's
Citrated Blood Agar
Add 5 parts per 100 of fresh defibrinated
human blood to Bailey Hormone Agar Constituents:
(1673). This medium is recommended to 1. Distilled water 1000.0 cc
preserve stock cultures of pneumococci and 2. Veal 500.0 g.
streptococci. 3. Glucose 1.5 g.
4. NaCl 6.0 g.
1896. Harvey's Ascitic Fluid Blood Agar
5. Agar agar 14.0 g.
3. Blood, defibrinated, sheep... 200.0 cc. of distilled water and 500.0 g. of lean
(3) Dissolve 3, 4, and 5 in 900.0 cc. dis- Use: Isolation of Spirochaeta icterohaemr-
tilled water rhagiae. Inoculate with suspected
(4) Add (2) to (3). blood (material) and cover with a thin
(5) Tube. layer of sterile paraffin oil. Author
(6) Add 2.0 to 3.0 cc. of a mixture of reported that growth took place at any
equal parts of sterile rabbits blood temperature from 10C. to 37C. Growth
and citrate saline solution to 4.0 cc. was nearly invisible and about 1.0 to 1.5
of the sterile agar jelly. cm. below the surface.
(7) Heat the mixture to 45C. for 30 Reference: Noguchi (1917 p. 761).
minutes.
and allow to stand until a 1900. Sparkar's Citrated Blood Agar
(8) Slant,
considerable water of condensation (Listen)
has collected, before inoculation. Constituents:
Sterilization: Sterilize (5) method not 1. Water 2000.0 cc.
given. 2. Meat 1000.0 cc.
Use: Cultivation of trj^panosomes. 3. Peptone 40.0 g.
Authors reported that medium afforded 4. NaCl 20.0 g.
rapid growth and the vitality of the 5. Agar 60.0 g.
cultures were not diminished. Gates 6. Blood (human)
cultivated meningococci on a similar Preparation
medium. (1) Boil 1000.0 g. of meat in 2 liters of
Variants : Gates prepared a similar medium water for 40 minutes.
as follows: (2) Filter.
(1) Prepare a veal infusion agar. (3) Dissolve 3, 4, and 5 in sterile (2).
(2) Add 1.0% glucose to (1). (4) Draw one volume of human blood
(3) Adjust to pH = 7.4. into a flask containing 4 volumes
(4) Sterilization not specified. sterile citrated saline(0.5% sodium
(5) Just before tubing add 5.0% sterile, citrate and 0.85% NaCl).
unheated citrated horse plasma. (5) Mix the blood and saline thoroly.
(Fresh rabbit serum may be em- (6) Heat in a water bath at 65C. for 30
ployed instead of plasma.) minutes.
References: Wolbach, Chapman and (7) Remove precipitate by filtering thru
Stevens (1915-16 p. 109), Gates (1919 p. sterilized filter paper.
322). Add 1.0 cc. of (7) to 10.0 cc. of
(8) melted
at 50C.
(3)
1899. Noguchi's Serum Plasma Agar
(9) Slant.
Constituents: Sterilization: Sterilize (2) at 15 pounds
1. Ringer's solution. . . 300.0 cc. pressure in the autoclave
2. Serum rabbit 100.0 cc. Use: Cultivation oi B influenzae (Pfeiffer).
.
(6) Method of sterilization not given. (3) Draw horse blood into flasks con-
(7) Medium is a faint yellow in color taining sterile sodium citrate solution
and transparent. to make the final concentration 1.5%.
(b) Harvey prepared a medium as (4) After have settled, the
corpuscles
follows: plasma is removed and sterile distilled
(1) Add 10.0 cc. of blood to 1.0 cc. water added to make up the original
sterile 10.0% sodium citrate. volume of the blood.
(2) Add 1.0 cc. of (1) to a test tube of (5) Heat sterile (2) to 90C. and add
melted infusion agar at 45C. sterile laked blood up to 1.0 to 2.0%.
(see variant (v) medium 1661 for Sterilization: Method not specified.
preparation) Use: Isolate influenza bacilli.
(3) Rotate the test tubes between the Variants: Harvey prepared a similar
hands to distribute the blood thru medium as follows:
the agar. (1) Mix 5 parts infusion agar at 45C. (see
(c) Harvey gave the following method of variant (v) medium 1661 for prep-
preparation: aration) with 2/lOths part of citrated
(1) Add 10.0 cc. of pigeon's blood to blood at 45C. (one part 10.0%
90.0 cc. of a 1.0% citrated 0.85% citrate in 0.95% sterile salt solution
(2) Heat 30 minutes at 65 to 70C. (2) Place in a water bath at 80C. until
(3) Filter thru thick filter paper. the agar has become chocolate in
(4) Mix 1.0 cc. of the filtrate from (3) color.
with 10.0 cc. of melted infusion, (3) Slope.
cooled to 45C. with a reaction References: Hirsch and McKinney (1919
slightly alkaline to litmus. (See p. 605), Harvey (1921-22 p. 77).
variant (v) medium 1661 for prep-
1902. Harvey's Glucose Blood Agar
aration of agar.)
(d) Soparkar (Harvey) prepared a similar Constituents:
medium as follows: 1. Distilled water 1000.0 cc.
(10) Clarify with white of egg. dilution of soda solution was not
(11) Bring the volume to its orginal specified.)
amount. (5) Pour into plates.
(12) Estimate and make the reaction (6) Smear 1.0 cc. of blood over each plate.
0.6% acid to phenolphthalein. Sterilization: Not specified.
(13) Add to the melted nutrient agar, Use: Cultivation of influenza bacilli.
(16) Keep until required for use in the tap water for 10 minutes.
ice chest. (2) Filter.
(17) Prepare sterile 10.0 cc. centrifuge (3) Add 10.0 g. Bacto peptone, 5.0 g.
tubes each containing 2.0 cc. sterile NaCl, and 25.0 g. agar and dissolve.
2.0% sodium citrate. (Heat).
(18) Have in readiness corks or rubber (4) Adjust to +1.0 (pH = 7.6-7.8).
bungs, contained in alcohol to fit the (5) Flask and sterilize.
centrifuge tubes. (6) Cool to 45-50C. and add 10.0%
(19) Fill up the centrifuge tubes with (100.0 cc.) sterile whole rabbit
human blood sterilely aspirated. blood. Shake.
(20) Replace the wool plugs of the centri- (7) Pour, under sterile conditions, into
fuge tubes by corks after burning sterile test tubes or Petri dishes.
off the alcohol. (8) Slant tubes.
(21) Centrifuge. (b) Twort and Twort prepared a similar
(22) Prepare with sterile precautions: medium as follows:
Centrifuged blood fluid at 45C. (1) Prepare infusion agar and infusion
(21) 75; melted nutrient agar (13) broth.
contained in the test tubes at 45C. (2) Sterilize the broth in a flask.
4. (3) Add 5.0% rabbit blood, withdrawn
(23) Roll the test tubes between the from the vein of the ear with a
hands to mix. sterile syringe, to (2).
(24) Test sterility by incubating 48 hours. (4) Incubate (3) at 37 for one hour,
Sterilization: See step (14) under prep- shaking repeatedly to prevent the
aration. forming of too much clot.
Use: Cultivation of gonococci. (5) Melt infusion agar tubes and cool
Reference: Harvey (1921-22 p. 82). to 50C.
(6) Add about 20 drops of the fluid
1903. Grassberger's Blood Agar
from (4) to each tube of (5).
Constituents (7) Slant.
1. Infusion agar. (c) Harvey gave the following method
2. Blood. of preparation
Preparation: (1) Wash and scrub the finger to be
(1) Prepare beef infusion agar. used well with hot soap and water.
(2) Add 10.0% NaOH until blue litmus (2) Drop on to the finger absolute
paper is no longer blue. alcohol followed by ether to remove
(3) Distribute in 20.0 cc. lots. the alcohol.
(4) Add 20 drops diluted 10.0% soda (3) Congest the pulp of the finger by
solution to each flask so as to obtain a winding a bandage round the base.
normal agar (10.0 cc. N/1 soda solu- (4) Prick the congested pulp with a
tion, for 1000.0 cc. agar). (The sterile needle.
604 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Take up a drop of the blood which (9) Stopper tubes air tight with sterile
exudes in a sterile platinum loop. cork stoppers.
(6) Smear on the surface of an agar Sterilization: Sterilize in the autoclave.
slope. (Serum may be used in the Use: Cultivation of gonococci. The
same way.) author reported that the addition of 1.0%
(7) Cover the test tube with an India- glycerol or 1.0% glucose inhibited growth
rubber cap. just as did the absence of peptone.
(8) Test sterility before use by incuba- Reference: Ruediger (1919 p. 377).
tion 48 hours.
1905. Dieudonne's Alkaline Blood Agar
(d) Harvey also prepared a medium as
(Tanner)
follows
Add 0.25 cc. blood to 5.0 cc. melted
Constituents
(1)
agar infusion in a test tube. (See 1. Infusion agar 700.0 cc.
variant (v) medium 1661 for prep- 2. Blood, beef 150.0 cc.
(2) Roll the test tube between the Sterilization: Not specified.
(5) Mix one part of (4) at 45C. with five Use: Cultivation of hemoglobinophilic
parts melted infusion agar at 45C. bacteria.
(See variant (v) medium 1661 for Reference: Kristensen (1922 p. 231).
preparation.)
1912. Brown and Orcutt's Hemoglobin
Sterilization: Not specified.
Infusion Agar
Use: Isolation of B' influenzae.
Variants: Author reported that Brilliant Constituents
green which inhibits the gram + bacteria 1. Veal infusion agar.
may be added. 2. Hemoglobin.
Reference: Harvey (1921-22 p. 76). Preparation
(1) Prepare veal infusion agar.
1911. Kristensen's Hemoglobin Infusion (2) Defibrinate blood and wash re-
(1) Mince 25,000.0 g. beef (4) Add the hemoglobin solution thus
(2) Add 25 liters of water and allow to obtained to melted agar (amounts not
stand in the cold until the next day. specified) and pour in plates.
(4) Press the fluid from the meat. Use: To determine food requirements for
(5) Add about 16 liters of water to the Bacillus pyogenes.
meat, heat to boiling once more and Reference: Brown and Orcutt (1920 p. 223).
press the juice again from the meat.
1913. Esch's Alkaline Hemoglobin Infusion
(6) Mix the fluid from (4) and (5).
Agar (Tanner)
(7) Add 1.0% peptone and 0.5% NaCl.
(8) Adjust to pH about 7.8 to 7.9.
of Constituents
(9) Dissolve 2.0% agar in (8). (Agar 1. Infusion agar 85.0 cc.
may be omitted giving a liquid 2. Hemoglobin (horse) (Merck).. 5.0 g.
medium.) 3. KOH (N/2) 30.0 cc.
(10) Filter thru cotton wool and distri- Preparation
bute in 600.0 cc. flasks. (1) Dissolve 5.0 g. of Merck's horse hemo-
(11) The pH of the sterile agar should globin in 30.0 cc. of half normal KOH.
be about 7.2. (2) Steam for 30 minutes.
(12) Allow a flask containing defibrinated (3) Mix 15.0 cc. of (2) with 85.0 cc. of
blood to stand several hours to per- neutral (litmus) infusion agar.
mit the corpuscles to sink. (4) Pour in plates.
(13) Pour off the supernatant fluid and (5) Dry at room temperatures with covers
replace with distilled water. removed.
(14) Distribute the hemoglobin solution Sterilization: Not specified.
thus obtained in 20.0 cc. quantities. Use: Cultivation of Microspira cholerae.
(15) Store in the ice box until ready for Reference: Tanner (1919 p. 70).
use.
1914. Sherwood and Downs' Basal Serum
(16) Add 20.0 cc. of (15) to 600.0 cc. of
Agar
sterile melted (11) cooled to 40 to
50C. Constituents:
(17) Pour into plates. 1. Infusion agar 1000.0 cc.
Sterilization: Sterilize (10) in the steamer 2. Serum 20.0 cc.
on each of 3 successive days. 3. Andrade's Indicator
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 607
(7) Cool and solidify in slanting (3) Prepare 2.5% agar in the usual way,
position. adding 0.5% NaCl, 1.0% glucose,
(8) Inoculate tubes and place in a 2.0% nutrose and 2.0% Chapoteaut's
10.0% atmosphere of CO2. peptone. The reaction is slightly
(i) Kinsella, Brown and Garcia used the alkaline.
following medium for the isolation of (4) Distribute in 100.0 cc. lots.
gonococci: (5) To 3 parts sterile (4) add 1 part sterile
(1) Prepare 1.6% beef or veal infusion beef serum.
agar. (6) Mix well and pour into plates. The
(2) Adjust to pH = 7.6. reaction is slightly alkaline.
(3) Distribute into 100.0 cc. quanti- Sterilization: Method of sterilization of (4)
ties inErlenmeyer flasks and not given. Sterilize beef serum in
sterilize (method not given). 50.0 cc. lots by placing it at 60''C. for one
(4) When ready for use melt (3) and hour on 4 successive days.
add to each flask containing Use: Cultivation of meningococci. Author
100.0 cc. of hot agar (90-100C.) reported that after 18-24 hours at 37C.
30.0 cc. of (sterile) beef seriun. meningococci colonies were circular, light
(5) Mix thoroly, distributing the small transparent, grayish-blue and about
particles of coagulated serum thru 2-3 mm. in diameter.
the medium. Variants
(6) Pour into sterile Petri dishes, (a) Scott gave the following method of
cover with porous terracotta lids preparation:
and place in incubator over night (1) Mince the placenta and prepare a
(18 hours). boiled extract as with meat (exact
(7) Replace terra-cotta lids by glass method not given).
lids and the medium is read for (2) Add 2.0% nutrose, 1.0% glucose,
inoculation. 1.5% peptone (Chapoteaut) and
Authors reported that gonococci 2.5% agar. Dissolve by steaming.
reached maximum growth in 48 hours. (3) Adjust the reaction to a -1-8 (Eyre).
The type of serum used had little or (4) Flask in 75.0 cc. lots.
no influence on the value of the (5) Sterilize (method not given).
medium. Plates should never dry (6) When ready for use melt and cool
under the terra-cotta lids longer than to 50 C.
24 hours. (7) Add 25.0 cc. of sterile (filtered) ox
References: Veillon (1898 p. 22), Joos serum to each flask.
(1899 p. 303), Behrens (1914 p. 29), Ferry (8) Mix and pour at once into plates.
and Davis (1918 p. 295), Ball (1919 p. 82), (b) Klimmer specified the use of 2.0%
Harvey (1921-22 pp. 79, 80, 82), Jones agar instead of 2.5%.
(1922 p. 363), Fitch (1922 p. 234), Kinsella, References: Kutscher (1908 p. 287), Scott
Brown and Garcia (1923 p. 1). (1915-17 p. 466), Harvey (1921-22 p. 79),
Klimmer (1923 pp. 202, 225).
1917. Kutscher's Serum Placenta Agar
1918. Bezanfon's Serum Placenta Agar
Constituents:
1. Water 1000.0 cc. Constituents
2. Placenta 500.0 g. 1. Water 1000.0 cc.
3. Agar 25.0 g. 2. Placenta 500.0 g.
4. NaCl 5.0 g. 3. Agar 25.0 g.
5 Glucose 10.0 g. 4. Glucose 10.0 g.
6. Xutrose 20.0 g. 5. Peptone 20.0 g.
7. Peptone (Chapoteaut) 20.0 g. 6. NaCl 5.0 g.
8. Serum (Beef) 335.0 cc. 7. Serum 300.0 cc.
Preparation Preparation
(1) Cut fresh placenta into small pieces. (1) Soak 500.0 g. of finely chopped pla-
(2) Weigh the pieces and juice and add a centa in 1000.0 cc. of water in the
double weight of water. ice box over night.
610 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Adjust reaction to 0.3 to 0.5% acid to References: Noguchi (1918 p. 606), Harvey
phenolphthalein. (1921-22 p. 80).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 611
Preparation
(4) Keep this as a stock agar.
(1) Prepare sugar free agar. Add 0.1% cystine (or cystein hydro-
(5)
(2) Dissolve 2 and 3 in 1. (Dye strength chloride) and 1.0% glucose to (4)
such that medium is colored.)
when ready for use.
(3) Tube in 7.0 to 8.0 cc. lots.
(6) Place in the Arnold for 15 minutes
(4) Just before use add 2.0 cc. of sterile in flowing steam to melt the agar,
beef serum to each tube of sterile (3). and sterilize the cystine.
Sterilization: Sterilize for three successive Cool to 45 to 50C. and add 5.0%
(7)
days in Arnold. (Time not specified.) sterile horse serum.
Use: Isolation and differentiation of strep- (8) Tube in sterile tubes.
tococci. Author reported that Strept, Slant.
(9)
pyogenes formed deep red colonies with Incubate for 24 hours to test
(10)
pink border. Strept. viridans (Schott- sterility.
miiller) formed red colonies surrounded
Sterilization: Method of sterilization of
by large area of milky-like haze; Strept. (3) not given. Cystine sterilized in step
mucosus (Schottmiiller) pink centered
(6) above.
colonies with white border, edge irregu-
Use: Cultivation of Bacterium tularense,
lar with milky-like haze. Pneumococci gonococci and diphtheria bacilli.
formed red centered colonies with narrow Reference: Stitt (1923 p. 46).
white border, smooth edge sharply de-
fined. Other sugars and dyes were tried 1925. Elser and Huntoon's Basal Ascitic
but this combination gave best results. Fluid Agar
Reference: Todd (1910 p. 74). Constituents
1. Distilled water 1000.0 cc.
1923. Harvey's Telluric Acid Serum Agar
2. Lean beef 500.0 g.
Constituents: 3. Peptone 10.0 g.
1. Infusion agar 4. NaCl 5.0 g.
2. Serum (sheep) 50.0 cc. 5. Agar agar 25.0 g.
3. Telluric acid (1.0% solution). 9.0 cc. 6. Litmus solution (Kubel &
Preparation: (1) Mix 50 parts sterilized Tiemann) 15.0 cc.
sheep serum at 45C., 9 parts of a sterile 7. Ascitic fluid 335.0 cc.
1.0% telluric acid solution at 45C., and Preparation
1000 parts of melted infusion agar, cooled (1) Prepare basic litmus infusion agar
to 45 C., neutral red to litmus (see from 1, 2, 3, 4, 5, 6 and one of the
variant (v) medium 1661 for prepara- added nutrients in the same manner
tion).* exactly as given in medium 1677.
Sterilization: Not specified. Use 25.0 g. agar instead of 16.0 g.
Use: Enrichment medium for B. diph- agar.
theriae. (2) To the finished product which is
Reference: Harvey (1921-22 p. 92). cooled to 55C. add I its volume of
sterile ascitic fluid. The degree of
1924. Francis' Cystine Serum Agar (Stitt)
alkalinity of the ascitic fluid must be
Constituents previously determined and taken into
1. Beef infusion 1000.0 cc. consideration when correcting the
2. Peptone (1.0%) 10.0 g. reaction of the agar.
3. Agar (1.0 or 1.5%) . . 10.0 or 15.0 g. Sterilization: Not specified.
612 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(4) Add brom-thymol-blue in suffi- (7) Rinse the mortar with the remainder
cient amount to give fairly deep of the 25.0 cc. of water.
Torrey and Buckell (1922 p. 145). (2) Mix (1) with ascitic fluid (pericardic
or hydrocelenic fluid may be used)
1926. Gilbert and Humphrey's Tellurite in the ratio of 3 to 1.
Serum Agar Sterilization: Not specified.
(1) Melt sterile beef infusion agar and most. Holding the tubes by the
cool to 50 C. butt, pass the tubes thru the flame
(2) Add 50.0 cc. of sterile horse serum, three times longitudinally and cork
10.0 cc. of a sterile 20% glucose quickly.
solution, heated to 50C. (1) Prepare a 2.0% beef (or veal) infu-
Weigh out 250 mgm. KiTeOs on a sion agar in the usual manner.
(3)
chemical balance. Grind to a very (2) Adjust (1) to pH 7.6 (pH 7.4 after
finepowder. autoclaving) and sterilize in the
(4) Add about 10.0 cc. of water. Mix autoclave.
well. (3) Mix one part sterile ascitic fluid
614 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
with two parts sterile melted (2). (d) Mulsow used the following medium
(Pleuritic or hydrocele fluid may for the isolation of gonococci:
be used instead of ascitic fluid.) (1) Infuse one pound of ground lean
(4) Replace the cotton stoppers with beef in 500.0 cc. of distilled water
sterile corks. over night in the ice box.
(b) Harvey prepared a similar medium (2) Dissolve 10.0 g. peptone in the
as follows: juice while cool.
(1) Mix one part ascitic fluid with two Add 500.0 cc. of a
(3) 3.0% agar solu-
parts infusion agar (see variant tion, melted and cooled to 60C
,
(v) medium (1661) for prepara- to (2).
tion). Adjust to +0.9 to phenolphthalein.
(4)
(2) Sterilize in the water bath 30 min- Before the agar has cooled suffi-
(5)
utes at 56C. on five successive ciently to harden, it is heated in
days. the autoclave at 15 pounds pressure
(3) Test sterility before use by incu- for 25 minutes.
bation 48 hours. (6) Filter thru moistened cotton and
(c) Schwartz (Stitt) gave the following canton flannel.
method of preparation:
(7) Distribute in 100.0 cc. lots in
(1) Prepare beef or veal infusion agar flasks.
in the ordinary manner. When ready for use, sterile (7) is
(8)
(2) Adjust (1) to pH = 7.6 phenol- melted and cooled to 60C. and
sulphonephthalein being used as added to 50.0 cc. of ascitic fluid.
an indicator in the hot agar. (0.5 g. ofany carbohydrate (levu-
(3) Cool to 50"C. lose or maltose) may be added
(4) Add the whites of three fresh eggs along with 1 cc. of a 0.04% solution
to each liter of agar. of brom cresol purple.)
(5) Start with a low flame, boil for (9) Pour in sterile plates.
10 minutes. (10) Final reaction -1.2 to phenol-
(6) Strain thru cloth and filter thru phthalein or pH = 6.8.
paper. References: Scholtz (1899 p. 5), Schwartz
(7) Tube in 5.0 to 6.0 cc. quantities and Davis (1920 p. 1125), Harvey (1921-
and autoclave at 10 pounds pres- 22 p. 83), Stitt (1923 p. 45), Mulsow
sure on 3 successive days. (1925 p. 421).
(8) Sterilization reduces the pH to 7.4.
Add sterile ascitic,
1928. Kiefer's Ascitic Fluid Agar (Abel)
(9) pleuritic or
hydrocele fluid to each tube. One Constituents
part fluid to two parts agar. 1- Water lOOO.O cc.
(10) Seal the tubes with sterile rubber 2. Meat 500.O g.
stoppers and slant. 3. Agar (3.5%) 35.0 g.
(11) Cork the tubes and store in the 4. Peptone (5.0%) 5O.O g.
incubator. 5. Glycerol (2.0%) 20.0 g.
(12) Discard all contaminated tubes. 6. NaCl (0.5%) 5.0 g.
(13) Inoculate when the medium is at 7. Ascitic fluid 1000.0 cc.
body temperature. Preparation
(14) After inoculation hold the tube (1) Chop 500.0 g. of fat free meat and
horizontal by the butt with the add to a liter of water at 50C.
agar slant up. Pass longitudi- (2) Keep at 50C. for 30 minutes and then
nally thru the Bunsen flame about boil for 30 to 45 minutes.
three or four times and cork (3) Filter or strain the fluid from the
quickly. This reduces the pres- meat.
sure within the tube. (4) Make up the fluid to 1 liter.
(15) The agar tubes should have about (5) Dissolve 3, 4, 5 and 6 in (4).
0.5 cc. of water of condensation in (6) Neutralize.
the lower angle of the slant. (7) Cool to 50C.
615
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(1921-22 p. 83).
1. Whey 200.0 cc.
2. Ascitic fluid (5.0%) agar
1929. Torrey and Buckell's Urine Ascitic 3. Nutrose (0.3%) agar 400.0 cc.
Fluid Agar 4. Urea 2.0 g.
Preparation
Constituents
2000.0 cc. (1) Warm 200.0 cc. of cow's milk to 60C.
1. Distilled water
1250.0 g. (2) Add 5.0% ascitic agar drop by drop.
2. Veal, fat-free
20.0 g. (Composition or method of prepara-
3. Peptone (Difco)
40.0 cc. tion not given) the milk being shaken
4. Urine, fresh
100 to cause precipitation of the casein.
5. NaCl g-
(3) Filter thru filter paper.
6. Glvcerol 40.0 cc.
36.0 g. (4) To filtrate add 10.0% caustic soda to
7. Flaked agar
1000.0 cc. slightly alkaline.
8. Ascitic fluid
9. lodin-green (5) Add urea (2.0 g.).
Preparation: (6) Mix sterile (5) with 0.3% nutrose agar.
chopped lean veal in (Composition or method of prepara-
(1) Place finely
tion not given.) One part fluid (5)
water and bring slowly to a boil.
20 minutes with to 2 parts agar at 45C.
(2) Allow to simmer
(7) Slant or plate as desired.
occasional stirring.
Incubate to insure sterility.
flannel.
(3) Strain thru cotton
(8)
Sterilization: Sterilize by heating at
(5)
(4) Cool and remove
the fat.
Place in double boiler over a satu- 60C. for 30 minutes on each of three
(5)
successive days.
rated brine bath and raise the
temperature to about 60C. Use: Cultivation of gonococci. Author
min- reported that the medium was not as
(6) Add 3, 4, 5, 6 and 7 and boil 45
satisfactory as blood agar or blood broth.
utes to dissolve materials.
Adjust reaction to pH = 6.9, using Ordinary broth or peptone water may be
(7)
10.0% Na2C03. mixed with the fluid or liquid medium.
Boil 30 minutes longer.
Reference: Watabiki (1916 p. 734).
(8)
(9) Remove from brine and add distilled 1931. Esch's Maltose Ascitic Fluid Agar
water to 2 liters.
Filter thru canton flannel and tube
Constituents
(10)
1. Infusion agar
750.0 cc.
in 10.0 cc. amounts.
2. Peptone. Witte (1.0%) 7.5 g.
(11) In preparing plates 5.0 cc. of ascitic
616 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
and 1.0 cc. of a solu- (13) Add 20.0 cc. of a freshly prepared
sterile beef bile
solution of 0.1 g. Crystal violet B
tion of 1.0 g. of malachite green I
(Hochst) in 60.0 cc. distilled water. Hochst in 100.0 cc. sterile warm dis-
tilled water.
It is preferable to mix 20.0 g. of agar
with 0.05, 0.1, 0.15, 0.2, etc., cc. of a (14) Pour into plates.
that inhibits the colon organism but Bad. typhi colonies were small, of bluish
not the typhoid organism. Add a color and glossy.
Filter off the meat water and boil (2) Express the juice and make up to
(2)
one hour. one liter.
(3) Coagulate albumin either by vigor-
(3) Filter.
Add 3, 4, 5 and boil one hour. ous boiling 10 minutes or by heat-
(4)
Filter. ing at 120C. in the autoclave.
(5)
60.0 g. of fine agar, boil 3 hours Filter.
(6) Add (4)
(or autoclave one hour) and make (5) Dissolve 10.0 g. Witte's peptone,
slightly alkaline to litmus. 10.0 g. nutrose and 5.0 g. NaCl in
c.p. lactose.
paper.
Filter.
(9) Boil (8) for 15 minutes. (8)
(10) Mix hot (9) and (7). (9) Add 30.0 g. agar to (8).
(10) Heat in autoclave at 120C. for
(11) Again adjust to slight alkalinity.
Add 4.0 cc. of a hot, sterile 10.0% 30 minutes, or over flame until
(12)
water free soda solution. agar is dissolved.
618 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
by the addition of 10.0% sodium (6) Add soda solution unti the reaction
is slightly alkaline to
litmus.
hydroxide
Boil again for an hour and filter hot
(19) Add 4.0 cc. hot 10.0% sodium (7)
in the sterilizer
hydroxide and 20.0 cc. hot sterile , , ,
blood and that has been allowed to 1943. Lubenau's Caffeine Whey Agar
stand undisturbed.
Boil for a few minutes. Constituents
(8)
Distilled water 1000.0 cc.
(9) Place in a flask with a patented 1.
Beef 500.0 g.
stopper and heat for one hour in 2.
3. NaCl 100 g-
streaming steam.
Peptone 60.0 g.
(10) Allow to cool slowly so that the 4.
Agar 40.0 to 60.0 g.
sediment and turbidity may settle 5.
to the bottom. Use only the top 6. Litmus whey 900.0 cc.
7. Caffeine 6%, solution
medium.
clear
Sterilization: Not specified. Preparation
Boil 500.0 g. of finely chopped lean
Use: Selective medium for typhoid bacilli. (1)
Variants: The author gave the following beef with one liter distilled water for
variants: 30 minutes.
the medium (2) Filter and make up to one liter.
(a) If caffeine is desired in
prepare a solution of caffeine in water (3) Add and 5 to (2) and dissolve by
3, 4,
(preparation not given) to hot sterile Variants Schloffer reported that urine may
:
S
(8) no n
T^a 110.0
Add
"^ '
cc. off a sterile T -^ "l"L
6.0% caf-
^'^''"^ * ^' ^ 80=C. to sterilize.
References: Schloffer (1893 p. 657), Ghon-
feme solution to (7). Mix well. Schlagenhaufer (1893 p. 619)^'
(9) Pour in plates.
Sterilization: Method of sterilization of (5) ^^'^^- dicker's Glycerol Sputum Agar
^"*gi^en. Constituents:
Use Detection of typhoid bacteria.
: 1. infusion agar (2.0%) 200 cc
Authors reported that typhoid colonies 2. Sputum inn n
were colorless.
3. Glycerol :.:::::;:::::::
Reference: Lubenau (1907 p. 248).
" so cc'
Preparation: "
m
(1) MiTlOOO
Mix n
1000.0 cc. off f . milk
fresh -H
u
with
^^^ ^"^ ''^
solidify.
^^' "^^'^ *^^ly ^^d allow to
'
^ '
50 cc'
3. Sputum 50 l
Use: Cultivation of gonococci. 4 NaCl 5
Reference: Harvey (1921-22 p. 95). 5^ Glucose '.[[[[]][][[[[[[][][] o.5 g.'
Glycerol
1945. Schloffer's Urine Infusion Agar ^ 12.0 g.
^ 7. Peptone 10?^
Constituents:
Preparation:
1. Infusion agar (2.0%) 200.0 cc. (1) See medium 1746 for preparation of
^2- Urine 100.0 cc. lung infusion agar.
Preparation:
(2) Boil slowly for one hour.
(1) Prepare a 2.0% infusion agar.
(3) Filter while hot
(2) Obtam urine under aseptic con- (4) Adjust to neutral or |% acid.
Sterilization Sterilize on 3 successive days
/o^ aI^^^^^' .,
(3) Mix
two parts sterile agar with one in streaming steam
:
Agar 25.0 g
arable is used he obtained vigorous
7.
Heart, ox 500.0 g.
growth of pasteurella and salmonella 8.
also.
9. Pea extract
Preparation
References: Costa and Boyer (1922 p. 857),
(1) Dissolve 2, 3, 4, 5 and 6 in 1 by steam-
van Saceghem (1923 p. 968).
ing (500.0 cc. of Douglas' trypsinized
1949. Capaldi's Egg Yolk Agar broth and 100.0 cc. of Cole's trypa-
mine may be used instead of 600.0 cc.
Constituents:
of Bacto peptone solution).
1. Nutrient agar.
(2) Soften 25.0 g. of fiber agar by soaking
2. Egg yolk.
in 0.25% acetic acid for 15 minutes.
Preparation
Wash the agar free from acid and
(1) Break an egg, separate the white
squeeze thru lint to get rid of as
from the yolk and place the yolk in a
sterile Petri dish.
much water as possible.
(3) Heat (1) and (2) on a water bath for
(2) Prepare nutrient agar.
20 to 30 minutes. Then boil over a
(3) Place a glowing glass rod on a spot
free flame for 15 minutes to complete
on the covering of the egg yolk.
the solution of the agar.
(This sterilizes the covering of the
naturally sterile yolk.) (4) Adjust to pH 7.6 and keep at 56C.
624 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(5) Add 500.0 cc. of water to 500.0 g. of (5) Evaporate to the volume by boiling.
fresh ox heart and immerse in a (6) Allow to stand two hours in the
56C. water bath shaking the con- steamer.
tainer frequently. (7) Filter the thick liquid thru a hot
(6) When the extract has reached a water funnel.
temperature of 39 to 42C. place on (8) Distribute into sterile test tubes.
a 37C. water bath and allow to ex- Sterilization: Sterilize in the steamer.
tract for 2 hours. Use: Cultivation of diphtheria, cholera
(7) Strain thru butter muslin. influenza and other pathogenic forms.
(8) Add (7) to (4) in five 100.0 cc. lots Variants: Besson prepared a similar me-
allowing 5 minutes between each dium as follows:
addition. Keep the agar at 56C. (1) Collect egg yolk under aseptic con-
(9) Heat (8) after all the infusion has ditions.
been added on a water bath for (2) Mix one volume of (1) with 3 volumes
20 minutes. of sterile water.
(10) Adjust to pH 7.6. (3) Melt tubes of nutrient agar.
(11) Cool to 56C. and add the whites of (4) Add each tube of
2.0 cc. of (2) to (3)
2 eggs. under aseptic conditions.
(12) Heat on a water bath for 20 minutes. (5) Slant.
(13) Pass thru butter muslin and filter References: Nastiukof! (1893 ^33 and 34),
thru English Chardin paper at 55C. Besson (1920 p. 55).
(14) Adjust to pH 7.6.
1951, Bezanfon and Griffon's Glycerol Egg
(15) Transfer, for storage into bottles of
Yolk Agar
which con-
200.0 cc. capacity, each of
Constituents
tains 10.0 to 15.0 cc. of pea extract.
1. Nutrient agar 100.0 cc.
Sterilization: Sterilize for 15 minutes at
2. Glycerol 6.0 g.
100 C. It is necessary to have the
3. Egg yolk 50.0 cc.
steamer at this temperature before plac-
Preparation
ing the medium in the sterilizer. The
(1) Prepare nutrient agar.
medium must not be over-heated, es-
(2) Add 6 parts of glycerol to 100 parts (1)
pecially after the addition of the heart
(3) Tube.
extract, hence all glassware, butter mus-
(4) Melt sterile (3) and cool to 50C.
lin, paper, etc. are to be Steamed before
(5) To each tube add one-half the volume
use.
of sterile egg yolk under aseptic con-
Use: Culture medium for gonococci.
ditions.
Reference: Tulloch (1922 p. 348).
(6) Mix thoroly and slant to cool.
1950. Nastukoff's Egg Yolk Nutrient Agar Sterilization: Method of sterilization of
(2) Add 0.5 cc. of a 10% NaOH solution tion not given in the abstract.
and 100.0 cc. of egg yolk to 1000.0 cc. Sterilization: Method not given.
distilled water. Use: Diagnosis of diphtheria.
(3) Prepare nutrient agar. Reference: Pergola (1918 p. 101) taken
(4) Mix equal parts of (2) and (3). from (1919 p. 57).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 625
1953. Lipschiitz's Egg Albumin Agar 3. Egg albumin (10.0% solution) 400.0 cc,
6.0 cc. of N/1 NaOH. (4) Mix the clear opalescent fluid with
Mix one part (1) with five parts agar. an equal amount of 3.0% agar.
(2)
Sterilization: Not specified. Sterilization: Not specified.
(2) Extract the sperm by pressing the (2) Slant. The tube should contain a
Funiculus sperm aliens between two small quantity of water of condensa-
fingers and remove the sperm, thus tion or add a little bouillon.
pressed out with a sterile platinum (3) Add to each slant \ g. of tissue or an
loop. equivalent amount of stomach or
(3) Streak the sperm on sterile agar other fluid or intestinal contents of
slants. the fetus.
(4) Incubate to prove sterility. (4) Seal the tubes with sealing wax.
Sterilization: Not specified. Sterilization: Not specified.
Use: Cultivation of influenza bacilli, tuber- Use: Isolation of Vibrio felus.
cle bacilli and others. Reference: Smith and Taylor (1919 p. 301).
Variants: A similar medium was prepared 1963. Noguchi's Ascitic Fluid Tissue Agar
as follows:
Constituents
(1) Prepare nutrient agar.
1. Nutrient agar (2.0%) 200.0 cc.
(2) Free testicular material of its capsule.
2. Ascitic fluid 100.0 cc.
(3) Wash the outer surface with alcohol
3. Tissue (rabbit testicle or
and ether.
kidney)
(4) When dry cut cross sections with a
Preparation
sterile knife.
(1) Prepare 2.0% nutrient agar.
(5) Remove some of the liquid from the
(2) Adjust (1) to slightly alkaline.
cross sections by means of a strong
(3) Place small pieces of fresh tissue
platinum loop.
(preferably rabbit testicle or kidney,
(6) Streak the liquid on sterile solidified
but human placenta, sheep testicle or
agar slants.
other sterile organs will suffice) into
(7) Incubate 10 hours to prove sterility.
a tube measuring 2 by 20 cm.
References: Cantani jun (1897 p. 601).
(4) Mix 2 parts melted (2) with one part
1961. Pettersson's Brain Ascitic Fluid Agar ascitic (or hydrocele) fluid and add
Constituents 15.0 cc. to each tube of (3).
1. Nutrient agar. (5) After solidification add a layer of
2. Ascitic fluid. sterile paraffin to prevent evap-
3. Brain. oration.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 627
(1) Prepare serum agar. (d) Loewe and Strauss cultivated organ-
(2) Melt (1) and pour to the height of isms causing epidemic encephalitis
about one inch into the bottom of on a medium prepared as follows:
a 200.0 cc. flask. (1) Mix one part sterile 2.0% nutrient
(3) Drop in sterile bits of tissue. agar with four or five parts ascitic
(4) Inoculate. fluid. Ascitic fluid should be ster-
(5) When the agar has solidified, cover ile, contain no bile or fibrin, and
(3) Dissolve 2.0% peptone and 0.5% (4) Cool to 55C. and add the white of
agar in (2). an egg. Mix well.
(4) Add 5.0 cc. of a 10.0%, NaoCOs solu- (5) Boil 20 minutes at 115C.
tion to give a slightly alkaline (6) Distribute as desired.
reaction. Sterilization: Sterilize for 20 minutes at
(5) Distribute in tubes. 113 to 114C.
(6) Cool sterile (5) to 45C. and to each Use: General culture medium.
tube add a small piece of sterile dog Variants: Besson (Tanner) prepared the
kidney under aseptic conditions. medium as indicated:
Sterilization: Sterilize (5) in the autoclave. (1) Dissolve 80.0 g. of gelatin in bouillon.
Constituents :
in 10.0 cc. nutrient agar.
1. Water 1000.0 cc. (5) Take 1.0 cc. of one of the extracts
2. Nutrient agar (2.0%) obtained and add to 5.0 cc. of sterile
3. Beef 500.0 g. nutrient agar.
Preparation Kligler reported that heat destroyed
(1) Chop 500.0 g. of fat free beef in a the growth stimulating material in
meat chopping machine. the extract. Best growth in (4) (b),
(2) Mix (1) and 1000.0 cc. of water, and no stimulated effects in (4) (e)
allow to stand for 18 to 24 hours in and (f).
1970. Esch's Hydrolyzed Meat Agar (2) To 150.0 cc. of defibrinated beef blood
(Kohlisch and Otto) add an equal part of N/1 KOH.
(3) To 7 parts melted (1) add 3 parts
Constituents (2), mix well and pour into plates.
1. Nutrient agar (4) Dry the plates for several days at
2. Beef 500.0 g.
37C. or for 4 minutes at 60C.
3. NaOH (normal) 250.0 cc.
Sterilization: Sterilization of agar not
Preparation specified. The laked blood alkali mixture
(1) Dissolve 500.0 g. of beef in 250.0 cc. maj^ be sterilized in the autoclave.
of N/1 NaOH solution in an aluminum Use: Enrichment medium for cholera
kettle by heating. Stir constantly. Author reported that cholera
vibrio.
Solution is complete after from about vibriogrew very luxuriantly on this
10 to 15 minutes. medium. B. coli was inhibited.
(2) When cool filter thru a towel. Variants
(3) Mix 3 parts sterile (2) with 7 parts (a) Sineff and Drosdowitsch gave the
neutral nutrient agar. following method of preparation:
(4) Pour into plates.
(1) Collect beef blood in a sterile
(5) at 60 for 30 minutes.
Dry enamel container and defibrinate it
(6) Allow the plates to stand
open at with a sterile egg beater.
room temperature for 24 hours. (2) Strain thru a fine tin sieve.
Sterilization: Sterilize in streaming steam Mix equal parts of the filtrate from
(3)
for one hour. Method of sterilization of (2) and a normal solution (5.61%)
agar not given. of KOH.
Use: Elective medium for cholera vibrio. (4) Sterilize for one-half hour.
Variants (5) Mix 7 parts sterile agar and 3
(a) The author used pike instead of beef. parts (4) (sterile alkaline defibri-
(b) Stitt prepared the medium as follows: nated blood).
(1) Treat 500.0 g. chopped beef with (6) Pour into sterile Petri dishes.
250.0 cc. of normal NaOH until dis-
(7) Dry at 37C. for 24 hours.
integrated. (b) Hofer and Hovorka prepared a sim-
(2) Filter thru cloth. ilar medium as follows:
(3) Sterilize (method not given). (1) To 3.0% melted nutrient neutral
(4) Add about one part of (3) to about agar add 4.0 cc. of defibrinated
2.5 or 2 parts of nutrient agar. beef blood, and 16.0 cc. of N/1
(5) Dry the plates. KOH.
References: Kohlisch and Otto (1915 p.
(2) Boil.
435), Stitt (1923 p. 50). (3) Distribute into 10.0 cc. lots.
(4) Prepare a 0.1% solution of crystal
1971. Wellman's Placenta Infusion Agar
violet in distilled water.
Add Wellman's Placenta Infusion (see
(5) To each 10.0 cc. of (3) add 0.5 cc.
medium 1377) to 2.0% nutrient agar. of (4).
(6) Pour into Petri dishes.
1972. Orcutt and Howe's Fat Blood Agar
(7) Place the plates in the incubator
Same as medium 960 but solidified by the for 24 hours with covers partly re-
addition of agar. moved and then at room tempera-
tures for 12 hours with covers on
1973. Dieudonne's Alkaline Blood Agar
before use.
Constituents (c) Lentz prepared the medium as
2. Blood, defibrinated beef 150.0 cc. (1) Mix fresh defibrinated beef blood
30.0 cc. distilled water. Add to (a) Klimmer prepared the medium as
70.0 cc. neutral agar. follows:
(d) Roddy gave the following method of (1) Mix 150.0 cc. of fresh defibrinated
preparation: beef blood, with 150.0 cc. of a 12.0%
(1) Obtain ox blood from the slaughter soda solution.
house. (2) Shake well.
(2) Collect the blood in small sterile (3) Allow to stand for 1 to 6 days.
bottles containing glass beads. (4) Steam for 60 to 90 minutes.
(3) Shake until the blood is defi- (5) Mix 30 parts (4) with 70 parts
brinated. sterile neutral agar.
(4) Mi.x equal volumes of (3) and (b) Park, Williams and Krumwiede gave
normal sodium hydrate solution. the following method of preparation
(5) Sterilize (3) in a steam sterilizer (1) Mix equal parts defibrinated beef
for 15 minutes on each of 2 succes- blood and 12.0% Crystalline soda
sive days. solution.
(6) Add 1 part of (5) to 7 parts sterile (2) Steam in the Arnold for 30 minutes.
nutrient agar. (3) Prepare 3.0% agar neutral to litmus
(7) Tube. (about pH 6.6).
(8) Slant. (4) Mix 3 parts (2) with 7 parts (3).
(9) Do not have the cotton plugs Sterilization not specified.
tighter than necessary. (5) Pour into Petri dishes (15.0 cc. in
(e) Stitt used normal NaOH instead a 10 cm. dish).
of KOH. (6) Allow to harden, uncovered but
References: Dieudonne (1909 p. 108), Sineff protected by paper.
and Drosdowitsch (1909 p. 429), Hofer (7) Plates can be used after drying
and Hovorka (1913 p. Ill), Lentz (1915 30 minutes.
p. 425), Roddy (1917 p. 45), Ball (1919 References: Pilon (1911 p. 331), Klimmer
p. 83), Klimmer (1923 p. 218), Stitt (1923 (1923 p. 218), Park, Williams and Krum-
p. 50), Park, Williams and Krumwiede wiede (1924 p. 130).
(1924 p. 130).
1975. Fildes' Pepsinized Blood Agar
1974. Pilon's Alkaline Blood Agar Same as 961 but using nutrient agar in-
stead of bouillon.
Constituents:
1. Nutrient agar (4.0%) 70.0 cc. 1976. Carpano's Hemolysed Blood Agar
2. Blood, defibrinated 15.0 cc. Constituents
3. NaaCOs (crystalline 12.0% solution) 1. Nutrient agar (2.5%) 1000.0 cc.
Preparation 2. Hemolysed defibrinated blood
(1) Mix defibrinated blood and a 12.0% Preparation
crystalline Na2C03 solution in equal (1) Prepare 2.5% nutrient agar (peptone
parts. broth with 2.5% agar).
(2) Prepare 4.0% nutrient agar. (2) Neutralize to phenolphthalein.
(3) Neutralize (2). (3) Adjust the reaction by the addition
(4) Add 3 parts non-sterilized to (1), of 4.0% normal HCl.
7 parts melted (3)and mix thoroly. (4) Add sterile, defibrinated blood natu-
(5) Pour into Petri dishes and allow the rally hemolysed (details of prepara-
plates to solidify. tion not given in the abstract) to the
Sterilization: If the plates are to be used solidified agar.
at once sterilization is not necessary due Sterilization: Not given.
to high alkali content. If the plates are Use: Cultivation of gonococci.
to be kept several days, draw blood under Reference: Carpano (1919 p. 599) from
aseptic conditions and use sterilized agar. (1920 p. 176).
632 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
1977. Mandelbaum's Lactose Blood Agar (3) To 95.0 cc. of (2), add 5.0 cc. of a2.2%
solution of neutral sodium oleate.
Constituents Add defibrinated or citrated blood to
(4)
1. Nutrient agar. while it is still hot (temperature
(3)
2. Lactose. amount of blood not
or specified).
3. Rosolic acid. Sterilization: Not specified.
4. Blood, defibrinated human. Use: Cultivation of Pfeiffer's bacilli.
Preparation Reference: Stitt (1923 p. 43).
(1) To a tube containing 8-10.0 cc. nu-
trient agar is added 0.3 g. lactose. 1980. Esch's Ascitic Fluid Blood Agar
(2) Heat to 100 C. Constituents
(3) Cool to 50C. 1. Nutrient agar 60.0 cc.
(4) Add 0.3 cc. 1.0% alcoholic rosolic 2. Blood, defibrinated sheep 20.0 cc.
acid solution. 3. Ascitic fluid 10.0 cc.
human
(5) Add 1.0 cc. defibrinated sterile 4. Maltose 1.0 g.
blood (Probably animal blood may 5. Bouillon 3.0 cc.
be used). Preparation
(6) Mix and pour into Petri dishes. (1) Prepare nutrient agar containing
(7) Stand 24 hours, or dry | hour at 37C. 1.0% Witte's peptone.
Sterilization: Not specified.
(2) Prepare bouillon.
Use: Isolation of colon typhoid group. (3) Mix 60.0 cc. of (1) that has been
Reference: Mandelbaum (1912 p. 306). cooled to 50C. with 20.0 cc. sterile
defibrinated sheep blood, 10.0 cc.
1978. Thompson's Glucose Plasma Agar
ascitic fluid and 1.0 g. of maltose dis-
Constituents solved in 3.0 cc. of bouillon.
1. Nutrient agar (2.5%) 1000.0 cc. (4) Pour into sterile Petri dishes.
2. NaCl 9.0 g. Sterilization: Not specified.
3. CaCh 0.25 g. Use: Cultivation and isolation of meningo-
4. KCl 0.42 g. cocci. Author reported that typical
5. Glucose (2.5%) 25.0 g. colonies developed after 8 hours.
6. Plasma (human) References: Esch (1909 p. 153), Klimmer
Preparation (1923 p. 225).
(1) Prepare nutrient agar (2.5%) in the
1981. Listen's Trypsinized Casein Blood
ordinary way using 1.0% Witte pep-
Agar
tone (Omit the NaCl).
(2) Adjust to +6 acid. Constituents
(3) Add 2, 3, 4 and 5 to (1). 1. Distilled water 100.0 cc.
(4) Tube in 4.0 cc lots. 2. Blood 20.0 cc.
(5) Melt sterile (4) in boiling water and 3. Trypsinized casein 100.0 cc.
cool to 50C. Then add 1.0 cc. of 4. Agar (3.0%) 400.0 cc.
human plasma to each tube. Mix and Preparation
slant. (1) A 200.0 cc. capacity flask, containing
Sterilization: Method of sterilization of (4) a few glass beads and 100.0 cc. dis-
not given. tilled water, is sterilized and kept
Use: Cultivation of gonococci. ready.
Reference: Thompson (1917 p. 869). (2) 20.0 cc. of human (or rabbit) blood,
removed with due precautions to
1979. Avery's Oleate Blood Agar (Stitt)
avoid contamination, are added to
2. Sodium oleate (2.0% soln.). . . 5.0 cc. shaken to prevent the formation of
3. Blood, defibrinated or citrated large fibrin masses and the blood
Preparation soon lakes.
1 Prepare nutrient agar. (3) 5.0 cc. of an alcoholic pancreatic ex-
(2) Adjust (1) to pH = 7.2 to 7.5. tract are then added to the flask.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 633
this extract being prepared locally the tube is sloped, and the agar
from goat's pancreas, according to allowed to cool.
the method recommended by Cole (12) Agar slopes thus prepared are trans-
and Onslow (Lancet (1916, see parent and have a slightly more
medium 1130.) Pig's pancreas was golden color than ordinary agar.
not used for various reasons. Sterilization: Final sterilization not spe-
(4) Next 5.0 cc. of the enterokinase solu- cified.
tion are added with a view to hasten Use: Cultivation of B. m/uensae (Pfeiffer).
the trypsinization process. This The author reported that agar slopes con-
enterokinase solution is a very dilute taining the digested blood gave almost
watery extract of the duodenal as good results as those containing in
mucous membrane of goats and sheep addition casein digest (stock broth of
which has been macerated in chloro- Cole Onslow). Many involution
and
form water. forms, however, were developed on this
(5) Lastly, 1.5 cc. of pure chloroform simpler medium, but the addition of
are added to the flask as a pre- sodium phosphate eliminated these. A
servative and it is plugged with an culture of B. influenzae inoculated on
India rubber cork to prevent the this medium showed a perceptible growth
evaporation of the chloroform. after six hours incubation at 37C., and
(6) The flask is then placed in the incu- after 24 hours incubation a luxuriant
bator at 37C., after having shaken growth was obtained. This growth had a
it well in order to mix the contents characteristic translucency. Individual
thoroly. isolated colonies on this medium meas-
(7) On the second day the flask will be ured from 1.0 mm. to 2.0 mm. in 24 hours.
found to contain a well-settled sedi- Reference: Liston (1918-19 p. 419).
ment with a clear supernatant red-
1982. Bernstein's Basal Blood Agar
dish fluid. The flask is shaken again
to resuspend the sediment and put Constituents
back into the incubator. The shak- 1. Nutrient agar 150.0 cc.
ingis repeated on the third day, but 2. Blood, beef 10.0 cc.
after that the flask is allowed to Preparation
stand undisturbed in the incubator (1) Prepare nutrient agar with 1.0% of
until the eighth day. one of the added nutrients.
(8) On the eighth day the flask is care- (2) Draw 400.0 cc. of beef blood directly
fully removed so as not to disturb into sterile Erlenmeyer flasks of
the sediment and the clear super- 500.0 cc. capacity containing 35.0 cc.
natant fluid is removed with aseptic of a 1.0% solution of ammonium
precautions. In case the sediment oxalate in distilled water.
has not settled firmly at the bottom, (3) Shake for one or two minutes.
as much of the clear fluid should be (4) Add volume formalin and
0.5 cc. of 40
removed as possible and the re- allow the flask to stand undisturbed
mainder filtered thru sterile filter for an hour.
paper, making arrangements to (5) Distribute the blood into sterile
maintain sterility. Erlenmeyer flasks and dilute with
(9) Broth prepared from casein after twice its volume of sterile (0.9%)
the method recommended by Cole saline solution.
and Onslow (see medium 1130) is (6) Allow the blood to stand for 24 hours
kept ready on hand. to 48 hours at room temperatures
(10) Tubes, each containing about 4.0 cc. before use.
of 3.0% agar, are kept ready steri- (7) Seal the flasks and keep on ice until
lized. Theagar is melted and cooled ready for use.
to a temperature of about 45C. (8) To 15.0 cc. of sterile melted (1), add
(11) One cc. of the stock broth and 1.0 cc. 1.0 cc. of (6).
of the blood fluid are well mixed; (9) Pour into sterile plates.
634 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
nose, typhoid colonies showed umbili- are pipetted to 100.0 cc. of melted
cated colonies with lines radiating from and cooled to 50C. nutrient agar.
the center. Colon colonies did not show (9) Keep the blood agar at 50C. for one
these lines. On maltose typhi were hour before pouring into plates, if to
deeply pigmented, almost black; colon be used at once. Or pour into plates
colonies were white. On dextrin ty- and incubate at 37C. over night.
phoid colonies caused precipitation of the This drives off the ether, and if the
medium and were black. Colon colonies plates are incubated over night, tests
caused hemolysis and were white. sterility.
(5) Transfer the blood with aseptic pre- (6) Stopper firmly and shake well.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 635
(3) Allow about 25.0 cc. of blood to (1) Prepare nutrient agar.
pass directly from the jugular vein (2) Adjust the agar to pH = 7.5.
of a goat into the melted agar or (3) Boil fresh rabbit blood for two
gelatin which has been cooled to minutes in a water bath.
about 45C. (Dog and chicken (4) Centrifuge.
blood gave good results also.) (5) Add 0.5 cc. of the resulting clear
Pour the blood medium into sterile pale pink or yellow fluid to 20.0 cc.
(4)
Petri dishes. of the broth or two or three drops
(e) Savini and Savini-Castano cultivated to 5.0 cc. of the melted agar.
influenza bacilli on a hematin agar (h) Levinthal (Klimmer) cultivated in-
prepared as follows: fluenza bacilli on a medium prepared
(1) Allow about 100.0 cc. of blood to as follows. Klimmer reported that
coagulate. best results were obtained using a
and place into a flask containing 3.0% agar and cool to 70C.
100 to 150.0 cc. of N/1 NaOH or (2) Mix 50.0 cc. of fresh human or dog
and sterilize (method not (3) Heat (2) to boiling over a wire gauze
(5) Filter
given). until the agar begins to raise in the
(6) Dry the plates for 24 hours at 37C. 3. Blood, horse 50.0 cc.
and then for 24 more hours at room 4. Glucose 2.0% 3.0 g.
temperature. Preparation
Sterilization : Method not given. (1) Mix one part horse blood with two
0.75 normal KOH solution. Hog and (4) Melt the glucose agar and cool to
horse blood gave as good results as did 60C.
beef blood. (5) Mix equal parts (1) and (4).
dark brown or brownish violet, dry and (3) Mix the tubes without shaking.
hard to remove from the agar. Pneumo- (4) Cool in a slanting position.
cocci colonies were larger than strepto- (c) Bezangon and Griffon (Besson) gave
cocci, about 5 mm. in diameter, colonies the following method of preparation:
were dark brown or brownish, forming a (1) Add 5.0% glycerol to nutrient agar.
smooth, coherent mucilaginous mem- (2) Liquify sterile (1) and cool to 45C.
brane. A light yellow decolorization of (3) Add about 1.0 cc. of fresh rabbit
the place when pneumococci
medium took blood. (A 1.0% solution of com-
growth was luxuriant. If agar and blood mercial hemoglobin may replace
were mixed at 50C. a clear ruby red the blood.)
transparent medium was obtained. (4) Mix well without shaking.
Heating above this temperature gave a (5) Slant and cool.
darker medium. When mixed at 60C. (d) Cantani (Besson) prepared a medium
the medium was not transparent in thick as follows:
[layers. (1) Mix equal parts of sterile blood and
References: Bieling (1921 p. 262), Klimmer sterile glycerol.
(1) Draw blood under aseptic condi- 3. Glycerol 2.0 or 3.0 cc.
tions directly from a vein into a 4. Nutrose 0.8 g.
sterile Erlenmeyer flask containing 5. Agar (2.0% peptone)
glass beads, using a sterile needle Preparation
and sterile tube. (1) Mix 15.0 cc. of hog blood, 30.0 to
(2) Shake the flask to defibrinate the 40.0 cc. of water, 2.0 to 3.0 cc. of
blood. glycerol and 0.8 g. nutrose.
(3) Prepare 3.0% agar containing 2.0 (2) Shake the mixture constantly and
to 3.0% glycerol. boil for 15 minutes.
(4) Mix one part (2) with 4 or 5 parts (3) Repeat the boiling and shake on the
sterile (3), cooled to 50C. following day for 15 minutes.
(5) Distribute into sterile test tubes. (4) When desired for use heat to 50 to
(b) Besson (Tanner) gave the following 60C. and mix with an equal quantity
method of preparation: of sterile 2.0% peptone agar.
(1) Melt tubes of glycerol agar and cool (5) Pour into plates.
(2) Add 1.0 CO. of blood from a rabbit's Use: Cultivation of gonococci.
artery. Reference: Abel (1912 p. 162).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 639
(2) Mix 1.0 cc. sterile (1) with 7.0 cc. Constituents
nutrient agar. 1. Distilled water.
(3) Mix sterile (2) with 85.0 cc. neutral 2. Soda (18.0% solution) 10.0 cc.
agar. 3. NaCl (0.85% solution) 10.0 cc.
Sterilization: Sterilize (2) in streaming 4. Hemoglobin extract (Pfeuffer) 3.0 g.
steam. Preparation
Use: Isolation of cholera vibrio. (1) Place 80.0 cc. of melted neutral nu-
Variants trient 3.0% agar in an Erlenmeyer
(a) Costa gave the following method of flask.
preparation (2) Add 10.0 cc. of 18.0% soda solution
(1) Pulverize crystalline blood in a and boil this mixture for about 10
mortar. minutes.
(2) Add distilled water slowly to (1). (3) Cool to 50C.
This forms sort of a sticky mixture. (4) Dissolve 3.0 g. hemoglobin extract
(3) Add 100.0 cc. of a normal KOH (Pfeuffer) in 10.0 cc. of 0.85% NaCl
solution to (2). solution.
(4) Heat in the autoclave for 30 (5) Mix (4) and (3) thoroly.
minutes. (6) Pour into 7 Petri dishes and allow to
(5) Filter. stand with covers removed until
(6) Sterilize by heating in the auto- solidification has taken place.
clave at 100C. for several hours. (7) Place the dishes in an incubator with
(7) Mix volumes of (6) with 7 volumes
3 cover removed for 20 to 30 minutes to
of sterile melted agar under aseptic remove the water of condensation.
conditions. Sterilization: Not specified.
(8) Pour into sterile Petri dishes. Use: Selective medium for cholera vibrio.
(b) Kohlisch and Otto prepared the Author reported that medium was trans-
medium as follows: parent and dark brown. Some cholera-
(1) Rub 15.0 g. of Merck's horse haemo- like vibrio and other organisms were
globin in a mortar. inhibited on this medium.
(2) Mix 15.0 cc. of N/1 NaOH with Variants: Klimmer prepared the medium
(1), and add 15.0 cc. distilled in the following manner:
water. (1) Dissolve 3.5 g. of Pfeuffer's hemo-
(3) Sterilize for one hour in the globin extract in 10.0 cc. of physio-
steamer. logical salt solution.
(4) To 15.0 cc. of (3) add 850.0 cc. of (2) Add 10.0 cc. of a 5.5% solution of
neutral agar. water free soda and 2.0 cc. of KOH
(5) Mix thoroly. solution (strength not given) to (1).
(6) Pour in plates. (3) Steam for 15 minutes.
(7) Dry the plates at room tempera- (4) Cool to 50C.
tures for one hour. (5) Add (4) to 80.0 cc. of 3.0% melted
(c) Klimmer used the following method nutrient agar, neutral to litmus and
in preparing the medium: cooled to 80 or 90C.
(1) Dissolve 50.0 g. of Merck's hemo- (6) Mix thoroly.
globin in 300.0 cc. of half normal (7) Pour in plates.
KOH by heating in a steamer for (8) Dry thoroly in the incubator.
an hour. References: Kabeshima (1913 p. 203),
(2) To hot (1) add 1700.0 cc. of melted Klimmer (1923 p. 220).
hot neutral agar.
Pour into
(3) plates. 2000. Besson's Glycerol Hemoglobin Agar
References: Esch (1910 p. 559), Costa (1912 (Tanner)
p. 846), Kohlisch and Otto (1915 p. 438), Constituents:
Klimmer (1923 p. 218). 1. Glycerol agar.
2. Hemoglobin.
1999. Kabeshima's Alkaline Hemoglobin
Preparation
Agar
(1) Melt tubes of glycerol agar and cool
Constituents to 40C.
1. Nutrient agar (3.0%) 80.0 cc. (2) Add 1.0 cc. of a solution of hemoglobin
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 641
(1) Add 0.1 cc. of an 18.0% NajCOa solu- that has been obtained under aseptic
tion to 1000.0 cc. of neutrient neutral conditions and heated to 60 C.
agar. (4) Pour into sterile plates or sterile
globin extract in 1000.0 cc. of 0.9% Similar media were used for the cultiva-
salt solution. tion of various organisms by a number of
(4) Distribute in petri dishes and remove investigators.
the bubbles by flaming with a Bunsen Variants
Burner. (a) Miihlens and Hartmann cultivated
(5) When cool remove the covers and Spirochaeta dentium and Bacillus
place in the incubator for 30 minutes. fusiformis on a medium prepared as
(6) Use when dry. follows:
Sterilization: Not specified. (1) Prepare nutrient agar with a
Use: Cultivation of cholera vibrio. slightly alkaline or neutral re-
Reference: Kabeshima (1916 #225) taken action.
from (1917 p. 395). (2) Boil half filled tubes of (1) for
about 30 minutes in a water bath to
2002. Finger, Ghon and Schlagenhaufer's free the agar of oxygen.
Dialyzed Serum Agar (3) Heat horse serum for about 30
Constituents
minutes in a water bath at a tem-
perature from 58 to 60C.
1. Nutrient agar.
(4) Cool (2) and (3) quickly to 45C.
2. Blood Serum (Dialyzed).
Preparation (5) Mix two parts of the agar with one
human blood serum in part of the serum.
(1) Place sterile
(6) Inoculate while the medium is still
parchment sack.
sterile
in the sack in a liquid state.
(2) Suspend the blood serum
(7) Solidify by placing the tubes in
in a sterile cylinder containing sterile
cold water.
water.
48 hours and (b) Carrel mixed serum with one-fifth
(3) Change the water every
its volume of 2.0% agar and used the
continue the dialysis until the water
medium for the cultivation of tissue.
is colored by the albumin that
(c) Besson prepared the medium as
dialyzes thru the parchment.
follows:
(4) Mix the dialyzed blood serum with
Melt tubes of sterile nutrient agar.
agar in the same manner as in the (1)
Cool to 45 to 50C.
preparation of blood serum agar. (2)
642 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(3) Add one-half or one-third the vol- 2005. Muhlen's and Hoffman's Glucose
ume serum to each tube.
of sterile Serum Agar (Stitt)
(4) Mix
thoroly by rolling the tube be-
Constituents
tween the palms of the hands.
1. Nutrient agar (3.0%) 100.0 cc.
(5) Slant or pour into a Petri dish.
2. Glucose (0.5%) 0.5 g.
(d) Dopter and Sacquepee gave the fol-
3. Serum (Horse) 100.0 cc.
lowing method of preparation:
Preparation
(1) Add 0.5 to 1.0 cc. of serum to an
(1) Fill sterile test tubes one-third full
agar slant.
with horse serum.
(2) Incline so that the entire surface is
(2) Add an equal amount of a 3.0% agar
covered with the serum.
containing 0.5% glucose which has
(3) Allow to stand for 12 to 24 hours.
been melted and cooled to 50C. to
(e) Klimmer mixed one part gelatin agar
sterile (1).
with one-half part serum at a tem-
(3) Keep at 55 for two hours.
perature of 45C.
Sterilization: Sterilize (1) on 3 successive
References: Muller (1906 p. 520), Muhlens
days at 55C.
and Hartmann (1906 p. 86), Carrel (1912
Use: Cultivation of treponemata. The
Besson (1920 p. 53), Dopter and
p. 393),
medium was inoculated while still liquid
Sacquepee (1921 p. 138), Klimmer (1923
and incubated under anaerobic condi-
p. 200), Kligler and Robertson (1922
tions.
p. 315).
Reference: Stitt (1923 p. 54).
solution, 2.5 cc. of a freshly prepared (2) Add 2 or 3.0 cc. of glycerol to (1).
sodium sulfite solution and 3.0 g. of (3) Add 0.8 or 0.9 g. nutrose to (2).
potato starch (Merck) stirred up in (4) Heat over a free flame to boiling,
and steam in a
5.0 cc. water, to (1) shaking constantly. The turbid fluid
(3) To a 50.0 cc. of beef serum diluted (5) Liquefy 2.0% peptone agar and cool
with 5 parts water, add 0.5 cc. of a to about 50C.
10.0% NaOH solution and boil for (6) Mix equal parts (5) and sterile (4).
30 minutes in the steamer. (7) Pour into sterile Petri dishes.
(4) Mix two parts (2) with one part (1). Sterilization: Heat (4) until sterile. With
(5) Distribute into sterile test tubes. fresh serum about 20 minutes is required.
(6) Pour sterile (5) into sterile Petri With old serum a longer time is necessary
dishes, seeing that the starch is well and it is often best to heat on several suc-
distributed. Dry the plates before cessive days. Sterilization of agar not
use. given.
on three successive
Sterilization: Sterilize Use: Cultivation of gonococci.
days for 30 minutes each day in the Variants: Klimmer used alkaline nutrient
steamer. agar.
Use: Elective medium for cholera vibrio. References: Wasserman (1898 p. 300),
cholera-like vibrio all gave one of the (2) Obtain nasal secretions by blowing
two indicated types of red colonies. the nose into sterile gauze.
Wheat starch gave the same results but (3) Place into saline solution or alcohol
2010. Meyer's Tuberculin Agar (4) Rub up the deposit with 10.0% KOH
solution added drop by drop to pre-
Constituents :
vent e.xcess of alkali.
1. 5.0% glycerol bouillon with Add HCl to (4) until a precipitate
(5)
heavy growth of tubercle begins to appear. Add this fraction
bacilli 200.0 cc.
to the clear supernatant fluid
2. Glycerol bouillon (4.0 or
from (3).
5.0%) 200.0 cc.
(6) Add sufficient glycerinated com-
3. Agar (2.0%) 8.0 g.
mercial trypsin to serum (horse
4. Serum (10.0%) 40.0 cc.
generally) to partially neutralize
Preparation : the antitryptic power. (Requires
(1) Tuberculin flasks containing about from 2.0 to 8.0 cc. of trypsin per
250.0 cc. of a5.0% glycerol peptone 100.0 cc. depending on the strength
bouillon, with a heavy growth of
of the trypsin, 2 to 4.0 cc. of Fair-
human bovine tubercle bacilli,
or child & Co. and Digestive Ferments
about 6 to 8 weeks old are steamed in Co. trypsin being sufficient and 5.0
a Board of Health sterilizer for two to 8.0 cc. of Allen and Hanbury's
hours. trypsin).
(2) Cool and filter thru fine filter paper
(7) Filter (6) thru a porcelain candle of
and centrifuge to eliminate every medium porosity and distribute in
tubercle bacillus. sterile flasks.
(3) Mix the clear liquid with an equal Melt sterile (1) and add 4.0 cc. of (5)
(8)
volume of the same 4.0 or 5.0%
for each 100.0 cc. of agar.
glycerol peptone bouillon and 2.0%
(9) Cool to between 65 and 60C. and
agar isadded and dissolved. add 15.0 cc. of trypsinized serum per
(4) Filter and correct the reaction to 1.5.
100.0 cc. agar.
Distribute in 9.0 cc. quantities in
(5) (10) Mix thoroughly.
test tubes. Tube in sterile tubes.
(11)
(6) Before being slanted 1.0 cc. (or 10.0%) (12) Slant.
of fresh sterile horse or cattle serum Incubate to test sterility.
(13)
is added to each tube of sterile agar.
Sterilization: Method of sterilization of (1)
Sterilization: Sterilize (5) in the autoclave. and (5) not given.
Use: Cultivation of Bacillus para tuber- Use: Cultivation of B. diphtheriae.
culosis.
Reference: Douglas (1922 p. 263).
Reference: Meyer (1913 p. 175).
4. Trypsin Preparation :
(3) Allow the insoluble portion of (2) Sterilization: Sterilize (3) in the autoclave.
to settle and pour off the clear Method of sterilization of glucose agar
supernatant layer. not given.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 645
the autoclave.
Variants
diphtheriae. The (a) Symmers and Wilson cultivated
Use: Isolation of B.
meningococci on a similar medium
authors used this medium in preparing
prepared as follows:
"little plate" cultures on slides.
(1) Prepare nutrient
agar containing
Reference: Frost, Charlton and Little
3.0% Chapoteaut's peptone.
(1921 p. 30).
646 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Add 1.0% sugar and some litmus levulose, galactose, mannitol, dul-
solution to (1). citol, sucrose, lactose, inulin with
(3) Steam for 10 minutes on 3 suc- Kubel-Tiemann's litmus and steri-
cessive days to sterilize. lize. (Method not given.)
(4) Add one part of sterile ascitic fluid (2) Add (1) to ascitic agar so that the
to two parts (3). agar content is 1.0%.
(5) Tube and slant. (3) Final sterilization not given.
(6) Incubate 2 days to test sterility. Eastwood reported that meningococci
(b) Gordon (Abel) used a medium pre- gave acid (red colonies) with glucose
pared as follows to study fermenta- and maltose only.
tion of meningococci and gonococci. References: Salomon (1908 p. 2), Symmers
(1) Prepare 10.0% solution of any de- and Wilson (1909 p. 10), Abel (1912
sired carbohydrate, alcohol, etc., in p. 159), V. Przewoski (1912 p. 15), Hancken
Kahlbaum's litmus solution and (1916 p. 368), Eastwood (1916 p. 408).
boil two minutes.
2016, Rosenow's Glucose Ascitic Fluid Agar
(2) When cool add 0.5 cc. of normal soda
solution to each 10.0 cc. of (1). Constituents
(3) Mix 3 parts 3.0% nutrient agar with 1. Nutrient agar (2.0%). 1000.0 cc.
one part ascitic fluid. 2. Glucose 2.0 to 10.0 g.
(4) Add 1.5 cc. of (2) to each 13.5 cc. 3. Ascitic fluid 300.0 cc.
of (3)_. Preparation
(5) Pour into plates. (1) Prepare 2.0% nutrient agar with a
(c) V. Przewoski used the following me- reaction of 0.4 to 0.6% acid to phenol-
dium to determine fermentation phthalein.
ability of diphtheria and pseudo (2) Dissolve 2 in (1).
diphtheria: (3) Tube in 7.0 to 8.0 cc. lots.
(1) Prepare nutrient agar. (4) Boil sterile (3) to drive off o.xygen
(2) To add 5.0
100.0 cc. of liquid (1) cc. and cool to 50C.
litmus solution (Kahlbaum), 2.0 cc. (5) Add 2 to 3.0 cc. heated ascitic fluid
ascitic fluid and 1.0 g. of one of (60C. for 24 hours) to each tube.
the following: (6) Cool to 40C.
fructose mannitol Sterilization: Method of sterilization of
glucose lactose (3) not given. See step (5) above for
mannose inulin sterilization of ascitic fluid.
dulcitol Use: To isolate organism causing rheuma-
(d) Hancken studied the fermentation tism, author added some of rheumatic
ability of meningococci on a medium material to medium and mixed well;
prepared as follows: plunged tube into cold water to "set"
(1) Prepare nutrient agar. and incubated at 37C. Author reported
(2) Dissolve 2.5 g. of any desired carbo- that largest number of colonies developed
hydrate sugar or alcohol in 50.0 cc. between 1.5 cm. from the top and 3.5 cm.
of Kahlbaum's litmus solution. from the bottom. A similar medium was
Boil for a few minutes. used for the cultivation of meningococci
(3) Melt the sterile agar and cool to and gonococci by Tilmant and Carrien.
55C. and add 3.3 cc. ascitic fluid Variant: Tilmant and Carrien cultivated
and 4.0 cc. of (2) to 9 or 10.0 cc. of meningococci and gonococci on a medium
the agar under aseptic conditions. prepared as follows:
(4) Pour into sterile plates. (1) Collect about 100.0 cc. of ascitic fluid
(e) Eastwood studied the fermentation as carefully as possible.
of carbohydrates, alcohols, etc., on (2) Estimate the albumin content and
Lingelsheim's medium prepared as dilute with physiological saline so
follows: that there will be 3.0 g. of albumin
(1) Prepare a 10.0% solution of one per liter.
of the following: maltose, glucose, (3) Add 1.0 cc. of a 10.0% soda solution
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 647
for each 200.0 cc. of fluid and sterilize Sterilization: Not specified.
4. Bouillon (sugar free) 800.0 cc. (3) After a time test the sterility of the
(5)
distilled water. (2) Allow to stand several hours.
(6) Dissolve 15.0 g. pure inulin in (3) Add 0.5 to 0.75 cc. of (2) to tubes of
nT7^TnT!''^!T"'^'-
^ " ^-^^^ ^^^^^ ^^l^^^ite green
(1)Add 0.5 to 1.0 cc. of. ,
sterile ascitic crystals (Hochst.). Mix thoroly
fluid to agar slants. (5) Pour in plates.
(2) Incline so that the entire surface Sterilization: Method of sterilization of
is covered with the serum.
agar not given. Sterilize the bile by
Allow to stand for 12 to 24 hours.
(3) boiling
References: Vellion (1898 p. 24), Thoinot Use: Enrichment of typhoid group. Klim-
and Masselin (1902 p. 36), Besson (1920 mer reported that colon colonies were
p 53), Bezangon (1920 p. 119), Dopter inhibited. Paratyphoid colonies grew
P- ^^^^' Klimmer luxuriantly as glassy, milk-like colonies
n^^noo 2y?l^^^^^^
(1923 p. 226), Stitt (1923 p. 42). References: Abel (1912 p. 132), KliLe;
(1923 p. 214).
2020. Herrold's Phosphate Ascitic Fluid
Agar 2022. Hasting's Milk Agar
Constituents: Constituents:
1. Nutrient agar 300.0 cc. ^- ^'"trient agar 1000.0 cc.
2. Na2HP04 '
' 2. xAIilk 100.0 to 120.0 cc.
3. Ascitic fluid 100.0 cc. Preparation:
Preparation: (1) Prepare nutrient agar.
(1) Prepare nutrient agar substituting ^2) Melt (1) and cool to 50C.
Na2HP04 for NaCl. (3) Add 10.0 to 12.0% skimmed milk
(2) Heat ascitic fluid at 56C. for one * (2).
hour. (4)Tube and slant or pour in plates.
(3) Add one part heated ascitic fluid to Not specified.
Sterilization:
three parts melted (1). Use: To demonstrate proteolysis. Hast-
(4) Tube or pour into plates. igs reported that if proteolysis takes
Sterilization: Not specified. place the opaque medium clears. More
Use: Cultivation of streptococci for agglu- advantageous than gelatin to determine
tination and absorption studies. proteolysis for milk agar may be incu-
Reference: Herrold (1922 p. 80). bated at a temperature greater than
20C. Used also to cultivate fowl diph-
2021. LoefHer's Dye Bile Agar (Abel) theria bacilli and B. vulgaricus.
Variants
Constituents
(^^ .^j^U^^ reported the colonies of fowl
1. Nurient agar (3.0%) 1000.0 cc. diphtheria bacilli appeared after
^."Jt^ose 10.0 g. 40 hours when inoculated on the fol-
Z-
(b) Hammer specified the use of albumin Di. Digested by means of minced stomach.
urine from a nephritis patient. Not containing animal fluids.
El.
References: Finger, Ghon and Schlagen- Martin and Loiseau's Peptic Digest
haufer (1894 p. 14), Hammer (1895 p. 859). Agar 2035
2028. Eberson's Yeast Infusion Dujarric's Orange Juice Peptic
Agar
Digest Agar 2036
Constituents Stickel and Meyer's Peptic Digest
1- Water 100.0 cc. Agar 2037
Nutrient agar (1.0%, with
2.
Harvey's Basal Peptic Digest Agar. 2037a
2.0% peptone) 100.0 cc. Besredka and Jupille's Egg Stomach
3. Potassium phosphate (0.4%) 0.4 g. Digest Agar 2038
4. Yeast, bakers or brewers 10.0 g. E2. Containing animal fluids.
Preparation
Sellards and Bigelow's Veal Digest
(1) Macerate 10.0 g. of bakers or brewers Blood Agar 2039
yeast in 100.0 cc. of water for 20
Martin's Basal Stomach Digest Veal
minutes.
Infusion Agar 2040
(2) Steam the suspension for 2 hours at a
Harvey's Defibrinated Blood Digest
temperature not exceeding 100C.
Agar 2041
(3) Clarify by adding Merck's dialized
Harvey's Pepsin Digest Serum Agar. 2042
iron (5.0% ferric hydroxide) and
Martin's Digest of Ascitic Fluid
filteringthru glass wool.
Agar 2043
(4) Add an equal amount of 1.0% agar
D2. Digested by means of pepsin.
containing 2.0% peptone and 0.4%
Harvey's Pepsinized Blood Agar. 2044
potassium phosphate to (3). . .
D2. Not containing digests of casein or (2) Filter and heat residue with 200.0 cc.
milk products. 10.0% HCl until no Biuret is given.
El. Containing digests of mutton or other (3) Heat in open vessel to drive off HCl.
meats. (4) Neutralize with NaOH.
Harvey's Trypsinized Ox Heart (5) Filter.
and Peptic Meat Digest Agar 2074 (a) Boil over free flame under a reflux
B2. Enzymes of plant origin employed. condenser for 8 hours, 50.0 g. crude
Kligler's Yeast Autolysate Agar... 2075 casein in 200.0 cc. 10.0% HCl.
van Steenberge's Yeast E.xtract (b) Distill solution by frequently add-
Agar 2076 ing water.
Jotton's Yeast Autolysate Agar 2077 (c) Neutralize residue with NaOH.
Couret and Walker's Autolyzed Tis- (2) Prepare edestin product by:
sue Agar 2078 (a) Heat 40.0 g. pure re-crystallized
edestin over a free flame with
2029. Robinson and Rettger's Lactalbumin 10.0% HCl under a reflux con-
Agar denser, until it no longer gives
Constituents Biuret test.
1. Water 1000.0 cc. (b) Heat for several hours in open dish
2. Lactalbumin 20.0 g. to drive outHCl.
3. Agar 15.0 g. (c) Neutralize with NaOH.
652 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
white of an egg and heat at 115 for Use: General culture medium.
30 minutes. Reference: Dujarric (1916 p. 843).
654 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
2037. Stickel and Meyer's Peptic Digest Carry out step (1) thru (6) as
Agar given above.
Constituents: (7) Sterilize at 10 pounds pressure for
1. Water, tap 4000.0 cc. 15 minutes in the autoclave or for
2. Pig's stomach, minced 400.0 g. 30 minutes at 100C. on 2 suc-
3. Liver, minced (beef pla- cessive days.
centa or blood clots) 400.0 g. (8) Inoculate (7) with 1.0% of a
4. HCl (Baker Chemical Co.). 40.0 g. 24 hour old broth culture of B.
5. K2HPO4 8.0 g. saccharolyte or B. coli and incu-
6. Agar 80.0 g. bate for 12-18 hours at 37C.
Preparation (9) Steam 20 minutes.
(1) Wash clean and mince fine five or (10) Adjust to desired reaction.
more large pigs' stomachs. Mix an (11) Add 5 and 6 to (10).
equal amount of clean pig or beef (12) Autoclave at 10 pounds pressure
liver, cheap fat-free beef, placenta for 45 minutes or heat in double
or blood clots. boiler to 100C. until the agar
(2) Mix one of 3, and 4 in 1 at 50C.
2, is dissolved.
and keep at 50C. for 18-24 hours. (13) Restore the volume lost by evap-
(3) Make a Biuret and trj-ptophane oration.
test. When both are + the digest (14) Adjust to slightly alkaline to lit-
is yellowish green and contains very mus or pH = 7.3 by using 2N
littleundigested debris. KOH or NaOH.
(4) Transfer to large bottles and steam (15) Cool to 60C. and add white of egg
for 10 minutes at 100C. to stop with crushed shells or for sake of
digestion. economy, ordinary beef or sheep
(5) Strain thru cotton or preferably serum in the quantity of 25-50 cc.
store over night in the ice chest and per liter.
(9) Make a biuret and tryptophane (8) Filter thru well-wetted thick filter
test, when both are +, the digest paper while hot.
is yellowish green and contains (9) Steam 30 minutes.
very little undigested debris. (10) Filter again thru well-wetted thick
(10) Transfer to large bottles and filter paper.
steam for 10 minutes to stop (11) Mince finely, fat free veal, and add
digestion. 500 g. to 1000 cc. water.
(11) Strain thru cotton or preferably (12) Incubate for 18 hours at 37C.
store over night in ice chest and (13) Pour the mi.xture on a thick clean
decant after 24 hours. cloth.
(12) Warm (11) to 70C. and neutralize (14) Collect the fluid, and that obtained
with 2N Na2C03 to litmus. by squeezing the cloth and its
(13) Filter into a flask. contents.
(14) Add 5 and 6 to (13). (15) Mix (14) with an equal volume of
(15) Adjust to desired reaction using pepsin digest solution (10).
litmus or preferably to a definite (16) Heat to 70C.
H-ion concentration (pH = 7.0 (17) Make the reaction neutral to litmus.
to 7.5). (18) Add 7 cc. of normal NaOH per liter.
(16) Clear (15) by adding 5-10.0% of (19) Filter thru well-wetted thick filter
the decanted beef serum. Steam paper.
45 to 60 minutes. (20) Add sufficient agar to give a solid
(17) Remove from steamer and
(16) medium.
allow the clot to form as a compact (21) Steam 30 minutes.
mass. Decant or better centri- (22) Filter while hot thru well-wetted
fuge the medium to remove it. thick filter paper.
(18) Sterilize at 100 for 30 minutes Sterilization : Method of sterilization of the
on 2 successive days. agar base not given.
Reference: Stickel and Meyer (1918 Added nutrients The author used the agar
:
(2) Heat slowly at first and then boil for (15) Add one of the added nutrients.
30 minutes. Sterilization: Method of sterilization not
(3) Concentrate to one liter. given.
(4) Filter and dissolve 2.0 g. of agar for Use: Cultivation of meningococci.
every 100.0 cc. of filtrate. Added nutrients:
(5) Make slightly alkaline and boil for (a) Defibrinated blood. The blood was
25 minutes. prepared and added as follows:
(6) Filter and distribute in tubes. (1) Add three parts distilled water to
(7) Add 4.0 cc. of egg stomach digest one part defibrinated blood.
medium 1007 for prepara-
solution (see (2) Add 0.5 cc. of a 10.0% NaOH per
tion) each tube of sterile (6).
to 100.0 cc. (1).
(Ordinary peptone agar may be used.) (3) Sterilize in the autoclave at 112C.
Sterilization: Sterilize (6) at 115. (The alkali prevents coagulation.)
Use: Cultivation of gonococcus, whooping (4) Mix one part (3) with two parts
cough bacillus, pnemnococci, tubercle basal agar at 80C. The blood may
bacilli and other forms difficult to cul- be made just slightly alkaline to
tivate. litmus by the addition of HCI
Reference: Besredka and Jupille (1914 before the addition to the agar.
p. 577). (b) Serum.
(1) Add 1.0 cc. formalin to 500.0 cc.
2039. Sellards and Bigelow's Veal Digest horse serum.
Blood Agar Add 1.0% ammonia
(2) to (1) to neu-
Same as medium 1000 but solidified by the tralize to litmus.
addition of 2.0% agar. (3) Sterilize at 110C. for 15 minutes.
(4) Mix one part (3) with 3 parts
2040. Martin's Basal Stomach Digest Veal basal agar.
Infusion Agar (Harvey) (c) Serum (alkaline). Same as for (a)
Constituents above, but use serum instead of
1. Water 1000.0 cc. blood.
2. Veal 500.0 g. Variants: Harvey used the basal medium
3. Stomach digest solution 1000.0 cc. without any additions.
Preparation Reference: Harvey (1921-22 pp. 74, 81, 99).
(1) Mince finely fat-free veal.
(2) Add 500.0 g. to 1000.0 cc. water.
2041. Harvey's Serum Digest Agar
(3) Place 18 hours at 37C. Constituents:
(4) Pour the mixture onto a thick, clean 1. Distilled water.
cloth. 2. Veal, fat free.
(5) Collect the fluid which drains thru 3. Minced stomach.
the cloth together with that obtained 4. HCI.
by squeezing the meat in the cloth. 5. Formalin.
(6) Mix the fluid collected with an equal 6. Ammonia.
volume of stomach digest solution 7. Serum.
(see medium 998) 8. Agar.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 657
thick paper.
filter Use: General culture medium.
(23) Mix one part defibrinated blood Reference: Harvey (1921-22 p. 77).
(8) Place in a mortar and knead it with (11) Add 2.5 g. NaCl, 0.125 g. of CaCls to
alcohol (amount not specified). each liter of the broth.
(9) Dry in a vacuum desiccator. (12) Sterilize at 115C. for one hour.
(10) Prepare a 2.0% agar solution in (13) Filter and place 100.0 cc. together
0.5% HCl. with 4.0 g. desiccated agar (prepared
(11) To 90.0 CO. of (10) add 4 to 5.0 g. of according to Cunningham, Ind.
(9) and exactly 10.0 cc. of a 10.0% J. Med. Res. April, 1919) into each
(20) Add the sterilized solution of agar Add chloroform to prevent bacterial
in quantities of 30.0 cc. to casein growth.
digest already sterilized in quanti- (2) Boil (2) and filter thru cotton.
ties of 70.0 cc. in round quart (3) Add various amounts of sterile (2)
bottles. to an agar solution containing 20.0 g.
(21) Sterilize in autoclave. agar and 5.0 g. NaCl per liter.
Reference: Norris (1919-20 p. 541), Harvey Sterilization: Sterilize (2) in the autoclave.
(1921-22 p. 116). Sterilization of agar or final sterilization
not specified.
2053. Norris' Simplified Caseinogen Agar
Use: To study growth requirements of
Constituents Unna-Ducrey bacillus. Authors re-
1. Tap water (or saline) . 1000.0 cc. ported that the medium did not support
2. Caseinogen (10.0%).. 100.0 g. growth.
3. Washing soda (0.8%) . 8.0 g. Reference: Teague and Deibert (1922
4. Agar 20.0 to 40.0 g. p. 70).
5. Pancreatic extract
2055. Sierakowski's Trypsinized Casein
(0.5%) 5.0 cc.
Lactose Agar
Preparation
(1) Digest in an 0.8% aqueous solution Constituents
of washing soda, 10.0% of caseinogen 1. Water 1000.0 cc.
with 0.5% pancreatic extract at 2. Trypsinized casein (10.0%) 100.0 g.
37C. for 24 hours. 3. Lactose (1.0%) 10.0 g.
(2) Concentrate the products of digestion 4. Brom thymol blue 0.3 g.
on the water bath, yielding a paste 5. Agar (2.0%) 20.0 g.
of the consistency and color of Preparation
Liebig's meat extract. This may be (1) Prepare a tryptic digest of casein and
further dried in a desiccator over adjust to pH = 7.0, Method of
H2SO4 yielding a brittle resinous preparation not given.
mass which may be powdered. (2) Dissolve 10.0% (1), 1.0% lactose, 2.0%
(3) To 1000.0 cc. of saline or tap water agar and 0.3 parts per 1000 brom
add 100.0 g. (10.0%) of (2) to 40.0 g. thymol blue in water.
agar. Sterilization: Not specified.
(4) Adjust reaction to +10. Indicator Use: Isolation of colon-typhoid group.
not specified. Author reported that B. coli and lactose
Sterilization: Method not given. fermenters gave yellow colonies. Ty-
Use General culture medium. The author
: phoid and non-lactose fermenters gave
suggested the term "trypsinoids" for the blue colonies. Staphylococci and other
digest (2). B. typhosus grew well on organisms resisting the gram stain were
this medium. Brewer's yeast and fresh inhibited.
yeast were treated in like manner but Reference: Sierakowski (1923 p. 1003).
caseinogen was the more nutritive of
2056. Harvey's Trypsinized Caseinogen
the two.
Blood Agar
Reference: Norris (1919-20 p. 706).
Constituents
2054. Teague and Deibert's Trypsinized
1. Distilled water 100.0 cc.
Casein Agar
2. Blood (human or rabbit) 20.0 cc.
Constituents 3. Trypsinized caseinogen agar. 400.0 cc.
1. Water 100.0 cc. Preparation
2. Casein (10.0%) 10.0 g. (1) Add 20.0 cc. sterile human or rabbit
3. Trypsin blood to 100.0 cc. distilled water in a
4. Agar solution flask furnished with a rubber cork
Preparation and containing glass beads.
(1) Prepare a 10.0% casein solution and (2) Shake vigorously to prevent forma-
digest with trypsin at 37C. for 8 days. tion of large fibrin masses.
662 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Add 5.0 cc. pancreatic extract, 5.0 cc. (11) Mix one part (10) and two parts
(3)
enterokinase solution, and 1.5 cc. water.
chloroform. (12) Make distinctly acid to litmus by
Shake to mix. the cautious addition of strong
(4)
Incubate 8 days with daily shaking hydrochloric acid.
(5)
the first three days. (13) Add agar 2.0%,.
Remove the flask without disturbing (14) Steam 150 minutes.
(6)
the sediment. (15) Filter, while hot, thru well-wetted,
Pipette off for use, with sterile pre- thick, filter paper by placing filter
(7)
cautions, as much clear supernatant funnel, stand, and receptacle for
Preparation
2058. Teague and Deibert's Serum Tryp-
(1) Prepare a suspension of one part
sinized Casein Agar
casein in 10 parts water in a well
stoppered bottle. Constituents:
that sheep serum medium gave no growth. (10) Distribute in quantities equal to
Remaining media gave good or excellent 10.0 g. of the original casein into
growth. flasks.
Variants: The author added various (11) When ready for use add water to
amounts of the following blood cell infu- make 1000.0 cc. to each sterile flask
sion to the medium prepared as given and dissolve 3.0 g. commercial meat
aVjove extract, agar to give the desired
(1) Add 2.0 cc. of red blood cells (obtained consistency, and 1.0% galactose in
by centrifuging defibrinated blood each flask.
(rabbit) and removing the serum) to Sterilization: Sterilize (10) in the auto-
10.0 cc. of normal NaCl solution. clave. Final sterilization not specified.
(2) Keep the temperature at 100C. for Use: Cultivation of Lactobacillus acid-
3 minutes. ophilus and Lactobacillus bulgaricus.
(3) Shake the tubes and allow to cool. The authors reported that any other
(4) Centrifuge and obtain the super- carbohydrate might be used instead of
natant fluid. galactose, but galactose favored the
Reference; Teague and Deibert (1922 growth of the organisms most. This me-
p. 70). dium is slightly inferior to a medium
5. Blood (citrated laked) 10.0 cc. (2) Filter thru thick filter paper.
Preparation (3) Sterilize (method not given).
(1) Mince finely an averaged sized ox (4) Mix 5.0% of (3) with the agar ob-
heart. tained at step (18) in the medium
(2) Add to 400.0 cc. tap water. given above. 25.0% rabbit or horse
(3) Heat slowly to 75C. serum may be added if desired.
(4) Allow to cool to 45C. Reference: Harvey (1921-22 pp. 75, 77,
Note: Add 1.0 cc. 5.0% copper 2. Mutton, fat-free 2.0 lbs.
(9) Filter thru muslin to remove fat. Keep (11) immersed for 1 to
Sterilization:
(10) Add 1.0 cc. glacial acetic acid per 2 hours in boiling water or in a steam
pot. sterilizer to bring the medium into
(11) Sterilize by heating up to 110C. solution and to sterilize.
and then cool. Use: Desiccated nutrient medium.
(12) Filter to remove mince. Variants: Vardon prepared a similar me-
(13) Make
slightly alkaline to litmus and dium as follows:
then add 2.5 g. NaCl and 0.125 g. (1) Wash a quantity of agar fiber in tap
CaCl2 per liter. water containing 0.25% acetic acid.
(14) Sterilize at 115C. for one hour. (2) Wash in several changes of tap water
(15) Filter thru paper. until the washings are neutral to lit-
(16) Put 100.0 cc. broth into each whiskey mus paper.
bottle. (3) Place the washed fiber on perforated
(17) Sterilize at 120C. for one hour. or wire gauze trays and cover with
(18) Add 4.0 g. desiccated agar to each thin muslin.
bottle. (4) Expose the agar in the trays to the
Sterilization: Sterilize (18) at 120C. for sun to dry.
one hour. (5) Prepare a double strength^ (composi-
Use: Growth of organisms for producing tion not given) tryptic digest of
vaccines. The quantities employed mutton.
yielded about 15 or 16 bottles. Authors (6) Filter thru paper and measure the
reported this medium inferior to medium filtrate.
2052 for vaccine production. (7) Adjust the reaction of the filtrate to
Reference: Norris (1919-20 p. 542). pH = 8.0.
extract, the sod. chloride, and the powder to produce a single strength
calcium chloride, ^xhis addition medium. As a percentage this
was found by trial to be necessary would be given as 4.0% of powder
in order to give a final medium of to be added to the water.
pH = 7.0 to 7.2. Variations in the (22) Add the requisite amount of water.
mode of manufacture from that (23) Place in an autoclave for 30 minutes
here given would demand trials to at 120C. to dissolve the powder.
determine the exact amount of addi- (24) Distribute the liquid nutrient agar
at the extremity of the cone. This References: Harvey and Iyengar (1921-22
extremity may be cut off and rejected p. 365), Vardon (1923-24 p. 429).
make a single test tube of medium (7) Cut the agar out of the receptacle
or several tubes. If the requisite
and into slices.
amount is weighed out with pre- (8) Pass slices thru meat mincing ma-
caution into each test tube sepa- chine with a finely perforated out-
let disc.
rately, or roughly measured out, the
sterilization required, if the test (9) Spread minced nutrient agar on
metal or other type of trays.
tubes are themselves sterile, need
not under ordinary circumstances (10) Dry in hot air oven or any other
convenient way.
be more than is required to dissolve
Store powder in sterile glass stop-
the agar in the water. As an exam- (11)
may be added in the form of a strong (16) Add, while liquid, 2.0 cc. 10.0%
sterilized solution and added to the freshly prepared sodium sulphite.
medium after sterilization.) (17) Pour plates or prepare slopes.
Sterilization: Sterilize 20 minutes at 100C. Sterilization: See step (15) above.
on each of 3 successive days. Use: Desiccated culture medium.
Use: Desiccated medium for colon typhoid Reference: Vardon (1923-24 p. 432).
group.
Reference: Harvey and Iyengar (1921-22 2066. Deycke and Voigtlander's Tryptic
p. 366). Meat Agar
Constituents
2065. Vardon's Desiccated Aronson's Agar
1. Water 3000.0 cc.
Constituents 2. Glycerol 190.0 g.
1. Tryptic digest of mutton. . 1000.0 cc. 3. Pancreas
2. Agar (9.0%) 9.0 g. 4. Meat 200.0 g.
3. Na2C03 (10.0% anhydrous 5. NaOH (3.0%) 300.0 cc.
solution) 12.0 cc. 6. Agar to solidify
4. Sucrose (20.0% solution) . . 10.0 cc. Preparation
5. Dextrin (20.0% solution) . . 10.0 cc. (1) Allow the finelj' chopped pancreas
6. Fuchsin, basic (sat. alcoholic of hogs to remain on ice for 24 hours.
solution) 0.8 cc. (2) Mix with 40.0 g. glycerol and
Preparation 160.0 cc. water.
(1) Add 9.0% washed fiber agar to (3) Press out the fluid from this mixture.
1000.0 cc. of double strength tryptic (4) Dissolve 200.0 g. meat in 300.0 cc. of
digest of mutton. (See variant of 3.0% NaOH solution.
medium 1715.) (5) Filter (4) and neutralize.
(2) Heat for 60 minutes at 120C. to (6) Add 0.25% NaaCOs (siccum) to (5).
(3) Add 12.0 cc. 10.0% anhydrous so- (8) Add 50.0 g. of (3) to (7).
dium carbonate to the nutrient agar. (9) Incubate for 7 to 10 hours at 37C.
(4) Steam 15 minutes at 100C. (10) Neutralize with HCl.
(5) Add while hot 10.0 cc. 20.0%o sucrose, (11) Dilute with water to 3 liters.
10.0 cc. 20.0%, dextrin, and 0.8 cc. (12) Add 150.0 g. glycerol and the usual
saturated alcoholic solution of basic amount of agar to (11).
fuchsin. Sterilization: Not specified.
(8) Prepare an equal volume of 4.0% (2) Heat 1500.0 cc. of water to boiling.
agar solution in the stock infu- (3) Drop 750.0 g. of (1) into (2), piece
sion (5). by piece, stirring constantly.
(9) Cool (8) to 70C. and mix with (7). (4) Boil strongly and remove from the
(10) Heat in the steamer for one hour. fire.
(11) Allow to set and stand over night. (5) Remove the meat and run thru a
(12) Melt the following morning and chopping machine.
strain through lint. (6) Cool the water to 37C. and add
(13) Suspend the residual meat and fine 1.5 g. NaaCOa per liter.
coagulum from (4) and (7) in a (7) Put the chopped meat in 2 liter
quantity of N/100 HCl equal to the Erlenmeyer flasks, 550.0 g. per flask.
weight of the original raw meat. (8) Add (6) to each flask, filling the
(14) Raise to 100C. and autoclave at flask to the neck.
130C. (25 lbs. pressure) for 30 (9) Add 3.0 g. pancreatin, 10.0 cc. chloro-
minutes. form and 10.0 cc. toluol to each flask.
(15) Allow to cool to 37C., incubate 24 (10) Cork tightly and shake well.
hours, to test sterility. (11) Incubate at 37C. over night.
(16) Add 2.0% of sterile pancreatic ex- (12) Shake the next day and add more
tract and incubate for 5 to 15 hours. pancreatin unless the fluid shows a
(17) Add 0.8% anhydrous NaoCOs in the yellow color and particles of meat
form of a sterile 32.0% solution. look smaller.
(18) Allow digestion to continue until a (13) Continue digestion for four or five
Sorensen figure of not less than 20 days at room temperature, or for
isproduced. (Titrate the digest in two or three days in the incubator.
the presence of neutralized formalin, The meat should be finely divided
using phenolphthalein as an indi- at the end of this time.
cator, and express the result in cc. (14) Decant the liquid thru cheese cloth.
of N/10 NaOH required to neu- (15) Add anequal volume of water to the
tralize the amino acids in 10.0 cc. residue, shake well and again decant.
of the filtered digest.) (16) Place the meat on cheese cloth and
(18) Filter thru absorbent cotton and (13) Mix acid and agar thoroly and leave
paper until clear. for 15 minutes.
(19) Mix the filtrate with an equal (14) Pour off the water and wash thoroly
amount of water. until the agar is free from acetic
(20) Solidify (19) by the addition of 1.5% acid.
agar in the usual manner. (15) Add the agar obtained from (14) to
Sterilization: Sterilize at 15 pounds pres- 1000.0 cc. of (10).
sure for 30 minutes. (16) Add 0.125 g. CaCU.
Use: General culture medium. (17) Autoclave for 45 minutes to dissolve
Reference Park, Williams and Krumwiede
: the agar.
(1924 p. 119). (IS) Neutralize to phenolphthalein by the
addition of N/10 KOH
while hot.
2069. Gordon and Hines' Trypagar (19) Cool to 60C. and add the white of
2 eggs beaten up with the crushed
Constituents shells.
1. Water (20) Autoclave again at 118C. for
2. Pea flour 100.0 g.
75 minutes or heat in the steamer for
3. NaCl 100.0 g.
2 hours.
4. Heart bullock 500.0 g.
(21) Filter.
5. Agar 20.0 g.
(22) Add 2.0% of sterile (3).
6. CaCl2 0.125 g.
(23) Distribute as desired.
Preparation Sterilization: Sterilize (23) in the ordinary
(1) Add a liter of water to a 100.0 g. of
way.
pea flour and NaCl.
100.0 g.
Use: Cultivation of meningococci. Wood
(2) Mix thoroly and steam for 30 min- used a similar medium for the cultivation
utes, stirring occasionally.
of diphtheria bacilli.
(3) Allow to settle and filter the super- Variants: Wood added 0.3 cc. of a sterile
natant liquid.
1.0% telluric acid solution to 10.0 cc. of
(4) Add 1000.0 cc. of water to 500.0 g. of the agar for the cultivation of diphtheria
finely chopped bullock heart and
bacilli.
make slightly alkaline to litmus by Reference: Gordon and Hines (1916
the addition of 20.0% KOH
solution.
p. 682), Wood (1921 p. 562).
(5) Heat (4) slowly at 75 to 80C. for
5 minutes. Duval
2070. and Harris' Tryptic Digest
(6) Cool to 37C. and add 1.0% liquor Agar
trypsinae (Allen and Hanbury's)
and incubate at 37C. for two and Constituents:
one-half to three hours. 1. Water 1000.0 cc.
(7) Test for peptone using the Biuret 2. Agar (3.0%) 30.0 g.
test. 3. Tryptic Digest Solution, . . . 1000.0 cc.
(8) Render slightly acid to litmus by the Preparation
addition of glacial acetic acid and (1) Prepare a 3.0% agar solution in water.
boil for 15 minutes. (2) Tube.
(9) Leave over night in a cool place and (3) Melt sterile tube of (2) and cool to
siphon off the clear liquid in the 45C.
morning. (4) Mix equal parts of the sterile agar
(10) Make faintly alkaline to litmus. with Duval and Harris' Tryptic Di-
(11) Weigh out 20.0 g. agar and cut in gest Solution (See medium 1134).
thoroly. Add
a sufficient quantity agar solution not given. The Tryptic
of water to cover the agar and add Digest Solution is sterilized by filtering
2.5 g. of glacial acetic acid per liter thru a Berkefeld filter.
of water. Use: Cultivation of leprosy bacillus.
670 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Variants: If desired, glycerol, peptone, (6) Make the thick brownish fluid
salt, etc., may be added in the preparation slightly alkaline to litmus.
of this agar. (7) Add 1.0% pancreatic extract and
Reference: Duval and Harris (1911 p. 169). incubate at 37C. for 5-24 or 48 hours.
(8) When the process is sufficiently ad-
2071. Distaso's Trypsinized Serum Agar
vanced, render slightly acid with
Constituents glacial acetic acid and boil slowly
1. Water 1500.0 cc. 15 minutes.
2. Serum (beef or sheep). 500.0 cc. (9) Either filter or decant the clear
3. Agar 30.0 to 40.0 g. fluid which results on placing the
Preparation digest over night in a cool place.
(1) Mix equal parts (500.0 cc.) of water (10) Adjust the reaction as desired.
and sheep or beef serum. (11) Dissolve 3 and 5 in (10).
(2) Sterilize at 120C. for 15 minutes. (12) Adjust to desired reaction using lit-
(3) Digest for 24 hours at 60C. with a mus or preferably to a definite H-ion
pancreatic extract from a hog in the concentration (pH = 7.0 to 7.5).
presence of chloroform. Activate the (13) Clear (12) by adding 5-10.0% of the
extract with an extract of the upper decanted beef serum. Steam 45 to
portions of the small intestine. 60 minutes.
(4) Filter on paper. (14) Remove (13) from steamer and
(5) Add 30.0 or 40.0 g. of agar to 1000.0 cc. allow the clot to form as a compact
of water. mass. Decant or better centrifuge
(6) Sterilize (5). (Method not given.) the medium to remove it.
(7) Mix equal parts (4) and (6). Sterilization: Sterilize at 100 for 30 min-
(8) Tube. utes on two successive days.
Sterilization: Method of final sterilization Use: General inexpensive culture medium.
not given. Authors reported that this medium was
Use: Culture medium for tubercle bacilli, excellent for primary isolation of highly
strict anaerobes, B. proteus and others. parasitic organisms.
Reference: Distaso (1916 p. 600). Reference: Stickel and Meyer (1918 p. 81).
refrigerator. Preparation
Weigh the blood clots and mix (1) Wash clean and mince fine 5 or more
(3)
500.0 g. with 1 liter tap water. large pigs stomachs. Mince an
Place the mixture in an enameled equal amount of clean pig or beef
(4)
liver, cheap fat-free beef, placenta
pot, bring slowly to a boil and boil
slowly for 5 minutes stirring con- or blood clots.
stantly. (2) Mix 2, one of 3, and 4 at 50C. and
Strain fluid thru cheese cloth and keep at 50C. for 18 to 24 hours.
(5)
pass the residue thru a fruit press, (3) Make a biuret and tryptophane test.
cool to 37C. When both are + the digest is yel-
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 671
lowish green and contains very little (8) When the digest is sugar free add
undigested debris. 4.0 g. CaCOs and 80.0 g. agar.
(6) Cool to 37C. and add 1.0% pan- Reference: Stickel and Meyer (1918 p. 80).
(1) Free horse meat from all fat and (2) Digest for two days at 37C.
tendons. (3) Filter, and if positive to the
(2) Chop intosmall pieces with a Biuret test add 3.9 g. dry NajCOs.
knife, a scraper or meat grinding (4) Add 15.0 cc. of a glycerol extract
machine until there are only very of a hog's pancreas to (3).
loose particles. (5) Incubate for 6 hours at 37C.
(3) To 125.0 g. of (2) add 3.0 g. of (6) Sterilize in the steamer.
fresh Witte-Rostock pepsin 400.0 (7) Neutralize with HCl.
cc. distilledwater and 2.0 cc. of a (8) Add 1950.0 cc. water, 6.0 g. NaCl
50.0% HCl solution. and 39.0 g. agar to (7) and boil for
(4) Place in an Erlenmeyer flask and 3 hours in the steamer.
incubate at 37 C. (9) Filter thru cotton.
(5) Incubate for 2 days, shaking oc- (10) Distribute in flasks.
casionally and adjusting the re- (11) Sterilize.
action with the addition of a 50.0% (c) Harvey gave the following method of
HCl solution. preparation:
(6) Filter the grey white precipitate Mix 150.0 g. of finely
(1) minced horse
which has formed at the end of heart, 5.0 g. pepsin, 2.0 cc. of
this time.
50.0% HCl and 400.0 cc. of distilled
(7) Test a portion of the filtrate by water.
the Biuret reaction (violet colora- Leave
(2) to digest 2 days at 37 C
tion with KOH and CUSO4) which (3) Filter.
must be positive. Add 40.0 cc. of glycerol
(4) and 160.0
(8) Add 3.9 g. of NaaCOs (siccum) to cc. distilled water to a finely
the filtrate. chopped pig pancreas.
(9) Sterilize (Method not given). (5) Infuse(4) for 3 days in an ice chest,
(10) Cut a hog's pancreas into small adding a small piece of camphor.
pieces with a knife, and place in Add
(6) 3.9 g. anhydrous NaaCOj and
the ice box for 24 hours. 15.0 cc. of (5) to the filtrate from
(11) Add 40.0 cc, of glycerol and 160.0
(3).
cc. distilled water, and extract for
(7) Leave 6 hours at 37C.
several days in the ice box. (8) Sterilize at 100C.
(12) Press out the juice and add a small (9) Neutralize with HCl.
piece of camphor to the liquid. (10) Add 1950.0 cc. water, 6.0 g. sodium
(13) Add 15.0 cc. of (12) to (9) by means chloride, and 39.0 g. agar.
of a sterile pipette.
(11) Steam 3 hours.
(14) Incubate at 37C. for six hours. (12) Filter.
(15) Sterilize immediately in the auto- (13) Distribute into test tubes and
clave, and neutralize with 50.0% flasks.
HCl. (14) Sterilize.
(16) Add 1950.0 cc. of water, 6.0 g. (d) Deycke (Klimmer) gave the follow-
NaCl, 39.0 agar to (15). ingmethod of preparation
(17) Boil in the autoclave for 3 hours. Mix 125.0
(1) g. of finely chopped
(18) Filter thru cotton and distribute horse meat with 400.0 cc. distilled
into little flasks. water and 3.0 g. of Witte's pepsin.
(19) Sterilize as usual (exact method (2) Add 2.0 cc. of 50.0% concentrated
not given). HCl.
(b) Bosse (Kolle and Wasserman) pre- (3) Incubate at 37C. for 48 hours,
pared the medium as follows: adding more HCl if necessary.
(1) Mix 150.0 g., of finely chopped (4) Filter.
horse heart with 3.0 g. of Witte's Add
(5) 3.9 g. water free soda to the
pepsin (fresh) 2.0 cc. of 50.0% filtrate.
HCl and 400.0 cc. of distilled (6) Sterilize (method not given).
water. Chop the pancreas
(7) of a hog into
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 673
small pieces, and store in the ice (6) Make up the volume to 1500.0 cc.
box for 24 hours. (7) Solidify with agar.
(8) Add 40.0 cc. of glycerol and 100.0 References: Kligler (1919 p. 186), Abt and
cc. water to (7) and place in the Blanc (1921 p. 452), Harvey (1921-22 p.
ice box for several days. 120).
22, p. 100), Klimmer (1923 p. 221). Reference: van Steenberge (1920 p. 841).
for ^ hour and decant the partially and brain of healthy rabbits, guinea
clear fluid to an Erlenmeyer flask or pigs, kittens and human placenta,
(6) Cool the agar (exact temperature not (2) Seal and place in the thermostat at
given), and add one whole egg to 40 C. for 10 to 20 days.
(1) Keep 100.0 g. yeast at 50C. for 24 (6) Distribute 0.1 to 0.5 cc. of (3) (auto-
hours or until liquefaction is com- lyzed tissue) over the surface of each
plete by autolysis. agar slant.
(2) Dilute with water to 400.0 cc. (7) Keep several hours in the horizontal
(9) The agar may be melted, and cooled Pinoy's Flax Seed Agar 2101
to 50C. and the tissue juice added. Omeliansky and Ssewerowa's Flax
In this case it is necessary to break Fiber Agar 2102
the surface of the slants before using. Heider's Wheat Agar (Klimmer)... . 2103
Sterilization: Not specified. Klimmer's Oat Agar 2104
Use: Cultivation of amoeba. Bacto Corn Meal Agar (Dehydrated). 2105
References: Couret and Walker (1913 p. C2.* Fruits or their derivatives used.
253), Stitt (1923 p. 51). Milburn's Basal Prune Agar 2106
Reed and Cooley's Prune Agar 2107
SUBGROUP II-C. SECTION 8
Bacto Prune Agar (Dehydrated) . . . 2108
Basal or complete media containing agar Dombrowski's Raisin Must Agar . . . 2109
with constituents of plant origin (exclusive Perold's Grape Juice Agar 2110
of digests) of unknown chemical composi- Richter's Meat Infusion Wine Agar. . 2111
tion. (Animal products may also be Jenkins' Tomato Infusion Agar 2112
present.) C3. Tubers or their derivatives used.
Ai. Containing bacteria, yeast, other fungi Di. Not containing animal products.
or their derivatives. Graham-Smith's Potato Agar 2113
Gassner's Yeast Autolysate Agar. . . 2079 Dawson's Potato Juice Agar 2114
Harvey's Yeast Extract Agar 2080 Gaehtgens' Potato Agar 2115
Beijerinck's Yeast Water Agar 2081 Bacto Potato Dextrose Agar (De-
Duval's Parasite Agar 2082 hydrated) 2116
Gassner's Asparagin Yeast Water Kellerman and McBeth's Potato
Agar 2083 Agar 2117
Emulsion Agar. 2084
v. Eisler's Bacterial Lubinski's Glycerol Potato Agar. 2118 . .
2126
Peklo's Malt Infusion Agar 2090
Owen's Molasses Agar 2127
Fulmer and Grimes' Malt Infusion
Le Fevre and Round's Cucumber
Agar 2091
Juice Agar 2128
Rettger, Reddish and McAlpine's
B2. Leguminous plants or derivatives em-
Malt Extract Agar 2092
ployed.
Tanner and Deck's Acid Near Beer
Agar 2093 Ci. Seeds used.
Janke's Lager Beer Agar 2094 Bacto Lima Bean Agar (De-
hydrated) 2129
Sobel's Lactose Beer Agar 2095
Stutzer's Basal Legume Seed (In-
Difco Wort Agar (Dehydrated) 2096
fusion) Agar 2130
D2. Materials other than Di.
Wyant and Tweed's Pea Agar 2131
Meacham, Hopfield and Acree's Corn
Kaufmann's Jequirity Seed Agar. . . 2132
Meal Agar 2097
Maze's Sucrose Bean Agar 2133
Buchanan's Silage Agar 2098
Reed and Cooley's Lima Bean Agar. . 2134
Reed and Colley's Corn Meal Agar.. 2099 .
Thorn's Bean Agar (Tanner) 2135 (2) Heat the mixture 20 minutes at a
Stutzer's Bean Infusion Agar 2136 temperature not exceeding 50C.
Vogel and Zipfel's Legume Flour (3) Steam 2 hours.
Agar (Klimmer) 2137 (4) Filter and refilter thru well-wetted,
C2. Materials other than seeds used. thick, filter paper.
Bacto Bean Pod Agar (Dehydrated) 2138 . (5) Mix two parts (4), cooled to 15C.
Didlake's Soy Bean Root Agar 2139 with three parts melted 2.5% agar
Zipfel's Legume Leaf Agar 2140 solution of a reaction pH = 7.4.
Simon's Legume Agar (Klimmer and (Agar may have peptone and NaCl
Kriiger) 2141 in it if desired.)
Sterilization: Sterilize in the autoclave.
2079. Gassner's Yeast Autolysate Agar Use: Cultivation of meningococci. Harvey
Constituents: reported that with a semi-liquid agar
1. Water. (0.5%) viability can be preserved in stab
2. Yeast, Brewer's. cultures much longer than with a stiffer
3. Agar (3.0%). agar.
4. NaCl. Reference: Harvey (1921-22 p. 120).
Preparation
2081. Beijerinck's Yeast Water Agar
(1) Place about 10 liters of brewer's yeast
and wash with water. Allow
in a flask Constituents
to stand for 30 minutes and pour off 1. Water tap 1000.0 cc.
of time and remove the liquid from Use: To study the fermentation of glucose
of (6). 2. Agar
(8) Adjust to slightly alkaline to litmus. 3. Entameba coli
(1) Mix one part bakers or brewers yeast Sterilization: Method of sterilization of
Use: Cultivation of B. leprae. Inoculate (5) Heat the emulsion for an hour and a
the slant with an emulsion prepared from half at 56C. or boil for one-half hour.
leprous tissue. The author reported that (6) Add from 1.0 to 3.0 cc. of the emulsion
the organism grew without the presence to each sterile tube of agar melted and
of B. typhosus. cooled to 50C.
Reference: Duval (1910 p. 653). Sterilization: Methodof sterilization of
agar not given. See step (5) for steriliza-
2083. Gassner's Asparagin Yeast Water tion of bacterial emulsion.
Agar Use: To study growth of bacteria on killed
Constituents bacteria. Author reported that B. coli
(3) Dissolve 30.0 g. agar, 10.0 g. aspara- emulsion might be centrifuged for quite a
gin, and 5.0 g. NaCl in (2). long time and the supernatant, cell free
To each
80.0 cc. of (3) add 20.0 opalescent fluid be added to the agar, or
(4) cc.
0.5% solution of water blue.
of a the sediment be mi.xed with a volume of
Sterilization: Not specified. NaCl solution, equal to that removed
Use: To study the nitrogen requirements from the sediment, and added to the
for growth of the colon-typhoid and agar.
Steam until the precipitate turns Added nutrients The authors added 2.0%
:
(4)
white. ofany desired carbohydrate, alcohol, etc.
(5) Dilute about one-third with distilled Reference Park, Williams and Krumwiede
:
2091, Fulmer and Grimes' Malt Infusion (2) Boil to dissolve the agar,
Agar (3) Tube.
(4) Just previous to use add 0.4 cc. of
Constituents: 5.0% sterile solution of lactic acid to
1. Distilled water 1150.0 cc. each sterile tube of (3).
2. Malt, distillers 360.0 g. Sterilization: Method of sterilization of
3. Agar (1.5%) 15.0 g. agar or lactic acid not given.
Preparation Use: Cultivation of yeast causing sore
(1) Mash at 55 C. 360.0 g. malt with throat probably Endomyces or Monilia.
1150.0 CO. distilled water for 24 hours. Reference: Tanner and Deck (1923-24
p
(2) Filter thru towelling, then thru filter 285).
paper.
(3) Heat for 30 minutes under 15 pounds 2094. Janke's Lager Beer Agar
pressure.
Constituents
(4) Allow to stand 3 days and filter.
1. Lager beer.
(5) Add agar.
2. Gelatin.
(6) Heat in autoclave 30 minutes at 15
3. Alcohol.
pounds.
4. Agar.
(7) Filter thru absorbent cotton and
Preparation:
tube.
Sterilization: Sterilize in live (1) Evaporate lager beer to one-half its
steam for 30
original volume by steaming.
minutes on 2 successive days.
Use: Growth of yeast Sacch. cerevisiae and (2) Add water to its original volume.
Torula sphaerica. (3) Add 2.0% agar and 2.0% gelatin to
(2).
Reference: Fulmer and Grimes (1923 p
(4) Clarify with white of egg.
586).
(5) Filter thru cotton.
2092. Rettger, Reddish and McAlpine's (6) Make up to the original volinne by
the addition of water.
Malt Extract Agar
(7) Distribute in 10.0 cc. lots in Freuden-
Constituents: reich flasks.
1- Water 900.0 cc. Sterilization: Sterilize on each of 3 succes-
2. Malt extract powder, Difco . 100.0 g. sive days in the steamer.
Preparation Use: Cultivation of acetic acid bacteria
(1) Dissolve 2 in 1. from beers.
(2) Reaction is about pH = 5.5 to 5.6. Reference: Janke (1916 p. 6).
Sterilization: Not specified.
Use: Isolation of yeast from feces. 2095. Sobel's Lactose Beer Agar
Authors reported that the reaction was
too acid for the development of bacteria.
Constituents:
Congo red.
(5) Boil vigorously for several minutes
and add the amount of water lost by
evaporation.
(6) Pour directly into sterile Petri dishes
ORaANISM
680 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(1) Soak 1.0 pound of dry prunes in 1 liter (y) Knops' nutrient solution 0.25, 0.5,
of water for 12 hours. 1.0, 2.0 or 5.0%.
(2) Filter thru a towel and then cotton. (z) Knops' nutrient solution 0.25, 0.5,
(.5) Heat at 120C. for 30 minutes. (1) Wash, pare and slice potatoes.
(6) Add 40.0% NaOH to obtain a reaction (2) Heat one volume of (1) in two
of a minus 5 on the Eyres scale. volumes of water slowly for 2 hours.
and resterilize the medium in (3) Boil, after heating for two hours.
(7) Filter
the autoclave. (4) Filter thru cloth.
Add water to make up the loss due
(8) Readjust the reaction to a minus 5. (5)
Constituents
1. Water 500.0 cc. 2114. Dawson's Potato Juice Agar
2. Potato pulp 500.0 g.
Constituents
3. Agar 30.0 g.
Water 1000.0 cc.
1.
Preparation
2. Agar 20.0 g.
(1) Wash and peel potatoes and crush
3. Potato juice 500.0 g.
in a mincing machine.
Preparation
(2) Add water in the ratio of 1.0 g. of and
(1) Use 500.0 g. unskinned potatoes
potato pulp to 1.0 cc. of water.
1000.0 cc. water and obtain potato
(3) Allow to stand in a flask for 12 hours.
juice.
Filter thru a filter paper.
(4)
(2) Free juice from starch (method not
(5) The medium may be left in the given).
acid condition or to give an alka-
(3) Dissolve agar in (2).
line medium, neutralize the acid
Sterilization: Not specified.
with N/1 caustic soda, using litmus
Use: To show bacterial variation of B. coli.
as the indicator and then adding
Reference: Dawson (1919 p. 142).
3.0 cc. of alkali per liter.
(6) Treat agar as usual (treatment not Agar
2115. Gaehtgens' Potato
specified) and add 3.0% to (4).
Place in steam sterilizer at 100C. Constituents
(7)
1. Water 1000.0 cc
until agar is dissolved.
Potato 500.0 g.
(8) When cool add white of egg and 2.
steam sterilizer.
clarify in the 3. Agar
(9) Filter thru Chardin filter paper.
4. NaCl
(10) Tube. Preparation
Sterilization: Sterilize on 3 successive days (1) Carefully wash and peel 500.0 g.
Variants: Klimmer gave the following Use General culture medium. The author
:
2120. Weinzirl's Sucrose Potato Water Agar (9) Distribute into test tubes.
(10) Sterilize.
Constituents Harvey
References: Rochaix (1913 p. 604),
1. Water 800.0 cc.
(1921-22 p. 119).
2. Potato water 200.0 cc.
3. Sucrose 10.0 g. 2122. Schardinger's Hay Infusion Agar
4. Agar 20.0 g.
Constituents:
Preparation 1000.0 cc.
1. Water
(1) Mix 200.0 cc. of potato water and 800.0
2. Hay 30.0 to 40.0 g.
cc. water. 10.0 to 15.0 g.
3. Agar (1.0 to 1.5%) . . .
(3) Add 30.0 to 35.0 g. of agar to (2). see medium 1179, and a melted and cooled
(2) Boil a mixture of 30.0 g. (1), 10.0 g. 2127. Owen's Molasses Agar
Gigartina prolifera (Tsunomato)
Constituents:
and 1000.0 cc. water in a Koch
1. Water 1000.0 cc.
steamer for one hour.
2. Molasses (Final 75 Brix) . . . 160.0 g.
(3) Filter thru a fine cloth.
3. Agar 20.0 g.
(4) Add 1.0% to 1.5%, agar and 10.0 cc. Preparation
normal soda solution. final molasses 75
(1) Dissolve 160.0 g.
(5) Boil for 30 to 40 minutes.
Brix and 20.0 g. agar in 1000.0 cc. of
(6) Distribute into test tubes and
water.
sterilize (method not given).
Sterilization: Not specified.
(b) Harvey solidified 1000.0 cc. of a straw
Use: Bacterial count in cane sugar
decoction with 15.0 g. agar. (See
products.
medium 1178 for the preparation of a
Reference: Owen (1914 p. 338).
straw decoction.)
References: Schardinger (1896 p. 541),
2128. Le Fevre and Round's Cucumber
Tsujitani (1898 p. 667), Harvey (1921-
Juice Agar
22 p. 121).
Constituents
2125. Reed and Cooley's Spinach Agar 1. Cucumber juice 1000.0 cc.
Use: Cultivation and isolation of halo- 2131. Wyant and Tweed's Pea Agar
philic bacteria. Authors reported that
Constituents:
organisms grew just as well on meat
extract with 5.0% salt.
1- Water 4000.O cc.
Reference: Le Fevre and Round (1919 2. Peas (canned) 1000.0 g.
p 3- Agar
178). qqq g
4. Litmus
Preparation
2129. Bacto Lima Bean Agar (Dehydrated)
(1) Boil 3 in 1 until the agar is dissolved.
Constituents (2) Make up to water lost by boiling.
1. Distilled water (3) Mash 1000.0 g. canned peas in their
2. Lime bean infusion (dry)... 8.0 g. juice and add to (2).
3. Agar, Bacto 15.0 g. (4) Autoclave for 30 minutes at 15 pounds
Preparation pressure.
(1) Dissolve 23.0 g. of Bacto Lima Bean (5) Allow the agar to cool slowly. This
Agar (Dehydrated) in 1000.0 cc. of allows the hulls to sink to the bottom.
water, by boiling or autoclaving, (6) Cut away and discard the sediment
preferably the latter. The dry lima after the agar has solidified.
bean infusion is prepared from an (7) Melt the remaining clear agar and
infusion of 50 parts dried lima beans. color with litmus.
(2) Distribute as desired. Sterilization: Sterilize in the autoclave.
Sterilization: Sterilize by autoclaving for Use: Cultivation of organisms causing flat
20 minutes at 15 pounds pressure. sour in peas.
Use: General culture medium. Reference: Wyant and Tweed (1923 p. 12).
Reference: Digestive Ferments Co. (1925
p. 17). 2132. Kaufmann's Jequirity Seed Agar
Constituents
2130. Stutzer's Basal Legume Seed 1- Water lOO.O cc.
(Infusion) Agar 2. Jequirity seeds 10.0 g.
Constituents 3. Agar 1.5 to 2.0 g.
1. Distilled water 1000.0 cc. Preparation
2. Legume seed 10.0 g (1) Grind 10.0 g. Jequirity seeds in a
3- Agar 10.0 g. mortar.
Preparation (2) The peeled or shelled seeds now weigh
(1) Same as for medium 1166, but solidified about 8.0 g.
by the addition of 1.0% agar. (3) Add to 100.0 cc. of water.
Sterilization: Not specified. (4) Boil in a steam sterilizer for about 2
Use: Cultivation of nodule bacteria. hours.
Variants Zipfel prepared a similar medium
: (5) Cool and filter.
as follows (6) Dissolve agar in (5).
(1) Mix powdered legume seed
100.0 g. of (7) Reaction is neutral or slightly
with 100.0 cc. N/1 KOH in a mortar. alkaline.
(2) Add 5 liters of water and allow to (8) Distribute into test tubes.
stand for 24 hours. Sterilization: Sterilize in the usual manner
(3) Pour off the clear supernatant fluid. (Method not given).
(4) Neutralize with H3PO4 and make up Use: Cultivation of Bacillus pyocyaneus
to 5 liters volume. and many others. Author reported that
(5) To 1 liter of (4) add 30.0 g. of agar and the medium supported the growth of a
20.0 g. dextrose. great variety of organisms.
(6) Acidify by the addition of 10.0 cc. Reference: Kaufmann (1891 p. 66).
N/1 malic acid.
(7) Sterilization not specified. 2133. Maze's Sucrose Bean Agar
References: Stutzer (1900 p. 898), Zipfel Constituents
(1911-12 p. 10). 1. Water 1000.0 cc.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 689
Same as medium
but Phaseolus
2099, bean flour.
lunatus, lima bean meal was used instead Reference: Klimmer (1923 p. 229).
of corn meal.
2138. Bacto Bean Pod Agar (Dehydrated)
2135. Thorn's Bean Agar (Tanner) Constituents
Constituents 1. Distilled water
1. Water 1000.0 cc. 2. Agar Bacto 15.0 g.
2. Bean, white 200.0 g. 3. Bean Pod Meal Infusion
3. Agar as desired (dry) 7.5 g.
Preparation: Preparation
(1) Heat common white beans with 5 (1) Dissolve 22.5 g. of Bacto Bean Pod
volumes of water. Boiling is stopped Agar (Dehydrated) in 1000.0 cc. of
just before the swelling of the coty- distilled water by boiling or auto-
ledons rupture the seed coats. claving, preferably the latter. Bean
(2) Filter. This solution filters easily. Pod Meal is prepared by infusion of
(3) Add agar as desired. 20 parts bean pod meal.
Sterilization: Not specified. (2) Distribute as desired.
Use: General culture medium. Tanner Sterilization: Heat in the autoclave for 20
reported that this medium contained minutes at 15 pounds pressure.
sufficient nutrients to grow many species Use: General culture medium.
normally. This medium is poor in Reference: Digestive Ferments Co. (1925
available carbon. It may be desirable to p. 17).
add carbohydrates for certain species.
Reference: Tanner (1919 p. 50). 2139. Didlake's Soy Bean Root Agar
Constituents
2136. Stutzer's Bean Infusion Agar Water 1000.0 cc.
1.
Agar 2151
a neutral reaction, or add 10.0 cc. of N/1
B2.* Body fluids used.
malic or citric acid or 10.0 cc. of N/1
Ci. Blood employed.
NaOH to give an alkaline medium.
Reference: Zipfel (1911-12 p. 106). * See B3, page 691.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 691
using sheep serum, or without the addi- Sterilization: Sterilize in the steamer.
tion of serum. Use: Cultivation of diphtheria, cholera,
Variants: The authors added various influenza and other pathogenic forms.
amounts of sheep or rabbit serum, heated References: Nastiukoff (1893 ;^33 and 34),
at 55C. for 15 minutes to the medium as Rechtsamer (1895 p. 493).
prepared above.
Reference: Teague and Deibert (1922 p. 2145. Steinschneider's Egg Yolk Agar
70).
Constituents:
2143. Duval's Leprous Tissue Agar 1. Distilled water 800.0 cc.
2. Egg yolk 100.0 cc.
Constituents: 3. Agar (2.0% soln.)
1. Agar solution. Preparation :
2. Tissue (Leprous). Mix two parts of
sterile distilled water
(1)
Preparation with one part hen or lapwing's egg
(1) Prepare an agar solution. Composi- yolk obtained under sterile con-
tion not given, but the author speci- ditions.
fied that it should contain no peptone.
(2) Prepare a 2.0% agar solution.
(2) Adjust to 1.5% alkaline. Mix one part (1) with two parts
(3) (2).
(3) Place small bits of leprous tissue upon Distribute into tubes.
(4)
the surface of the sterilized and Slant and solidify.
(5)
solidified alkaline agar.
Sterilization: Final sterilization not given.
(4) Moist the surface with a thin suspen- Use: Cultivation of gonococcus. The
sion of an organism capable of oxidiz-
medium was not transparent.
ing nucleo-proteids such as B. typho- Variants: The author prepared a similar
sus, paraiyphosus, prodigiosus, py- medium as follows:
ocyaneus, dysenteriae, etc.
(1) Mix one part yolk of hen or lapwing's
(5) Incubate at 32C. to 35C. for two egg with three parts sterile distilled
weeks. water. Shake thoroly.
Sterilization: Sterilization of agar not To 20.0 g. of (1) add 10.0 cc. of a 20.0%
(2)
given.
Na2HP04 and 3 times, hence 90.0
Use: Indirect culture of B. leprae. cc. ofa 2.5 to 3.0% agar solution.
Reference: Duval (1911 p. 371). Pour into tubes and solidify (method
(3)
3. Na2HP04 2.0 g.
(Time not specified.)
4. Ammonium lactate 10.0 cc. (4) Add 3.0 cc. of 1.0% gentian violet
5. CaCOs 10.0 g.
solution when cool.
(5) Tube.
Sterilization: Sterilize at 12 pounds pres- 2150. Noyes' Gelatin Agar
sure for 15 minutes. Constituents:
Use: To study variation in morphology of 1. Water 1000.0 cc.
^
B. coli. 2. Agar (best) 15.0 g.
Reference: Scales (1921 p. 595). 3. Gelatin 7.5 g.
Preparation
2148. Scales' Egg Starch Agar (1) Dissolve 2 and 3 in 1.
(1) Strain a fresh whole egg thru four (b) Weiss prepared a similar medium as
thicknesses of cheese cloth four times. follows:
(2) Prepare a soluble solution of agar (1) Dissolve 15.0% gelatin in 500.0 cc.
1.5%, and 0.1% soluble starch in of water from the "Mlihlgraben der
distilled water Schwarzawa."
(3) Add 2.0 cc. of (1) to each 7.0 cc. of (2) Dissolve 1.5% agar in 500.0 cc.
(2).
water.
Sterilization: Sterilize at 12 pounds pres- (3) Mix (1) and (2).
of B. coli.
2151. Standfuss and Kallert's Bone Jelly
Reference: Scales (1921 p. 596).
Agar
2149. Despeignes' Egg Yolk Milk Agar Solidify medium 1360 with agar.
Constituents:
2152. Nicolle's Blood Agar
1. Distilled water
2. Milk 100.0 cc. Constituents
3. Glycerol 15.0 g. 1. Water 900.0 cc.
4. Agar 9.0 g. 2. Agar 14.0 g.
5. Gentian violet 3. NaCl 6.0 g.
6. Egg Yolk 4. Blood, rabbit
694 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(1) Dissolve 3.0 g. of agar in 100.0 cc. (b) Teague and Deibert reported that the
Ringer's solution. Unna-Ducrey bacillus would not
(2) Add a drop of dog blood to 2 to 3.0 grow on a medium prepared by mix-
cc. of dog serum diluted with 2 to 5 ing various amounts of sheep or
parts Ringer's solution. (Sera or rabbit serum, heated at 55C. for 15-
ascitic fluids of other animals may minutes with a 2.0% agar solution
be used.) containing 0.5% NaCl.
(3) To 9.0 cc. of (2) add 1.0 cc. of (1). References: Tanner (1919 p. 70), Harvey
(4) Heat at 56 water bath
to 58C. in a (1921-22 p. 79), Teague and Deibert
forabout 30 minutes. (1922 p. 70).
(5) Cover with a layer of liquid sterile
2155. Kanthack and Stephens' Serous
paraffin.
Exudate Agar
(b) Stitt cultivated Leptospira icteroides
(cause of yellow fever) on a medium Constituents:
prepared as follows: 1. Serous exudate (ser-
(1) Mix one part rabbit s erum and 3 um, ascitic fluid, etc.). 100.0 cc.
parts of Ringer's solution solidified 2. KOH 10.0% 2.0 cc.
partially by the addition of 0.3% 3. Agar (1.5 to 2.0%) . . . 15.0 to 20.0 g.
agar. 4. Glycerol (4.0 to
(2) Tube in tall tubes. 5.0%) 4.0 to 5.0 cc.
Introduce 1.0 cc. of yellow fever
(3) Preparation
patients blood into the lower part (1) To 100.0 cc. of a serous exudate add
of each tube. 2.0 cc. of 10% KOH solution. (It is
(4) Pour a thin layer of petroleum over always best to heat a small
first
(2) Leave overnight to allow for sep- 2157. Ecker's Bile Agar
aration of clot.
Constituents:
(3) Add 50.0 cc. N/1 sodium hydroxide
1. Bile (ox) 1000.0 cc.
per liter.
2. Agar 15.0 g.
(4) Steam 20 minutes.
Preparation
(5) Add 1.0agar per liter after
g.
(1) Dissolve 2 in 1 as rapidly as possible
making it into a paste or suspen-
by gently boiling.
sion with a little of the alkaline
(2) Filter and allow to cool and again boil
ascitic fluid.
and refilter without titration.
(6) Steam to dissolve the agar.
(3) Tube.
(7) Filter, while hot, thru thick, filter
Sterilization: Sterilize in autoclave for 3
paper by placing filter funnel,
minutes at 15 pounds pressure.
stand and receptacle for filtrate in
Use: To study effect of bile on B. typhosus.
the sterilizer.
The author reported that some strains
(8) Dissolve 10.0 g. glucose per liter
were completely inhibited while others
in the hot, filtered, nutrient agar
were only partially inhibited. Approxi-
(7). (Add 50.0 cc. glycerol per
mately the same results were obtained
liter also if desired.)
using 50.0% or 10.0% bile. (Bile diluted
(9) Distribute into test tubes.
with distilled water.)
(10) Sterilize.
Reference: Ecker (1918 p. 97).
(c) Harvey prepared a similar medium
from 20.0 g. glycerol, 20.0 cc. of 10.0% 2158. Raskin's Milk Egg Albumin Agar
NaOH, 20.0 g. agar and 1000.0 cc. of a
Constituents
body fluid (ascitic fluid, pleuritic
1. Milk 1000.0 cc.
fluid, hydrocele fluid, ovarian fluid,
2. Egg albumin
milk, urine, etc.).
3. NaCl 5.0 g.
References: Kanthack and Stephens (1896
4. Agar 5.0 to 7.0 g.
p. 609), (1896 p. 835), Harvey (1921-22 p.
Preparation
83).
(1) Stir thoroughly with a glass rod
100.0 g.albumin from fresh eggs in a
2156. Beck's Glycerol Serum Agar
flat bottomed dish and add drop by
(Klimmer)
drop concentrated NaOH until a
Constituents solid opaque gelatinous material is
1. Water 1800.0 cc. obtained.
2. Serum (beef) 200.0 cc. (2) Cut this solid egg albumin in small
3. KHSO4 10.0 g. pieces and place in distilled water.
4. MgS04 5.0 g. Do not allow the alkaline albuminate
5. Asparagin 4.0 g. to stand or the albuminate will
6. Glycerol 40.0 g. become liquid in several hours.
7. Agar (3.0%) 60.0 g. (3) Shake thoroughly and pour off the
Preparation water, continue this washing until
(1) Steam 200.0 cc. beef serum (con- the wash water is only slightly
taining no chloroform) with 1800.0 cc. alkaline.
of water for 1.0 to 1.5 hours. (4) Place the coagulated sodium al-
(2) Filter. buminate in a flask of distilled water,
(3) Add 3, 4, 5 and 6 to the filtrate and plug with cotton and heat in the
steam for 2 to 3 hours. steamer for 15 to 30 minutes. The
(4) Filter while hot. albumin dissolves.
(5) Add 3.0% fiber agar, and heat until (5) Filter.
dissolved. (6) Add 50.0 cc. glycerol and 5.0 to 7.0 g
(6) Tube. of finely divided dried agar to 1000.0
(7) Reaction should be slightly acid. cc. of fresh milk.
Sterilization: Not specified. (7) Allow to stand for 12 to 14 hours (at
Use: Cultivation of tubercle bacilli. room temperature in the winter)
Reference: Klimmer (1923 p. 224). and then boil for 75 to 90 minutes
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 697
over a free flame. In order to (12) Distribute into sterile test tubes.
place a lid over the container or Sterilization: Sterilization of (3) not given.
steam for 3 to 3.5 hours in a steamer. Use: Cultivation of typhoid bacilli and
The
casein coagulates slowly. others. The author reported that the
(11) After about 20 or 30 minutes remove separatory funnel and add 1.0 g.
the yellow top layer containing the NaOH (2.5 cc. solution of 400.0 g.
fat by means of a spoon. NaOH in a liter).
(12) Heat the fat-free portion to boiling (2) Shake well and allow to stand at
and add 1.0% of the filtrate from (5). about 18C. for 48 hours.
(13) Add soda to neutralize the reaction. (3) Remove the nearly transparent milk
(14) Add 0.5% NaCl if desired. from the bottom of the funnel and
(15) Filter until clear thru a paper in a add it to a second separatory funnel.
hot water funnel. (4) Add 250.0 cc. ether and shake well.
Sterilization: Not specified. (5) Allow to stand for 48 hours.
Use: Cultivation of pathogenic organisms. (6) Place the opalescent liquid now in a
Reference: Raskin (1887 p. 359). large sterile flask plug with cotton
and heat to 50C.
2159. Reinsch's Alkaline Milk Agar Place under the receiver of a water
(7)
separatory funnel and add 1.0 g. 2. Agar (1.0 to 1.5%) . . . 10.0 to 15.0 g.
(6) Remove the nearly transparent milk Use: General culture medium.
from the bottom of the funnel and Reference: Abbott (1921 p. 139).
add it to a second separatory funnel. Whey Agar
2161. Raskin's Casein
(7) Add 250.0 cc. ether and shake well.
(8) Allow to stand for 48 hours. Constituents
Place the opalescent liquid now in a 1. Milk 1000.0 cc.
(9)
large sterile flask, plug with cotton 2. Agar
and heat to 50C. Preparation
(10) Place under the receiver of a water (1) Allow 1000.0 cc. of skimmed (or
suction pump for 3 or 4 hours until whole) milk to stand 48 hours at
all the ether is evaporated. room temperature.
(11) Mix 1 part of (3) with 3 parts of (10) (2) Remove any cream that settles out
(3) Obtain the casein and thoroly press (6) Medium may be employed with a
it dry. neutral, basic, (by adding Na2C03), or
(4) Rub the casein to a powder and wash acid, (by adding lactic acid), reaction.
with 95.0% alcohol, and place in a Sterilization: Not specified.
flask of ether. Use: Stutzer and Hartleb attempted to
(5) Shake for 2 minutes and allow to cultivate the bacterium causing foot and
stand for 18 minutes. mouth disease. Other investigators used
(6) Pour off the ether and add fresh. similar media for the cultivation of a
Repeat this process 3 or 4 times. large number of organisms.
(7) Pour off but half the last portion of Variants
the ether and add alcohol (amount (a) Huss cultivated aroma producing
not specified). bacteria, Bacillus and
esterificans
(8) Shake for 5 minutes. Pseudomonas trifolii on a medium
(9) If drops of fat raise when alcohol is prepared by dissolving 20.0 g. agar
added, repeat the washing of the and 5.0 g. NaCl in 1000.0 cc. of whey.
casein with ether until the casein is (b) Meier made bacterial counts of milk
fat-free. and milk products in a medium pre-
(10) Collect the casein on a filter. pared as follows:
(11) Dry and heat for 5 to 20 minutes at (1) Dissolve 2.5 g. NaCl and 15.0 or
120 to 140C. (10.0 g.) agar in 500.0 cc. of water.
(12) Wash in a moderately concentrated (2) Mix equal parts (1) and whey.
alkali solution. (3) Neutralize by the addition of
(13) This gives a transparent material, KOH. Add KOH
until tumeric
which when dry becomes hard as paper is turned quite weakly brown-
stone. This casein will dissolve by ish-red.
heating in slightly alkaline water. (4) Sterilization not specified.
(14) Prepare 150.0 cc. of an 8.0% solution (c) Fulmer and Grimes used the following
of (13). medium for the cultivation of yeast
(15) Mix 350.0 cc of filtered
. whey contain- found in cream and butter.
ing 1.75% agar with (14). (1) Dissolve 1.5% agar in whey ob-
(16) Heat for 15 to 20 minutes at 60 or tained from skim milk by coagulat-
70C. Do not boil or the casein will ing the casein with rennet.
be coagulated. (2) Filter (1) thru absorbent cotton.
(17) Distribute in sterile test tubes. (3) Tube in 10.0 cc. portions.
Sterilization: Not specified. (4) Sterilize for 20 minutes at 15 pounds
Use : Cultivation or pathogenic organisms. pressure.
References: Raskin (1887 p. 359), Peifer
(5) When using as a plate medium, add
(1888 p. 568). 1.0 cc. of a1.0% tartaric acid solu-
tion to each Petri dish to keep down
2162. Stutzer and Hartleb's Whey Agar
bacterial growth.
Constituents
(d) Klimmer and Sommerfeld (Klimmer)
1. Whey 1000.0 cc.
made bacterial counts of milk on a
2. Agar 20.0 g.
medium prepared as follows:
Preparation
(1) Prepare clear agar solution using
(1) Separate the cream from milk by
20.0 g. agar, 7.0 g. NaCl and 1000.0
centrifugation.
cc. water.
(2) Heat to 40C. and add a little rennet.
(2) Sterilize (1) method not given.
(3) After the casein has coagulated heat
to 75C. and filter. (3) Add a rennet to milk obtained
little
To a liter of (3) add 20.0 g. of finely under the cleanest possible con-
(4)
chopped agar and heat in the steamer ditions and heat at 40C. until the
(5) Neutralize by adding soda (indicator (4) Separate the casein from the whey
not specified). by straining thru a straining cloth.
699
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
2167. Henssen's Organ Infusion Agar 2168. Mayer's Salivary Gland Agar
Constituents
Constituents:
1. Water 200.0 cc.
1- Water 500.O cc.
2. Kidney 100.0 g.
2. Salivary glands 500.0 g.
3. Agar 2.5 g.
3. Agar (1.5%) 15.O g.
Preparation
Preparation
(1) Remove the capsule from the kidney
(1) Chop fresh salivary glands in a meat
(dog, calf and hog were used) and rub
chopping machine.
to a pulp in a mortar with an equal
(2) Mix with an equal weight of water.
amount of water.
(3) Infuse on ice for 24 hours after strong
(2) Allow to extract for 3 hours.
stirring.
(3) Press thru fine pored linen.
(4) Press the mass in a pressing machine.
(4) Prepare a 2^% water solution of agar.
(5) Solidify sterile (6) by the addition of
(5) Heat(4)to40C.
1.5% agar (method not given).
(6) Mix equal parts of sterile (5) and Sterilization: Sterilize(4) in streaming
melted agar cooled to 40C.
sterile
steam for 30 minutes. Final sterilization
(7) Incubate to test sterility.
not specified.
Sterilization: Filter (3) thru a sterile clay
Use: General culture medium. Author re-
filter using a water suction pump to ported that meat extract media generally
facilitate the filtering. Method of ster-
gave better results.
ilization of agar not given.
Reference: Mayer (1899 p. 747).
Use: To study the effect of kidney infusion
on the growth of some organisms. The
2169. Graham-Smith's Heart Infusion Agar
author reported that the medium checked
the growth of the organisms studied, i.e., Solidify medium 1342 by the addition of
diphtheria bacilli, anthrax bacilli, B. coli, agar.
701
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Meat Infusion Agar (6) To each 80.0 cc. of (5) add 5.0 cc. of a
2170. Kutscher's
3.0% acid fuchsin solution and 0.0,
5. NaCl 3.0 g.
used, but all gave the same results.
Preparation
(b) Abel boiled the brain infusion for 45
(1) Remove the suprarenal capsule from minutes instead of 15 minutes as in
an ox immediately after death. above.
step (4)
(2) Chop to small bits and rub to a pulp. References: Ficker (1900 p. 593), Abel
To 100.0 g. of (2) add 200.0 cc. of
90), Kolle and Wasserman (1912
(3)
(1912 p.
water.
p. 410), Klimmer (1923 p. 224).
(4) Make (3) alkaline with soda and boil
(6) Prepare a 2.5% agar solution in 2181. Loeffler's Malachite Green Nutrose
distilled water and filter. Agar
(7) Mix equal parts of (6) and sterile (5).
Add 3.0% glycerol. Constituents
(8)
1. Water 2000.0 cc.
(9) Tube sterilized (8).
and Beef 1 lb.
(10) Cool to 45C. Mix well solidify 2.
3. Agar 30.0 g.
quickly in a slanted position.
704 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
sufficient moisture to prevent its drying Other authors used similar media for the
up under ordinary conditions and was cultivation of amoeba, parasitic flagellata
not liquefied by fast growing types. and ciliata, cladothrix and protozoa.
Reference: Williams and Burdick (1916 p. Variants
413). (a) Hiss omitted the NaCl.
(b) Musgrave and Clegg cultivated
2184. Burger's Basal Lead Acetate Agar
amoeba on a medium containing 20.0
Constituents g. agar, 0.3 toO.5 g. NaCl and 0.3 to 0.5
1. Water 1000.0 cc. g. beef extract per liter. The original
2. Agar 30.0 g. reaction of the medium being 1.5%
3. Lead acetate 1.0 g. alkaline before sterilization, and
4. Meat extract 4.0 g. 1.0% alkaline following sterilization.
Preparation The authors reported that in some
(1) Dissolve 2, 3 and 4 in 1. cases even smaller amounts of salt
(2) Suspend 1.0 g. of one of the added and extract were more desirable
nutrients in physiological salt solu- especially when amoeba were growing
tion and add 3.0% Na2C03. in company with some saprophytic
(3) Add (2) to melted (1), cooled to 56C. bacteria.
Sterilization: Not specified. (c) Linde isolated cladothrix {Clado-
Use : To study the production of hydrogen thrix dichotoma) on a medium con-
sulphide. taining 15.0 g. agar and 5.0 g. meat
Added nutrients: The author suspended extract per liter. Linde reported
1.0 g. of one of the following in physio- that contaminating forms grew but
logical salt solution: cladothrix soon overgrew them. The
cystine best solid medium for cultivation is
taurine 10.0 g. agar with 0.5 g. meat extract
sodium taurocholate in 1000.0 cc. water.
Reference: Burger (1914 p. 202). (d) Zikes cultivated Cladothrix dicho-
toma and Cladothrix natans on
2185. Hiss' Extract Agar
Linde's medium, containing 5.0 g.
Constituents meat extract and 10.0 g. agar per
1. Distilled water 1000.0 cc. liter.
2. Extract, Liebig's 5.0 g. (e) Malm (Besson) prepared the medium
3. NaCl 5.0 g. as follows:
4. Agar 15.0 g. (1) Dissolve 5.0 g. Liebig's meat
Preparation extract (or 20.0 g. Cibils) in 1000.0
(1) Dissolve agar in water by boiling cc. water.
over free flame for 30 to 45 minutes. (2) Soak 20.0 g. chopped thread
of
(2) Dissolve Liebig's extract and sodium agar in water for several
cold
chloride in (1). hours. Squeeze the water thru a
(3) Clarify by the coagulation of two egg cloth.
whites in (2). (3) Heat (2) in (1) at 100C. until the
(4) Filter thru cotton. agar is dissolved.
(5) Reaction is 0.75% acid to phenol- (4) Readjust the reaction if necessary.
phthalein. (5) Allow to cool to 55 or 60C.
(6) Tube in 10.0 cc. lots. (6) Beat the white of an egg in 100.0
Sterilization: Sterilize in the Arnold in the cc. of water and add to (5).
usual manner on three successive days. (7) Mix well.
Use: Differentiation of typhoid, colon (8) Autoclave at 120C. for one hour.
and allied forms. The author reported (9) Filter thru a moistened Chardin
that typhoid colonies were threaded. filter using a hot water funnel.
Constituents: Constituents
1. Distilled water 1000.0 cc. 1. Water 1000.0 cc.
2. Agar 15.0 g. 2. Meat extract, Liebig. 3.0 g.
3. Extract Liebig's 5.0 g. 3. Agar 30.0 g.
4. NaCl 5.0 g. 4. Sucrose 10.0 g.
5. Glucose 10.0 g. 5. Malachite green
Preparation (2.0% solution) 2.0 to 2.9 cc.
(6) Add the glucose after clearing. (7) Tube in about 10.0 cc. lots.
(7) Tube. (8) Sterilize the usual manner at
in
Sterilization: Method of sterilization not 100C. on 3 successive days.
given. References: Hiss (1897 pp. 681, 694),
708 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(1901 p. 728), (1902 pp. 151, 152, 156), 2. Ashes, wood 5.0 g.
Frost (1903 p. 340), Ball (1919 p. 80), 3. Agar 2.0 g.
Tanner (1919 pp. 51, 56) Heinemann 4. Maltose 2.0 g.
(1922 p. 34). Preparation
(1) Dissolve 2.0 g. agar and 2.0 g. maltose
2192. Stapp's Egg White Extract Agar in 100.0 cc. water by heat.
Constituents: (2) Add 5.0 g. wood ashes to 100.0 cc.
1. Water 500.0 cc. water.
2. Egg white (3) Boil and filter.
3. Meat extract 1.33 g. (4) Mix 100.0 cc. of (1), 40.0 cc. of (2),
4. NaCl 0.33 g. and 60.0 cc. of water or mix 100.0
5. Glucose 1.66 g. with 60.0 cc. (2) and add 40.0
cc. (1)
(6) Mix 200.0 g. of the filtrate, 0.33 g. medium 1377, and then adding 1.2,
NaCl, 1.66 glucose, 10.0 g. agar, 1.33 1.5 or 2.0% agar to solidify.
g. meat extract and 300.0 cc. water. (b) Lohnis prepared the medium as
(The meat extract may be omitted). follows:
(7) Dissolve by heating in the steamer. (1) Infuse 4.0 g. of wood ashes with
(8) Filter while hot. 1000.0 cc. of water.
Sterilization: Not specified. (2) Add 4.0% maltose and 2.0%
Use: Cultivation of Bac. cobayae, Bac. K2HPO4 to (1).
capri, Bac. guano, Bac. musculi, Bac. (3) Solidify with agar.
hollandicus. (4) Sterilization not specified.
Reference: Stapp (1920 p. 5). (c) Harvey dissolved 4.0 g. maltose, 5.0
g. wood ashes and 10.0 g. agar in
SUBGROUP II-C. SECTION 10
1000.0 cc. distilled water.
Basal or complete media containing agar (d) Percival prepared the medium as
with derivatives of soil, ashes, etc., but not follows:
containing digests. (1) Add 8.0 g. of well burnt wood ashes
Ai. Ashes or derivatives employed. to 500.0 cc. distilled water and boil
Harrison and Barlow's Wood Ash for one minute.
Agar 2193 (2) Filter thru two sheets of paper.
A2. Soil infusions or extracts employed. (3) Add 4.0 g. maltose and 4.0 g. of
Fremlin's Soil Infusion Agar 2194 agar to (2).
Gowda's Soil Infusion Agar 2195 (4) Heat until dissolved.
Conn's Soil Infusion Agar 2196 (5) Filter.
Lohnis' Mannitol Soil Infusion (6) Tube.
Agar 2197 (7) Sterilize in the usual way, method
Perotti's Dicyandiamide Soil Infu- not given.
sion Agar 2198 (8) Slant.
(e) Giltner gave the following method of
2193. Harrison and Barlow's Wood Ash Agar preparation:
Constituents: (1) Stir 5.0 g. of wood ashes (elm,
1. Water 200.0 cc. beech, maple) into 1000.0 cc. of
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 709
distilled water for two or three 2195. Gowda's Soil Infusion Agar
minutes only. Constituents:
(2) Filter. 1. Water 2000.0 cc.
(3) Add 1.0% washed agar.
2. Soil 1000.0 g.
(4) Heat in steam for 30 minutes. 3. K0HPO4 1.5 g.
(5) Add 1.0% commercial sucrose. 4. (XH4)2S04 1.5 g.
(6) Boil for 5 minutes over a free flame. 5. MgS04 0.75 g.
(7) Strain while hot thru several thick- 6. reoS04 0.02 g.
nesses of clean cheese cloth. This 7. NaCl 3.0 g.
may be filtered if desired.
8. Na.COs 1.0 g.
(8) Sterilize by the Tyndall method in
9. Agar
the steamer.
Preparation
References Harrison and Barlow (1907 p.
:
(1) Heat 1000.0 cc. of good rich garden a Chamberland filter, then sterilize on 3
soilwith a liter of tap water for 30 successive days in the autoclave under 15
minutes in the autoclave under pounds pressure for four hours. Sterilize
pressure of 1 atmosphere or boil (3), (4) and (5) separately,
method not
with 2 liters of water over a free given.
flame. Use: To study nitrification. Gowda re-
(2) Pour off the turbid liquid. ported that the ammonia was oxidized to
(3) Mix talc with the liquid. nitrites.
(4) Filter thru a double filter paper. Reference: Gowda (1924 p. 255).
(5) Make up the volume to 800.0 cc. if
(1913 p. 101), Giltner (1921 p. 370), (3) Mix (1) and (2).
(5) Filter until clear thru thick filter (2) Pour off the turbid liquid.
paper. (3) Mix talc with the liquid.
(6) Mix 100.0 cc. of (5) and 900.0 cc. of (4) Filter thru a double filter paper.
(1). (5) Make up the volume to 800.0 cc. if
addi- /, ,,
s j mnrv x-
(b) Meyer used a 10.0% gelatin solution,
x- i i
.
, ^
tion to gelatin j- x j n ^or / xx xxto culti"
ix-
none ooTiN adjustcd to 0.4% to attcmpt
i.
r V. u
gela- , or>r> n ,
t u
dissolving 200.0 g. of Gold Label
i
.j,.^. .,
tin.
,.,
Additional constituents, if any, in-
, ,
.
^. , ,^
gelatin in 1000.0 cc. tap water, adjust-
.
^ , x ^
.\..
Ai. Containing gelatin
,,.
and water only. , , , ing to 0.5% normal acid to phenol-
n x on n
'
. u-x /-I x- oirn . 1
phthalein /or> (20.0 to 30.0 cc. of normal
i e i
,
1
.
.,^, .
Phosphate ....^, ^^ . , J^ ,
x- u
'
2 Gelatin '
. 100.0 g. method of preparation
Preparation: (1) Dissolve 120.0 g. Gold Label,
Thoroly shake 100.0 g. gelatin in one 100-0 B^^to, or 100.0 g. U. S. Glue
(1)
water.
liter of
^^- gelatin in 1000.0 cc. distilled
water.
(2) Heat until the gelatin is melted.
(2) Clarify with white of egg.
(3) Neutralize (indicator not specified).
(4) Boil in the steamer. (3) Adjust the pH between 6.6 and 7.4.
(4) Sterilize.
(5) Filter.
Sterilization: Sterilize in the steamer on (0 Waksman studied the metabolism of
part gelatin to five parts of tap water. 2202. Stutzer and Hartleb's Phosphate
The reaction was adjusted to 0.5% Gelatin
acid to phenolphthalein.
Constituents:
References: Matzuschita (1902 p. 287),
Kufferath (1914 p. 559), Meyer 1- Water looo.O cc.
(1915
2. Gelatin lOO.O g.
p. 467), Conn (1916 p. 188), (1917 p. 42),
Committee 3. Potassium phosphate 1.0 g.
S. A. B. (1918 p. 116), Waks-
Preparation
man (1920 p. 22), Weiss (1920 p. 25),
Harvey (1) Dissolve 100.0
gelatin and 1.0 g.
g. of
(1921-22 p. 105), Committee
of potassium phosphate in water.
S. A. B. (1923 p. 10).
(2) Make up the volume to 1 liter.
Constituents
3. MgSO^. 1.0 g.
1. Gelatin
4. K2HPO4 1.0 g.
10.0%
Preparation: (1) Solidify one of the added
5. CaCU trace
nutrients by the addition of 10.0% Preparation
(1) Dissolve 2, 3, 4 and 5 in 1.
gelatin.
(2) Pour sterile (1) into plates.
Sterilization : Method not specified.
Use: Cultivation of intestinal bacteria.
Sterilization: Not specified.
Use: Cultivation of Mycobacteria. The
Added nutrients: The author used one of
author reported that the plates were
the following:
slightly turbid due to a precipitate of
Beer wort
Mg3(P04)2. After 8 days mycobacteria
Urine
caused a clearing of the medium around
Straw Infusion
the colony.
Reference: Matzuschita (1901-02 p. 214). Reference: Vierling (1920 p. 202).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 13
Gelatin 2210
2205. Beijerinck's Starch Gelatin
C2. Not containing carbohydrates; other
organic carbon used. Constituents
Sohngen's Petroleum Gelatin 2211
1. Water 1000.0 cc.
Weisser's Glycerol Gelatin 2212
2. Gelatin 100.0 g.
Proskauer and Beck's Glycerol Gela- 3. KH2PO4 0.5 g.
(Klimmer)
tin 2213
4. Starch (soluble) 1-0 g.
Fermi's Phenol Gelatin 2214
Preparation
B2. Inorganic nitrogen salts added.
(1) Dissolve 2, 3 and 4 in 1.
Sohngen's Ammonium Chloride
(2) Pour into plates.
Malate Gelatin 2215
Sterilization: Not specified.
Woltje's Basal Sucrose Gelatin
Use: Detection of quinone production.
(Zikes) 2216
The author seeded the medium by pouring
Mortensen's Sucrose Ammonium a culture of S. chromogena in medium 250
Sulphate Gelatin 2217
over the plate. After a few days the
Higgins' Lactose Ammonium Suc- gelatin was melted and the quinone was
cinate Gelatin 2218
extracted with benzol.
V. Tubeuf's Sucrose Ammonium Ci- Reference: Beijerinck (1900 p. 10).
trate Gelatin (Malenkovic) 2219
Cohn's Ammonium Tartrate Gelatin 2206. Waksman's Starch Gelatin
(Klimmer) 2220
Kossowicy's Ammonium Phosphate Constituents
Sucrose Gelatin (Will) 2221 1. Distilled water 1000.0 cc.
V. Tubeuf's Citric Acid Ammonium 2. Gelatin 150.0 g.
Nitrate Gelatin 2222 3. Starch 10.0 g.
714 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Constituents
Reference: Bobilioff-Preisser (1916 p. 395).
(4) Heat in the autoclave for 15 minutes Use: Isolation of Microspira desulfuricans
at 110. and Microspira aestuarii.
(5) Filter.
Variants: The author added 30.0 g. NaCl
(6) Tube in 9.0 cc. lots. for the isolation of Microspira aestuarii.
(7) Just before use add 1.0 cc. of a 10.0% Reference: van Delden (1903-04 p. 88).
KI solution to each tube (liquefied). 2231. Frankel's Lactate Asparagin Gelatin
Sterilization: Final sterilization not spe-
(Klimmer)
cified.
Use: Isolation of typhoid bacilli. Char- Constituents:
rin and Dissard cultivated Bacillus 1. Water 1000.0 cc.
Constituents: Constituents
1. Water ~.
1000.0 cc. 1. Water 100.0 cc.
Preparation: (1) Dissolve 2, 3, 4, 5 and 6 B2. One or more of the additional constit-
in 1.
uents organic.
Sterilization: Not specified. Ci. All additional organic constituents of
Use Cultivation of organism causing water
:
known chemical composition.
rust in flax (Plectridium pectinovorum). Di. No additional nitrogen supplied.
Author reported that if HaS be produced El. Only one type of organic carbon added.
the medium was turned brown. Fi. Carbohydrates employed.
Reference: Goslings (1904 p. 392). C 1. Monosaccharides used.
Utz's Glucose Peptone Gelatin 2246
2234. Sullivan's Nitrate Asparagin Gelatin
Beijerinck's Glucose Peptone Gela-
Constituents: tin 2247
1. Water 1000.0 cc. Pfeilerand Lentz's Ringer Solution
2. Asparagin 10.0 g. Peptone Gelatin 2248
3. MgSOi 0.2 g. Vierling's Glucose Peptone Gelatin. 2249
4. CaCl2 trace Sear's Glucose Peptone Gelatin 2250
5. K0HPO4 1.0 g. Go. Disaccharides used.
6. NaCl 1.0 g Utz's Lactose Peptone Gelatin 2251
7. KNO3 0.2 g. Molisch's Sucrose Peptone Gelatin
8. Gelatin 100.0 g. (Smith) 2252
Preparation: (1) Dissolve 2, 3, 4, 5, Trommsdorff's
6, Sucrose Peptone
7 and 8 in 1.
Gelatin 2253
Sterilization: Method not given. G3. Polysaccharides used.
Use: Used to determine liquefaction of Molisch's Dextrin Peptone Gelatin 2254 . .
El. Animal derivatives (exclusive of ex- Hi. Basal media employed with the addi-
tracts and infusions) employed. tion of other materials.
em- Rosenberg's Basal Extract Gelatin. 2293
Fi. Cells, tissues or their derivatives
Biirger's Basal Fuchsin Sulphite
ployed.
Extract Gelatin 2294
Deycke's Peptone Alkali Albumin
2269 Tausz and Peter's Basal Ragit
Gelatin
2270 Gelatin 2295
Kotlar's Pancreas Gelatin
2271 Buchan's Basal Litmus Extract
Harde's Tissue Gelatin
Gelatin 2296
F2. Body fluids employed.
H2. Complete media.
Pergola's Nitrate Alkaline Blood
Bacto Nutrient Gelatin (Dehy-
Gelatin 2272
drated) 2297
Fs. Secretions or excretions employed.
Heinemann's Peptone Extract Gela-
Gi. Milk or its derivatives added. tin 2298
Matzuschita's Peptone Milk Gelatin 2273
Frost's Peptone Extract Gelatin 2299
Raskin's Whey Peptone Gelatin. . . . 2274
Kohn's Peptone Extract Gelatin .... 2300
Appel's Peptone Whey Gelatin 2275
Buchan's Neutral Red Peptone Ex-
Meier's Whey Peptone Gelatin 2276
tract Gelatin 2301
Wigger's Whey Gelatin 2277
von Varon's Nitrate Extract Gela-
G2. Milk or its derivatives not employed. tin 2302
Piorkoweki's Peptone Urine Gela- G2. Containing additional organic constit-
tine 2278 uents.
Heller's Urine Peptone Gelatin 2279 Henneberg's Glucose Extract Gela-
E2. Animal extracts or infusions employed. tin 2303
Gi. Additional
.
if
References: Kita (1913 p. 446), Mat- (2) Boil over a free flame for a minute.
zuschita (1902 p. 286, 288). (3) Cool to about 50 to 60C.
(4) Dissolve the gelatin in (3) without
2237. Banning's Basal Peptone Gelatin further heating.
Same as medium 1535, but solidified by (5) Correct the acid reaction by the
the addition of 70.0 g. gelatin instead of addition of concentrated Na2C03.
10.0 g. agar. (6) Transfer to a large flask.
(7) Add the white of an egg, mi.x well
2238. Harvey's Basal Neutral Red Peptone and steam for | hour.
Gelatin (8) Filter, using compressed air to
Constituents hasten filtration.
1- Water 1000.0 cc. (9) Distribute in 50 to 100.0 cc. lots in
2. Peptone 20 g flasks.
3. KOH(5.0%) 10.0 cc. Sterilization: Place in flowing steam for
4. Gelatin 75.O g. 10 minutes on 3 successive days.
5. Neutral red (1.0%) (0.5%) . 5.0 cc. Use: General culture medium. Other in-
Preparation vestigators used similar media for spe-
(1) Dissolve2, 3 and 4 in 1. cific purposes as indicated below.
(2) Steam 45 minutes. Variants
(3) Filter. (a) Jacobi stated that the medium might
(4) Add 5.0% of a 20.0% solution of one be diluted with distilled water when
of the added nutrients, and 0.5% of a ready for use.
1.0% neutral red solution. (b) Matzuschita dissolved 10.0 g. of
Sterilization: Sterilize for 10 minutes on Koch's peptone in 1000.0 cc. water
each of 3 successive days at 100C. and solidified with gelatin. He culti-
Use: Cultivation and differentiation of vated spore forming bacilli on this
colon group. medium.
Added nutrients: The author added 5.0% (c) Lieske isolated iron bacteria, Lepto-
of a 20.0% solution of any desired sugar. thrix ochracea on a medium made
Reference: Harvey (1921-22 p. 109). slightly alkaline by the addition of
(1) Dissolve 40.0 g. Chapoteaut's pep- (2) Adjust to a slightly alkaline reaction
tone, 5.0 g. NaCl, 30.0 g. gelatin and by the addition of soda.
5.0 g. glucose in 1000.0 water by Sterilization: Not specified.
boiling. Use: To study proteolytic action, lique-
(2) Make slightly alkaline. faction by mycobacteria. The author
(3) Filter. reported that growth was good, but lique-
(4) Distribute in bottles. faction did not take place. Same results
(5) Plug with cotton and cover the cotton if glucose be omitted.
Reference: Boekhout and Ott de Vries 2261. Remy's Phenolated Lactose Peptone
(1918 p. 130). Gelatin
Constituents
2258. Matzuschita's Glucose Glycerol 1. Distilled water. .. 1000.0 cc.
Peptone Gelatin 2. Asparagin 6.0 g.
(2) Adjust the reaction to slight alka- 2265. Eisner's Peptone Potato Gelatin
linity.
(Heinemann)
Sterilization: Method not specified.
Use: Cultivation of colon-typhoid group. Constituents:
The author reported that colon and 1. Water 1000.0 cc.
typhoid colonies could not be distin- 2. Potato 1.0 lb.
References: Raskin (1887 p. 358), Klimmer Use: To determine bacterial counts of milk
(1923 p. 172), Cunningham (1924 p. 102). and milk products.
Reference: Meier (1918 p. 436).
2275. Appel's Peptone Whey Gelatin
Constituents
2277. Wigger's Whey Gelatin
(9) Remove from the autoclave, and (d) Jensen (1898) cultivated denitrifying
loosen the solidified gelatin from bacteria on the following medium:
the walls of the container. (1) Prepare an infusion from 500.0 g.
(10) Upset the gelatin on a filter paper. meat and 1000.0 cc. of water.
(11) Cut off the turbid upper layer of (2) Dissolve 5.0 g. NaCl, 10.0 g. pep-
the gelatin cylinder with a thread tone and 200.0 g. of gelatin in (1).
or wire. (3) Adjust the reaction to a slight
(12) Cut the clear gelatin into pieces, alkalinity by adding soda.
place into a flask and melt. (4) Method of sterilization not speci-
(13) Distribute into test tubes. fied.
(4) Make slightly alkaline and cool (1) Mix 500.0 g. of finely chopped lean
a little. beef with one liter of water, and
(5) Add the white of an egg. allow to stand in the ice box for
(6) Place the kettle containing (5) in 12 to 24 hours.
boiling water, and stir with a (2) Press the liquid thru a towel and
spoon to insure equal heating. make up the volume to one liter.
(7) Adjust the reaction once more and (3) Boil in the steamer cooker for
heat to 100C. for 15 minutes. 30 minutes.
Leave the lid loosely on the kettle. (4) The infusion may be boiled for an
(8) Heat a water funnel to 60C. (not hour before removing the meat
higher) and filter quickly. and then filtered thru paper. If
(9) Distribute the filtrate into sterile the liquid is still red, boil again
culture tubes. for 15 minutes.
(10) Heat in boiling water or in flow- (5) Filter when cold to remove any fat.
ing steam at 100C. for 17 to 20 (6) Heat to boiling and add 0.5%
minutes. NaCl, 1.0% Witte's peptone and
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 731
10.0% gelatin (1.0 to 2.0% glucose (6) Heat over a small flame or on a
may be added). water bath until the gelatin is com-
(7) Neutralize carefully by the addi- pletely dissolved.
tion of concentrated Na2C03 until (7) Make slightly alkaline.
litmus paper is colored violet. (8) Heat minutes at 113-114C.
for 5
(8) Add soda solution as desired. in the autoclave and filter while
Generally 10.0 cc. of 15.0% soda hot thru Chardin paper.
solution is added per liter. (9) Distribute as desired.
(9) Boil for 30 minutes in the steamer. (10) Sterilize at 112C. for 25 minutes.
(10) Filter thru paper. (i) Frost (1903).
(11) The gelatin should be perfectly (1) Remove the fat and connective
all
clear. not cool to 40C., and
If tissue from
500.0 g. beef and
mix thoroly with the white of an mince or use hamburger steak.
egg. (2) Add 1 liter of distilled water to (1),
(12) Boil again for a short time. shake thoroly and set in the ice
(13) Filter. chest for 12 to 24 hours.
(14) Distribute in tubes or flasks. (3) Squeeze thru a cloth and add
(15) Sterilize on 2 successive days in enough distilled water to make one
flowing steam for 20 minutes each liter.
day, and for 10 minutes on the (4) Add 1.0% peptone (Witte) 0.5%
third day, or for 15 minutes on NaCl and from 10.0 to 15.0% of the
each of 3 successive days. Cool best gold label sheet gelatin.
quickly after each heating. (Add 10.0% gelatin in winter and
(g) Roux (Thoinot and Masselin 1902). 15.0% gelatin in summer.)
(1) Macerate 500.0 g. of finely chopped (5) Weigh the solution and the vessel.
beef in a liter of water for several (6) Heat until solution is complete.
hours. (7) Neutralize to phenolphthalein.
(2) Pass thru a linen cloth and express (8) Boil 5 minutes and restore the
the juice from the meat. weight lost by the addition of dis-
(3) Add 10.0 g. peptone, 5.0 g. NaCl tilled water.
and 100.0 g. of light colored gelatin (9) Test the reaction, and readjust if
to (2). necessary.
(4) Heat in a water bath not above (10) Boil until the albumin coagulates
60C. to dissolve the gelatin. and floats in the clear liquid.
(5) Add soda to make slightly alkaline. (11) Filter thru cotton supported on a
(6) Heat in steam at 100 for an hour. wire using a suction pump
coil of
NaCl and from 10.0 to 15.0% of (6) Add 10.0 g. peptone and 5.0 g.
the best gold label sheet gelatin. NaCl and stir with a small wooden
(Add 10.0% gelatin in winter and spoon.
15.0% gelatin in summer.) (7) Add the gelatin and stir until
(6) Weigh the solution and the vessel. dissolved.
(7) Heat until solution is complete. (8) Make up to 1 liter by the addition
(8) Neutralize to phenolphthalein. of (4).
(9) Boil 5 minutes and restore the (9) Add KOH until the reaction is
(4) Make up the fluid to a liter. (1) Add 1000.0 cc. distilled water to
(7) Add 1.0% Witte's peptone and (5) Throw on a thick cloth and press
0.5% NaCl. the meat free from juice.
Steam for one-half hour. (6) Filter the juice thru moistened
(8)
paper.
(9) Filter.
(10) Cool. (7) Make up to 1000.0 cc.
Adjust the reaction. (8) Add 10.0 g. Chapoteaut's peptone,
(11)
(12) Steam again for 30 minutes. 5.0 g. NaCl, and 80.0 to 130.0 g.
(13) Filter.
gelatin (extra) (80.0 g. in winter
(14) Add 100.0 g. of the best quality of and 130.0 in summer) to (7).
gelatin and soak 2 hours at room (9) Heat slowly in an enamelled
temperature. kettle.
(10) After solution is complete, boil
(15) Steam 5 minutes.
(16) Cool. for 2 or 3 minutes.
Make slightly alkaline by the
(17) Titrate and adjust the reaction. (11)
(5) Sterilize for 20 minutes at llO'C. (1) Add 1000.0 cc. of water to 500.0 g.
734 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
finely chopped fat and tendon cloth and make up the volume to
free beef. 1 liter.
(2) Allow to stand in the ice box for (4) Inoculate with a culture of the
12 hours, or heat at 50 to 55 for colon bacillus.
30 minutes. (5) Allow to stand at room tempera-
(3) Heat slowly to boiling". ture over night.
(4) Boil slowly for 10 minutes stirring (6) Boil and add 10.0 g. Witte's pep-
constantly. tone and 5.0 g. NaCl.
(5) Strain thru a cloth. (7) Weigh the saucepan and contents
(6) Heat slowly (not above 80C.) and heat to 60C.
and add 20.0 g. peptone, 5.0 g. (8) Make up the loss in weight by the
NaCl and 120.0 g. of gelatin per addition of water.
liter of filtrate. (9) Neutralize to litmus,
(7) Shake until solution is complete. (v) Klimmer (1923).
(8) Add soda solution to neutralize (1) Prepare meat infusion.
to litmus. (2) Dissolve 10.0 g. peptone, 5.0 g.
(9) Heat in the autoclave at 112 for NaCl and 100.0 g.
gelatin in (1)
10 minutes. by heating between 50 and 60C.
(10) Filter thru paper. Do not heat above 60C.
(11) Distribute. (3) Neutralize to litmus.
(12) Sterilize at 110 for 10 minutes. (4) Filter.
(Do not heat over 110C.) (5) Add 5.0 cc. of normal soda solu-
(13) Store in a cool place. tion.
^t) Harvey (1921-22). by heating for 40 minutes
(6) Sterilize
(1) Proceed as in the preparation of on each of 3 successive days for
infusion broth, see variant (bb) 15 minutes in streaming steam.
of medium 779, step (1) thru (9). (w) Park, Williams and Krumwiede
(2) Add 10.0 g. peptone, 5.0 g. sodium (1924).
chloride and 120.0 g. gelatin. (1) Dissolve 100.0 g. gelatin, 10.0 g.
(3) Heat gently with constant stirring. peptone and 5.0 g. NaCl in
(4) Steam or boil 45 minutes to obtain 1000.0 cc. meat juice.
complete solution. (2) Coagulate the albumin present in
(5) Bring the volume up to 1000.0 cc. the meat juice to clarify.
by the addition of hot water. (3) The coagulation of the albumin
(6) Adjust reaction to a definite pH present in the meat juice clears
value, or faintly alkaline to litmus the medium.
or 1.0% acid to phenolphthalein. (4) Filter thru cotton.
(7) Steam 30 minutes. (5) Tube and sterilize in the Arnold
(8) Filter, while hot, thru well-wetted, sterilizer 20 to 30 minutes on 3
thick filter paper by placing filter successive days.
funnel, stand and receptacle for (x) Park, Williams and Krumwiede
filtrate in the steam sterilizer, and (1924).
steaming till filtration is complete. (1) Add "10.0 g. (1.0%) peptone and
(9) Distribute into flasks or test tubes. sodium chloride 0.5 g.," to
(10) Sterilize. 1000.0 cc. of meat infusion.
(11) Cool the flasks or test tubes on (2) Heat nearly to boiling and add
finalremoval from the sterilizer. 100.0 g. gelatin. Dissolve with
(12) Store in a cool place, as little heat as possible.
(u) Pitfield (1922). (3) Adjust the reaction neutral to
(1) Cover 500.0 g. of finely cut fat litmus (pH = 6.8 to 7.0).
free beef with 1000.0 cc. water. (4) Cool to 50C. and clarify by heat-
(2) Shake well and place on ice over ing for 45 minutes in an Arnold
night. sterilizer.
(3) Squeeze out the fluid by means of a (5) Filter.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 735
Masselin (1902 pp. 30, 32), Frost (1903 4. Gelatin (best white French) 10.0 g.
(1921 p. 125), Dopter and Sacquepee (2) Add enough KOH to neutralize to
ties of gelatin might be used, that peptone and gelatin are dissolved
the gelatin may be dissolved directly and not allowing the temperature
in the nutrient medium. to rise above 60C.
(b) Hueppe (1885) prepared the medium (7) Heat over boiling water (or steam)
any desired nutrient medium. (9) Titrate after boiling one minute
(13) Filter thru absorbent cotton and (8) To each 100.0 cc. add 3.0 cc. of a
cotton flannel, passing the filtrate normal phosphoric acid and 2.0 cc.
thru the filter until clear. of 2.0% malachite green solution.
(14) Titrate and record the final Sterilization: Not specified.
reaction. Use: Isolation of typhoid bacilli from
(15) Tube, using 5.0 cc. in each tube feces.
in the case of gelatin. References: Loeffler (1906p. 289), Klimmer
(16) Sterilize 15 minutes in the auto- (1923 p. 213).
clave at 110 or for 30 minutes in
streaming steam on three successive 2283. Roux and Rochaix's Peptone Infusion
days. Gelatin
(17) Store in the ice chest in a moist Constituents
atmosphere to prevent evaporation. 1. Distilled water 1000.0 cc.
(b) Committee A. P. H. A. (1905) pre- 2. Beef 500.O g.
pared the medium as given in variant 3. NaCl 5.0 g.
(a), but sterilized for 5 minutes in 4. Peptone 10. g.
the autoclave at 120 C. or for 30 5. Sodium or potassium
minutes in streaming steam on each phosphate 2.0 g.
of 3 successive days. Cool quickly 6. Gelatin (15.0 to
on ice following heating. 18-0%) 150.0 to 180.0 g.
(c) Giltner prepared the medium as Preparation
follows: (1) Chop 500.0 g. of fat and tendon free
(1) Prepare meat infusion. beef into very small pieces.
(2) Dissolve 1.0% peptone and 15.0% (2) Add 1 liter of water and allow to
gelatin in a mixture of equal parts stand over night in the cold.
(1) and distilled water. (3) Press the juice thru a linen cloth
(3) Adjust the reaction to +1.0%. by means of a meat press.
(4) Sterilization not specified. (4) Make up the volume to 1000.0 cc.
References: Smith (1897 p. 480), Com- by the addition of water.
mittee A. P. H. A. (1901 p. 384), (1905 (5) Add 5.0 g. NaCl, 2.0 g. of sodium or
p. 107), Giltner (1921 p. 380). potassium phosphate and 10.0 g. of
peptone.
2282. LoefHer's Malachite Green Infusion
(6) Boil for an hour.
Gelatin
(7) Filter thru a linen cloth and then
Constituents thru paper.
1. Water 5000.0 cc. (8) Adjust the reaction slightly alkaline
2. Beef 4.0 lbs. to litmus by addition of KHCO3
3. Gelatin (15.0%) 750.0 g. solution, drop by drop.
4. Peptone (Witte 1.0%) 50.0 g. (9) Add 15.0 to 18.0% gelatin and heat
5. XaCl(0.5%) 25.0 g. on the water bath until solution is
6. Malachite green solution complete.
7. Phosphoric acid (10) Neutralize and make slightly alka-
Preparation line again to litmus.
(1) Chop four pounds clean lean beef. (11) Filter using a hot water funnel.
(2) Mix 5 liters tap water with (1). (12) Distribute in flasks or tubes.
(3) Add 15.0% = 750.0 g. gelatin, 1.0% Sterilization Sterilize on 2 or 3 successive
:
Use: Cultivation of Pseudomonas cerevi- (2) Prepare a 2.0% solution of congo red.
siae. The author reported that best (3) Add 4.0 cc. of (2) to every 50.0 cc. of
growth was obtained when the reaction (1), also add 0.83% brilliant green
was 1.0% N/1 alkali. and 0.5% glucose.
Variants: The author gave the following (4) Add 25.0 cc. of bromoform to 100.0 cc.
method of preparation: of sterile distilled water.
(1) Digest 1000.0 g. of horse meat with (5) Shake thoroly and allow to stand at
1 liter of water for 24 hours in the room temperature over night.
cold. Pipette some of the saturated solu-
(6)
(2) Boil for one hour in the steamer. tion into a sterile flask (do not dis-
(3) Cool and filter. Make up to 1 liter. turb the bromoform at the bottom of
(4) Add 40.0 g. of Witte's (siccum) pep- the flask, do not include any of the
tone, 20.0 g. glucose, and 10.0 g. of globules floating on the surface).
NaCl. (This is a stock bouillon.) (7) One cc. of (6) (saturated bromoform
(5) To 500.0 cc. of (4) and 100.0 g. gelatin. solution) is added to each 10.0 cc. of
(6) Neutralize and clarify.
(3) , (congo-red-brilliant-green-gela-
(7) Dilute with an equal volume of water. tin) just before use.
(8) Add N/1 acetic acid or N/1 NaOH to Sterilization: Not specified.
obtained the desired reaction. Use: Enrichment medium for typhoid
(9) Sterilization not specified. organisms.
Reference: Fuhrmann (1906 pp. 310, 316). Reference: Teague and Clurman (1916-17
p. 125).
2287. Choquet's Glycero Phosphate
Infusion Gelatin (Besson) 2289. Kowalski's Glycerol Lung Infusion
Constituents Gelatin
1. Beef juice 300.0 cc. Constituents
2. Gelatin (extra white) 35.0 g. 1. Water.
3. Peptone (Chapoteaut) 5.0 g. 2. Lung, calf.
4. Glycerol-phosphate of lime. 5.0 g.
.
3. NaCl.
Preparation 4 Potassium phosphate.
(1) Dissolve 2, 3 and 4 in beef juice. 5. NaSOi.
(2) Heat in the autoclave. 6. (NH4)2S04.
(3) Filter hot in the autoclave or use a 7. Sucrose.
hot water funnel. 8. Peptone.
(4) Tube. 9. Gelatin (10.0 to 15.0%).
Sterilization: Not specified. 10. Glycerol (8.0 to 10.0%).
Use: Cultivation of organisms from dental Preparation
caries. Grind one kilogram fresh
(1) calf lung.
Reference: Besson (1920 p. 40). (2) Add 2 liters water. Boil in glass
vessel 30 minutes.
2288. Teague and Clurman's Congo-Red
(3) Filter, pressing the meat.
Green Glucose Gelatin
Brilliant
(4) Dissolve 3, 4, 5, 6, 7 and 8 in (3).
Constituents Add 10-15.0% gelatin.
(5)
1. Infusion gelatin (5.0%) . . . 500.0 cc. Dissolve by heat.
(6)
2. Congo red (2.0%) 40.0 cc. Neutralize with
(7) equal parts of
3. Brilliant green 0.415 g. sodium and potassium hydrate.
4. Glucose (0.5%) 2.5 g. Make to
(8) 2.5 liters with distilled
5. Bromoform water.
Preparation Cool to 58C.
(9)
(1) E.xact method of preparation of 5.0% (10) Add the beaten whites of 4 eggs.
nutrient gelatin or composition not (11) Heat a few minutes.
given. It is to be prepared however
(12) Filter.
from meat infusion, not from beef (13) Add 8-10.0% glycerol to filtrate.
extract, and the reaction to be 1.0+.
(14) Distribute in tubes or flasks.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 739
dium gave as good results as the average (11) Add 20.0 g. lactose or glucose.
grade of serum media. (12) To prepare litmus glucose gelatin
Reference: Huntoon (1918 p. 171). add 2.0% glucose and enough
Kubel-Tiemann neutral litmus to
2293. Rosenberg's Basal Extract Gelatin
color it a purple tint before steri-
Constituents lization.
1. Water 1000.0 cc. References: Rosenberg (1886 p. 452), Ban-
2. Gelatin (10.0%) 100.0 g. ning (1902 p. 426), Percival (1920 p. 47).
3. Peptone (Koch) (4.0%) 40.0 g.
2294. Burger's Basal Fuchsin Sulphite
4. Meat e.xtract (0.5%) 5.0 g.
Extract Gelatin
Preparation
(1) Dissolve 2, 3 and 4inl. Constituents
(2) Dissolve one of the added nutrients 1. Distilled water 900.0 cc.
in (1). 2. Meat extract (Liebig's) 10.0 g.
(3) Gelatin may be neutralized by the 3. Peptone (Witte Rostock) .... 10.0 g.
addition of Na^COs or used without 4. NaCl 5.0 g.
adjusting the reaction. 5. Gelatin 100.0 g.
Sterilization: Not specified. 6. NaaSOa 2.5 g.
Use: Bacterial count of water. Used also 7. Fuchsin, ale. solution 5.0 cc.
as a general culturemedium. 8. Soda (crystalline) 1.0 g.
(3) Heat in a water bath until the (8) Add 10.0 g. of one of the added nu-
gelatin is dissolved. Shake from trients either in a solid state or in a
time to time. concentrated solution.
(4) Neutralize to phenolphthalein by (9) Cool to about 40-50C.
the addition of normal caustic soda. (10) Dissolve 1.5 g. of egg albumin in
(5) Add 10.0 cc. of normal HCl to each 25.0 cc. of luke warm water, and
1000.0 cc. of the medium. add to (9). Shake thoroly.
(6) Cool the medium to 40C. and (11) Boil in the steamer for about
slowly add the white of an egg 30 minutes.
well beaten up in a little water. (12) Filter hot, thru sterile paper at a
(7) Heat in the steam sterilizer or temperature of 50 or 60C. This
water bath. may be carried out in the steamer
(8) Filter the hot solution thru after the flame has been removed.
moistened hot filter paper into a May allow to filter over night.
sterilized flask. Use a hot water (13) Distribute in 100.0 cc. lots.
funnel for filtering. (14) When ready for use, melt sterile (13)
(9) Tube in 8 to 10.0 cc. quantities. and to each 100.0 cc. add 5.0 cc. of a
(10) Steam for 20 minutes on 3 suc- saturated filtered alcoholic solution
cessive days. of fuchsin and 02.5 g. of crystalline
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 741
20.0 g. Witte's Rostock's peptone, (2) Add 10.0 cc. of a 5.0% KOH solution.
10.0 g. NaCl, 200.0 g. gelatin, 2.0 g. (3) Tinge with litmus.
crystalline soda, 20.0 g. of the added Sterilization : Not specified.
nutrients, 10.0 cc. of a saturated Use: To study fermentation.
alcoholic fuchsin solutionand 5.0 g. Added nutrients: The author added 10.0 g.
NaoSOa instead of the amounts indi- ofany desired carbohydrate, alcohol, etc.
cated. Water under investigation Reference: Buchan (1910 p. 107).
may be added to the sterile medium.
2297. Bacto Nutrient Gelatin. (Dehydrated)
(b) Burger used the following amounts
of constituents in preparing a medium Constituents:
in the same manner as above. Dis- 1. Distilled water
tilled water 750.0 cc, Liebig's meat 2. Beef extract, Bacto 3.0 g.
e.xtract 25.0 g., Witte's Rostock pep- 3. Peptone, Bacto 5.0 g.
tone, 25.0 g., NaCl, 12.5 g., gelatin 4. Gelatin, Bacto 120.0 g.
250.0 g., saturated alcoholic fuchsin Preparation
solution 12.5 cc. NasSOj 6.25 g., (1) Dissolve 128.0 g. of Bacto Nutrient
crystalline soda 2.5 g., and 25.0 g. of Gelatin (dehydrated) in 1000.0 cc.
one of the added nutrients. Water distilled water by warming.
under investigation may be added to (2) If sterilized at 15 pounds for 20 min-
the sterile medium. utes pH = 6.6.
Reference: Burger (1916-17 p. 478). Sterilization: Sterilize for 20 minutes at
15 pounds pressure.
2295. Tausz and Peter's Basal Ragit Use: General culture medium. The au-
Gelatin
thors reported that the medium con-
Constituents: forms to the "Standard Methods"
1. Water 1000.0 cc. formula.
2. Ragit powder 22.0 g. Reference: Digestive Ferments Co. (1925
3. Gelatin 100.0 g. p. 10).
742 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
2298. Heinemann's Peptone Extract Gelatin (b) Bahr reported that Gartner's bacillus
Constituents: did not liquefy a medium prepared
1. Water (tap) 1000.0 cc. as follows
2. Meat extract 2.5 g. (1) Dissolve 200.0 g. gelatin, 10.0 g.
3. Peptone 10.0 g. peptone, 20.0 g. meat extract
4. Gelatin 100.0 to 120.0 g. (Cibil's) in 1000.0 cc. of water.
(1) Dissolve 2.5 g. meat extract in dium will titrate 0.3 with N/10
1150.0 cc. of tap water. (Added NaOH using phenolphthalein as an
150.0 cc. of water to be lost during indicator.
preparation.) (3) Sterilizationnot specified.
(2) Heat and when near boiling add 10.0 g. (c) Commiteee A. P. H. A. (1917) (1920)
peptone to (1).
gave the following method of prepa-
(3) When add 10.0 to 12.0% of the
boiling ration :
best Gold Label gelatin to (2). Add (1) Add and 5.0 g.
3.0 g. of beef extract
2 or 3 sheets of gelatin at a time, peptone to 1000.0 cc. of distilled
stirring constantly. water and add 100.0 g. gelatin,
(4) When solution is complete adjust the dried for 30 minutes at 105 C. before
reaction to alkaline to litmus or weighing.
neutral to phenolphthalein and then (2) Heat slowly on a steam bath to
add 0.5% normal HCl. 65C. until the gelatin is dis-
all
(5) Cool to 60C. and stir in the white of (May be soaked for 30
solved.
one egg dissolved in 30.0 cc. water. minutes before heating.)
(6) Heat over the flame on a piece of (3) Make up lost weight, titrate and
asbestos without stirring. Heat until if the reaction is not already be-
the egg albumin is completely coagu- tween +0.5 and +1.0, adjust to +1.
lated and forms a dry film on top. (4) Filter thru cloth and cotton until
(7) Add water to make up any loss in clear.
weight over 50.0 g. (5) Distribute in tubes in 10.0 cc.
(8) Filter thru paper or absorbent cotton quantities or in larger amounts as
previously moistened with hot water. desired.
(9) Tube in 10.0 cc. quantities. (6) Sterilize in the autoclave at 15
Sterilization Sterilize in the autoclave for pounds
:
(120C.) for 15 minutes
5 minutes at 120 C. or steam for 3 suc- after the pressure reaches 15
cessive days for 20 minutes each day. pounds.
Use: General culture medium. (d) Committee S. A. B. (1918) recom-
Variants mended the same medium as Com-
(a) Wherry used the following medium to mittee A. P. H. A. (1917) but clarified
determine effect of reaction and dry- with egg and adjusted to pH between
ness on liquefaction of gelatin by 6.6 and 7.4.
Cholera spirillum.
(e) Treece dissolved 12% gelatin, 2.0%
(1) Dissolve 200.0 Gold Label gela-
g.
peptone and 0.5% meat extract in
tin, 10.0 g. Witte's peptone and
water. The medium was used to
30.0 g. Liebig's beef extract in
determine fecal and non-fecal strains
1000.0 cc. water.
of the colon-aerogenes group. The
(2) Divide (1) into two 500.0 cc. lots. author reported that this medium
(3) To one lot add NaOH so that after correlated the fermentation of ado-
sterilization the final reaction is nitol. Positive results were indi-
-1-0.8.
cated by a line of 4 to 8 bubbles
(4) To the other portion add NaOH so extending down the line of isolation.
that the final reaction after steri- (f) Committee A. P. H. A. (1923) ad-
lization is +1.0. justed the medium to a faint pink
(5) Method of sterilization not given. with phenol red or to the required
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 743
tint with brom thymol blue instead gelatin in winter and 15.0% in
of titrating to +1.0% as in 1917. summer.)
(g) Committee A. P. H. A. (1925) pre- (5) Weigh the solution and the vessel.
pared the medium as follows: (6) Heat until solution is complete.
(1) Add 3.0 g. of beef extract, 5.0 g. (7) Neutralize the phenolphthalein.
peptone and 120.0 g. gelatin (un- (8) Boil 5 minutes and restore the weight
dried market product as stored in lost by the addition of distilled water.
the ordinary cupboard) to 1000.0 cc. (9) Test the reaction and readjust if
tilled water and adjust the reaction (12) Filter thru cotton supported on a
so that after the final sterilization coil of wire using a suction pump to
the pH value will be between 6.2 hasten filtration.
(1919 p. 77), Tanner (1919 p. 55), Com- 20.0 g. peptone, 5.0 g. NaCl and 5.0 g.
5. Gelatin (Gold Label (4) Add 900 parts (3) to 100 parts gela-
sheet) (10.0 or 15.0%) . 100.0 or 150.0 g. tin and allow to soak and soften.
(3) Mix 10.0 g. of egg albumin in (4) To 900.0 g. of (2) add 100.0 g. of
100.0 cc. of water and add to (2). gelatin.
(4) Add 3.0 g. meat extract, 10.0 g. (5) Steam until solution is complete
peptone and 5.0 g. NaCl to (3). (not longer than 30 minutes).
(5) Cover the container, place in the (6) Add 4.0% NaOH solution until
autoclave and heat at about 15 blue litmus paper is no longer
pounds pressure for 45 minutes. turned red.
(6) Adjust the reaction to +1.5% by (7) Boil for 15 minutes in the steamer.
the addition of NaOH or HCl. (8) Add 1.5 g. of crystalline (not
(7) Filter while boiling thru
hot effloresced) soda per liter.
plaited filter paper, washed with (9) Boil 30 minutes.
500.0 g. boiling water. (10) Filter.
(8) Distribute as desired. (11) Tube in 10.0 cc. quantities.
(9) Autoclave for 20 minutes at (12) Sterilize by a single heating at 15
10 pounds pressure. to 20 minutes in steam.
(10) Cool in a running water bath im- References: Frost (1903 p. 6), Stoklasa
mediately, (1908 p. 489), Abel (1912 p. 19), Day and
(j) Stitt prepared the medium as follows: Walker (1912-13 p. 435), Burger (1916-17
(1) Place 3.0 g. Liebig's extract, p. 477), Meier (1918 p. 435), Besson (1920
10.0 g. peptone and 5.0 g. NaCl in p. 40), Wolf and Shunk (1921 p. 325)
a mortar. Giltner (1921 p. 35), Stitt (1923 p. 38),
(2) Dissolve the white of one or two Klimmer (1923 p. 193), Park, Williams
eggs in 1000.0 cc. of water. and Krumwiede (1924 p. 177).
(3) Add (2), little by little, to (1),
2300. Kohn's Peptone Extract Gelatin
until a brownish color is obtained.
2. Beef extract (Lemco) 5.0 g. (5) Heat in the autoclave at 115 for one
3. Peptone (Witte) 10.0 g. minute.
4. Gelatin 100.0 g. (6) Filter thru Chardin paper.
5. Urea 20.0 g. (7) Distribute in tubes.
Preparation Sterilization: Not specified.
desired carbohydrate, alcohol, etc., to (d) Roddy gave the following method of
French gelatin in 1000.0 cc. of 2. Neutral red (Sat. Aq. soln. Grubler's
bouillon. or other indicator).
(2) Allow to soak for 5 minutes. Preparation: (1) Add 4 drops of sterile
(3) Heat.
saturated watery solution of Grubler's
(4) Stir until solution is complete.
neutral red to 10.0 cc. of sterile melted
(5) Sterilize in the steamer for 15 min- gelatin at 40C.
utes on each of 3 successive days.
Sterilization: INlethod of sterilization of
(e) Bezangon prepared the medium as
gelatin or neutral red solution not given.
follows:
Use: Neutral red test for coli organisms.
(1) Dissolve 100.0 g. of gelatin (126.0
Author reported that B. coli caused a
to 150.0 g. in summer) in 1000.0 cc.
decolorization and fluorescence of this
bouillon.
Coolto50"C.
medium after 6 hours. Other investi-
(2)
gators used similar media for purposes
(3) Add the beaten white of an egg.
as indicated.
(4) Adjust the reaction to slightly
alkaline. Variants
(5) Heat for 15 minutes at 110C. (a) Calandra added 4 drops of a sterile
(6) Filter while hot. 1.0% watery solution of brilliant
Tube. cresyl blue to 10.0 cc. of sterile nu-
(7)
(8) Sterilize at 110C. for 15 minutes. trient gelatin. The gelatin was
(f) Giltner added 3.0% NaCl to nutrient colored bright green. The medium
gelatinand adjusted to 2.0%. The was used to differentiate colon bacilli
medium was used for the cultivation
from typhoid bacilli. He reported
of phosphorescent that B. coli did not cause decoloriza-
halophilic or-
ganisms. tion after 24 hours. Typhoid in con-
methyl green, 4.0 cc. of chrysoidine 2312. Wurtz's Litmus Lactose Gelatin
and 3.0 cc. of fuchsin. Constituents
(3) Add 200.0 cc. of distilled water to 1. Nutrient gelatin 1000.0 cc.
(2) and allow to stand several 2. Lactose (2.0%) 20.0 g.
hours. The color should be green- 3. Litmus
ish blue. If not obtain the origi- Preparation
nal color by adding either blue, Add 20.0 g. lactose to sterile neutral
(1)
green or red dye. nutrient gelatin.
(4) Sterilize at 100C. (2) Tube.
(5) Add drops of (4) to a tube
7 to 10 Add sufficient tincture of litmus to
(3)
of sterile melted nutrient gelatin. each tube to give a violet color.
References: Heller (1905 p. 118), Calandra (4) Pour sterile (3) into plates.
Signorelli (1912 p. 472),
(1910 p. 570), Sterilization: Sterilize (3) at 100C.
Besson (1920 p. 60). Use: Differentiation of Bad. colt. The
author reported that Bad. coli fermented
2310. Matzuschita's Glucose Gelatin lactose with the production of acid.
Typhoid bacilli colonies were colorless,
Constituents
colon colonies red.
1. Bouillon 1000.0 cc.
Variants
2. Gelatin 100.0 g.
(a) Heinemann gave the following
3. Peptone 10.0 g.
method of preparation:
4. NaCl 5.0 g.
Dissolve 1.0% lactose in ordinary
(1)
5. Glucose 20.0 g.
10 or 12.0% gelatin.
Preparation: (1) Dissolve 2, 3, 4 and 5 in
(2) Distribute in 8.0 cc. quantities in
1000.0 cc. bouillon.
tubes.
Sterilization: Not specified.
(3) Add 1.0% sterile litmus solution to
Use: Cultivation of mammalian and
each tube before using (amount of
chicken tubercle bacilli. The author re-
solution not specified).
ported that the Mammalian and chicken
(b) Abbott prepared the medium as
types gave same kind of growth. A
follows:
white precipitate was formed in the nutrient gelatin so the
(1) Prepare
liquid gelatin (at 30C.). Other in-
requires
alkalinity is such that it
vestigators used similar media for pur- 1:20 normal H2SO4
0.1 cc. of a
poses as indicated below. neutralize 1.0 cc. of
solution to
Variants Indicator not specified.
medium.
(a) Rivas solidified medium 923 by the
(2) Add 2.0 to 3.0% lactose.
addition of gelatin. This medium
(3) Decant into test tubes.
was used to cultivate anaerobes. way (method
(4) Sterilize in the usual
(b) Frost added 1.0% glucose to nutrient
not given).
gelatin.
(5) Add sufficient sterile litmus tincture
(c) Worth cultivated members of the
to each tube to give a decided but
colon-typhoid group on nutrient
not intense blue color. Add the
gelatin of a reaction 0.4% or 1.0%
litmus under aseptic conditions.
acid to which was added 2.0% glu-
References: Wurtz (1897 p. 43), Heinemann
cose. He also prepared a medium Abbott
(1905 p. 127), Tanner (1919 p. 55),
adding calcium carbonate.
(1921 p. 142).
References: Matzuschita (1899 p. 128),
Rivas (1902 p. 836), Frost (1903 p. 64), 2313. Wurtz's Glycerol Gelatin
Worth (1919 p. 604).
Constituents
1. Nutrient gelatin 1000.0 cc.
2311. Ramond's Rubine Acid Lactose 60.0 g.
2. Glycerol (6.0%)
Gelatin
Preparation: (1) Add 6.0% sterile glycerol
Same as medium 1796 but solidified by the to sterile nutrient gelatin under aseptic
addition, of gelatin instead of agar. conditions.
750 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
Sterilization: Method not given. medium was then heated in the steam
Use: Cultivation of tubercle bacilli.
sterilizer.
Reference: Wurtz (1897 p. 46). (c) Geilinger
cultivated urea splitting
2314. Holz's Phenol Gelatin organisms on nutrient gelatin con-
taining 1.0% urea.
Constituents: (d) Cunningham determined the de-
1. Nutrient gelatin.
composition of urea on nutrient
2. Phenol (5.0% solution).
12.0% gelatin containing 2.0% urea.
Preparation
The medium was sterilized by the
(1) Prepare nutrient gelatin in the usual
intermittent method in the steamer.
manner. References: Stutzer and Hartleb (1897
p.
(2) Neutralize with 10.0% NaoCOs solu-
403), Hoffman (1912 p. 387), Lohnis
tion, using litmus as an indicator. (1913 p. 95), Geilinger (1917 p. 246),
(3) Distribute sterile (2) in 10.0 cc. lots Cunningham (1924 p. 143).
by means of a burette.
(4) Heat once more. 2316. Park, Williams and Krumwiede's
(5) Add from 7 to 12 drops of 5.0% phenol Meat Gelatin
solution to each tube. (3 drops = Constituents
0.1 cc.)
1. Nutrient gelatin.
(6) Pour in plates, or prepare roll 2. Meat.
cultures. Preparation
Sterilization: Sterilize by repeated
(2) (1) Drop pieces of meat into tubes of
heatings.
nutrient gelatin.
Use: Isolation of typhoid bacilli. Sterilization: Method not given.
Variants: Migula added 0.5 cc. of a 4.0% Use: General culture medium.
phenol solution to each tube (10.0 cc.) Reference Park, Williams and Krumwiede
:
of slightly alkaline nutrient gelatin.
(1924 p. 125).
References: Holz (1890 p. 144), Migula
(1901 p. 24). 2317. Worth's Glucose Liver Gelatin
Constituents
2315. Stutzer and Hartleb's Urea Gelatin
1. Nutrient gelatin 1000.0 cc.
Constituents: 2. Glucose (2.0%) 20.0 g.
1. Nutrient gelatin 1000.0 cc 3. Liver, rabbit
2. Urea (2.0%) 20.0 g. Preparation
Preparation
(1) Prepare nutrient gelatin and add 2.0%
(1) Add 2.0% urea to ordinary nutrient glucose.
gelatin.
(2) Adjust the reaction to -f-1.0% acid.
Sterilization: Method not given. (3) Add rabbit liver.
Use: Attempt to cultivate the bacterium Sterilization: Not specified.
of foot and mouth disease. Other in-
Use: Preservation of cultures.
vestigators cultivated soil bacteria and Reference: Worth (1919 p. 604).
urea splitters on similar media.
Variants 2318. Nastinkoff's Egg Yolk Nutrient
(a) Hoffman added 1.0% urea to 10.0% Gelatin
alkaline gelatin. The medium was Same as medium 1950 but solidified with
used for differential count of bacteria gelatin instead of agar.
from soil. He reported that if urea
was fermented the colonies were 2319. Pergola's Alkaline Blood Gelatin
surrounded by a halo of characteris- Constituents
tic biscuit shaped crystals.
1. Nutrient gelatin 700.0 cc.
(b) Lohnis studied urea decomposition 2. Blood, beef 150.0 cc.
on a medium prepared by adding KOH,
3. normal 150.0 cc.
1.0 cc. of a 15.0% aqueous solution
Preparation
of urea to tubes of gelatin. This (1) Collect beef blood in a sterile flask
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 751
(2) Mix an equal volume of (1) and N/1 obtain growth. Incubate at 15 to
KOH solution. 37C.
(3) Prepare nutrient gelatin. It may be References: MuUer (1906 p. 519), Ito and
necessary to increase the gelatin Matsuzaki (1916 p. 558).
2337
Goadby's Glycerol Potato Gelatin Wroblewski's Suprarenal Capsule
Infusion Gelatin 2364
.(Tanner) 2338
Zikes' Carrot Infusion Gelatin
Harde and Hauser's Fish Infusion
2339
Gelatin 2365
D4. Non leguminous plants or their
deriva- C2. Extracts specified.
vatives other than Dj to Dj used. Molisch's Meat Extract Gelatin .... 2366
Stutzer, Burri and Maul's Glucose
Hollborn's Meat Extract Gelatin. 2367
Mustard Seed Gelatin 2340
. .
Barthel's Basal Lupini Extract (1) Dissolve 10.0% gelatin in yeast water.
Gelatin 2349 Sterilization: Not specified.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 753
(1) Add 6.0% sucrose and 10.0% gelatin (b) Vierling studied urease production
to neutral yeast water. by mycobacteria on a medium
Sterilization: Not specified. prepared as follows:
Use: Cultivation of non-spore forming (1) Boil 200.0 g. pressed yeast with
saccharomyces. 1000.0 cc. of water.
2329. Heinemann's Beer Wort Gelatin 2330. Janke's Alcohol Beer Gelatin
Constituents Constituents:
1. Beerwort 1000.0 cc. 1. Lager beer 1000.0 cc.
Boil 1000.0 g. of plums with 1000.0 cc. equivalent to 1.0 g. H2SO4 per
(1)
liter.)
of water twice.
Add 10.0% gelatin to the juice of (1) (8) Heat in the autoclave for 10
(2)
without neutralization. minutes at 110C. to clarify.
(9) Filter.
Sterilization: Not specified.
Use: Cultivation of molds. (10) Tube in 9.0 cc. lots.
(b) Migula prepared the medium as (1) Mash 500.0 peeled and washed
g. of
(2) Boil (1) with nutrient gelatin (4) Cook for one hour.
(amount not specified). (5) Add 10.0% gelatin to (4).
(3) Add 2.5 to 3.0 cc. of 0.1 normal (6) Add 2.5 cc. of 1/10 normal NaOH
NaOH to each 10.0 cc. of (2). and 1.0% KI.
(4) Filter. (7) Sterilization not specified.
(method not given).
(5) Sterilize (e) Bezan^on gave the following method
(6) When ready for use, pour into a of preparation:
flask, add 1.0% KI and the water (1) Peel and grate 500.0 g. of potatoes.
under investigation. (2) Add (1) to a liter of water and
(7) Pour into plates. allow to soak for several hours.
(c) Smith prepared the medium as (3) Allow to settle.
follows: (4) Decant and filter.
(1) Grate 500.0 g. of pared potatoes (5) Autoclave for 10 minutes.
and place the pulp in the refrig- (6) Add 150.0 g of gelatin and melt on
erator in a porcelain dish over a salt water bath.
night. (7) Adjust the reaction to a slight
(2) Express the juice from the pulp. acidity.
(3) Filter several times thru a layer of (8) Sterilize at 150C.
absorbent cotton or thru animal (9) Filter.
charcoal. (10) Flask in 100.0 cc. lots.
(9) Add 50.0 g. gelatin to 400.0 cc. of the (1) Peel clean potatoes and grind in
filtrate, and heat for 45 minutes in the grinding machine.
the steamer. (2) Press out the juice by means of a
(10) Tube in 5.0 or 10.0 cc. lots in sterile hand press.
test tubes. (3) Add bone black and heat and
(11) The medium is clear and brown. finally filter while hot.
The reaction is acid. 10.0 g. re- (4) Add 10.0% gelatin and prepare the
quires 1.6 cc. N/1 alkali to neutralize potato water gelatin in the usual
to litmus. manner. (Method not given.)
Sterilization Sterilize on 3 successive days
: (Carrots may be handled in the
for 15 minutes each day in streaming same way. Heat the carrot juice
steam. in the steamer before filtering.)
Use: Isolation of Bacillus typhosus and (d) Eisner (Tanner) gave the following
Bacillus coli communis. nethod:
Variants (1) Peel and grate 500.0 g. of potato.
(a) Holz added 0.05% phenol to inhibit (2) Grind (1) and soak in distilled
molds, liquefiers and other forms. water for 12 hours.
(b) Smith prepared the medium as (3) Strain and allow to stand.
follows: (4) Filter to clear.
(1) Grate 500.0 g. of pared potato and (5) Make up to one liter.
place the pulp in the refrigerator (6) Dissolve 15.0% gelatin in (5).
in a porcelain dish over night. (7) Adjust the reaction to acid (indi-
(2) Express the juice from the pulp. cator not specified).
(3) Filter several times thru a layer of (8) Sterilize (method not given).
absorbent cotton or thru animal (e) Eisner (Besson) prepared the medium
charcoal.
as follows:
(4) The filtrate should now be titered Thoroly
(1) wash, grate and peel
with decinormal sodium hydroxide potatoes.
solution to determine its reaction
(2) Soak 500.0 g. of (1) in a liter of
and the amount of water which will water for 4 hours.
be required to be added to reduce Pass thru a sieve.
(3)
its acidity to the standard. In
(4) Allow to settle over night.
this quantity of water is now
(5) Decant.
boiled the amount of gelatin
Make up the decanted liquid to
(6)
required to make 10.0% proportion
1000.0 cc.
of the gelatin when the potato
(7) Dissolve 150.0 to 200.0 g. of gelatin
juice shall have been added, the in (6) by heating gently.
reaction of the gelatin being
(8) Boil for several minutes.
corrected to neutral point after
(9) Add soda solution until the re-
it has been dissolved (and before
action isbut only slightly acid.
adding the potato juice) by means Heat at 115 for 5 minutes.
(10)
of normal sodium hydroxide solu-
(11) Filter thru paper, in the autoclave
tion. This done and the bulk of
or use a hot water funnel.
the fluid corrected for evaporation
(12) Tube.
to that necessary to properly
(13) Sterilize at 110 for 20 minutes.
dilute potato juice, the latter is
References: Holz (1890 p. 159), Smith
slowly added to the gelatin and
(1902 p. 78), Will (1907 p. 435), Tanner
mixed, and the whole boiled for 5
(1919 p. 60), Besson (1920 p. 40).
to 10 minutes.
(5) Filter.
2338. Goadby's Glycerol Potato Gelatin
(6) Tube.
(Tanner)
(7) Sterilize by the intermittent
method. Constituents:
(c) Zikes (Will) prepared a medium as 1. Glycerol potato broth 1000.0 cc.
follows: 2. Gelatin 100.0 g.
758 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
macerate for 2 days and then boilan 2349. Barthel's Basal Lupini Extract
hour. Pour off the liquid, filterand Gelatin
evaporate until it contains 10.0%
Constituents
dry material.
1- Water goQ.O cc,
(2) Dissolve 2, 3, 4 and 5 in 1.
2. Gelatin lOO.O g.
(3) Add 2.0 cc. of (1) to (2).
3. Sucrose 5.0 g.
(4) Neutralize by the addition of NaOH 4. Lupine extract 2.5 cc.
using litmus as an indicator.
Preparation
(5) Add malic acid to give a distinct acid
(1) Dissolve 2, 3 and 4 in 1.
reaction.
(2) Add one of the added nutrients.
Sterilization: Not specified.
(3) Tube in 10.0 cc. lots.
Use: Cultivation of nodule bacteria from
Sterilization: Sterilize the fractional
leguminous plants.
method.
Variants: Klimmer prepared the medium
Use: To study formation of bacteroids by
using citric acid to adjust the reaction
Bacillus radicicola and other legmnes.
instead of malic acid.
Added nutrients: The author used the
References: Klimmer and Kriiger (1914 p.
following materials:
258), Klimmer (1923 p. 228).
(a) 0.1, 0.3 or 0.5%, caffeine ground up
2346. Zipfel's Glucose Legume Infusion with water and gum arable.
Gelatin (b) 0.1, 0.3 or 0.5 g. guanine ground up
with a water and gum arable.
little
Same as medium 2140 but solidified with
(c) 0.1, 0.3 or 0.5 g. guanidine from a
150.0 g. gelatin instead of 30.0 g. of agar.
watery solution. Acidify slightly by
2347. Kaufman's Jequirity Seed Gelatin the addition of tartaric acid.
(d) 0.1, 0.3 or 0.5% pyridine from a
Same as medium
2132 but solidified with
watery solution.
15.0% gelatin instead of 1.5 to 2.0% agar.
(e) 0.1, 0.3 or 0.5% chinoline from an
2348. Stutzer, Burri and Maul's Glucose alcoholic solution.
Alfalfa Gelatin Reference; Barthel (1921 p. 638).
Constituents
1. Water
2350. Nastiukoff's Egg Yolk Gelatin
1000.0 cc.
2. Alfalfa 400.0 g. Same as medium 2144 but solidified with
3. Glucose 20.0 g. 80.0 to 100.0 g. gelatin instead of 15.0 to
4. Gelatin 100.0 g. 20.0 g. agar.
5. Na2C03 1.0 g.
Preparation
2351. Deycke and Voigtlander's Albuminate
(1) Pour 1 of water over 400.0 g.
liter
Gelatin
green alfalfa without roots.
(2) Add 20.0 g. glucose. Same as medium 2034 but solidified with
(3) Steam for 90 minutes. gelatin instead of agar.
(4) Filter.
(5) Add 100.0 g. gelatin to the filtrate
2352. Raskin's Whey Albumin Gelatin
and proceed to prepare a sterile medi- Constituents
um in the usual manner. (Method 1. Whey 1000.0 cc.
not specified.) 2. Albumin 100.0 g.
(6) Do not neutralize but add 1.0 g. 3. Gelatin 60.0 to 100.0 g.
Na2C03 (Reaction slightly acid). Preparation
(7) Tube in 8.0 cc. lots. (1) Stir thoroly with a glass rod, the
Sterilization Not specified
: albumin of fresh eggs in a flat bot-
Use: Cultivation of Bacillus radicicola tomed dish, and add, drop by drop,
and other nodule bacteria. concentrated NaOH until a solid
Reference: Stutzer, Burri and Maul (1896 opaque gelatinous material is
p. 666). obtained.
760 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Cut this solid egg albumin in small 2354. Raskin's Whey Gelatin
pieces and place in distilled water.
Same as medium 2161 but solidified with
Do not allow the alkaline albumin to 12.0% gelatin instead of 1.75% agar.
stand or it will liquefy in several
hours.
2355. Reinsch's Fat-free Milk Gelatin
(3) Shake thoroly and pour off the water.
Continue this washing until the wash Constituents
water is only slightly alkaline. 1. Water 100.0 cc.
(4) Place the coagulated albuminate in a 2. Milk 500.0 cc.
flask of distilled water, plug with 3. Gelatin 20.0 g.
cotton and heat in the steamer for 15 Preparation
to 30 minutes. This dissolves the (1) Dissolve 20.0 g. of gelatin in 100.0
albumin. cc. water.
(5) Filter. This is to be a saturated (2) Place 500.0cc. of fresh cow milk in a
(3) Boil until the casein is completely all the ether is evaporated.
coagulated. (9) Add about 0.2% NaOH in order that
(4) Strain thru a linen towel. the acid gelatin does not cause a
(5) Pour in a suitable container so that precipitate of casein.
the fat may raise. (10) Add two to three parts of (9) to one
(6) Remove the fat that comes to the part (1).
2353. Giltner and Ludlum's Amniotic Fluid 2356. Abbott's Milk Gelatin
Gelatin
Constituents
Constituents
1. Milk 1000.0 cc.
1. Amniotic fluid.
2. Gelatin (10.0 to
2. Gelatin.
12.0%) 100.0 to 120.0 g.
Preparation
Preparation: (1) Solidify milk by the addi-
(1) Solidify amniotic fluid with gelatin.
tion of 10.0 to 12.0% gelatin.
Sterilization: Method not given.
Use: General culture medium. Sterilization: Not specified.
Reference: Giltner and Ludlum (1916 pp. Use: General culture medium.
91-92). Reference: Abbott (1921 p. 139).
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 761
Constituents
2358. Raskin's Casein Whey Gelatin
Meat Infusion
1. 1000.0 cc.
(Peifer)
2. Gelatin (10.0%) 100.0 g.
Constituents Preparation
1. Water 150.0 cc. (1) Prepare meat infusion.
2. Casein (8.0%) 12.0 g. (2) Dissolve 10.0% gelatin in 1 by boiling
3. Whey 350.0 cc. over a free flame.
4. Gelatin 42.0 g. (3) Add soda solution until the reaction
Preparation is only slightly alkaline.
(1) Prepare an 8.0% solution of pure (4) Filter.
fat-free casein. Sterilization: Method of sterilization not
(2) Prepare a filtered mixture of whey given.
with 12.0% gelatin. Use: Isolation and cultivation of Spirillum
(3) Mix 150.0 cc. of (1) with 350.0 cc. of undula majris. Also used by other
(2). investigators in making bacterial counts.
(4) Heat to 60C. for 15 minutes. Variants
(5) Distribute into sterile containers. (a) Fuller and Johnson made bacterial
Sterilization : Not specified. counts in water analysis on a medium
762 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
prepared by dissolving 120.0 g. of (3) Add (2) to moistened linen and press
gelatin in 1000.0 cc. of meat infusion the fluid thru.
(contains no peptone or salts), (4) Prepare a 20% watery gelatin solution
(b) Meier used the following preparation containing 1.0% NaCl.
for the bacterial count of milk: (5) Mix equal volumes of (4) and sterile
(1) Boil 500.0 g. of fat and tendon free (3).
(1) Exact method of preparation of meat (7) Sterilize. (Method not given.)
infusion not given. To be prepared (8) Filter 2 or 3 times to clarify.
2363. Kopp's Thyroid Infusion Gelatin 2365. Harde and Hauser's Fish Infusion
Gelatin
Constituents
1. Water 1000.0 cc. Same as medium 1235 but solidified with
2. Thyroid glands, sheep 500.0 g. 15.0% gelatin.
3. Gelatin 100.0 g.
2366. Molisch's Meat Extract Gelatin
4. NaCl 5.0 g.
Preparation Constituents
(1) Free sheep
thyroid glands from fat 1. Water 1000.0 cc.
and chop the glands into small pieces. 2. Meat
extract 0.5 g.
Schluter's Basal Isinglass Medium 2376 . 5.0% "caragheen" without any addi-
Marpmann Peptone Plant Tissue tion of nutrients supported the
Medium 2377 growth of parasitic and saprophytic
Matzuschita's Peptone Konbu fungi and air forms.
Medium 2378 Reference: Lehmann (1919 p. 426).
Preparation
Sterilization: Sterilize by the intermittent
ripe pieces of cartilage or ear
method.
(1) Boil
Use: To study fat decomposition. Author
muscle in water under an atmosphere
reported that other kinds of fat might be
pressure in an autoclave, or in small
similarly treated.
amounts in a pressure flask which
Reference: Giltner (1921 p. 365).
stand in 100.0% solution of Na2S04.
(2) Filter thru a filter paper.
Sterilization: Not specified. 2375. Mortensen's Gum Arabic Media
Use: Substitute for gelatin. Mortensen's solidified media 207, 329 and
Reference: Marpman (1891 p. 123). 560 with gum arable (method not given.)
765
766 CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS
(2) Dissolve 3 and 4 in (1). (3) Boil for one hour in the steamer
(3) Dissolve one of the added nutrients (4) Filter.
in (2) in any desired concentration. Add 3 and
(5) 4 to the filtrate.
(4) Distribute into test tubes, plug and (6) Boil.
slant.
(7) Neutralize (indicator not specified).
Sterilization: Method not specified. Sterilization: Sterilize in the steamer on
Use: To study growth of bacteria on acid from 2 to 5 successive days for 15 to 30
media. minutes. Incubate for 2 days at 37C. to
Added nutrients: test sterility.
Lactic acid Acetic acid Use: Cultivation of spore forming bacilli,
Tartaric acid HCl Clostridium hutyricum, Bacillus oedma-
Citric acid Alum tis maligni, Bacillus anthracis sympto-
Reference: Schluter (1892 p. 592). matici, Bacillus sporogenes, Bacillus
2377. Marpmann's Peptone Plant Tissue hotulinus. The Konbu is a Laminaria
likeJapanese sea weed.
Medium
Reference: Matzuschita (1902 p. 2S7).
Constituents
1- Water 700.0 cc. 2379. Puccinelli's Infusion Fucus Medium
2. Glycerol 40.0 g.
Constituents
3. Sphaerococcus confervoides . . 30.0 g.
4. Peptone (liquid Koch)
1. Meat infusion 200.0 g.
20.0 g.
2. Fucus crispus 6.0 g.
Preparation
Preparation
(1) Macerate 30 parts Sphaerococcus
(1) Method of preparation of meat
confervoides with 2 parts HCl and 1
infusion not given. Neutralize.
literwater for two hours.
Wash with water until the washings (2) Wash well, 6.0 g. of Fucus crispus in
(2)
water.
no longer turn blue litmus paper red.
(3) Boil (2) for one hour in (1) in a water
(3) Add to the residue of (2) 700 parts
bath or steamer.
water, 40 parts glycerol, 20 parts
peptone (liquid Koch) and 2 parts (4) Filter thru a funnel heated by a
flame or water funnel.
beaten egg-white.
(5) Distribute in tubes and sterilize.
(4) Boil for 20 minutes in a steam
cylinder.
Sterilization : Method not given.
Use: General culture medium.
(5) Neutralize.
Reference: Puccinelli (1890 p. 405).
(6) Filter thru a syrup filter, prepared
from a balloon flask with a filter
2380. Jacobi's Peptone Fucus Medium
layer of buck-skin and felt.
Sterilization: Not specified. Constituents
Use: General culture medium. Agar 1- Water 1500.0 cc.
substitute. 2. Peptone (Kemmerich's) .... 7.5 g.
Reference: Marpmann (1891 p. 123). 3. Peptone (siccum) 15.0 g.
4. Fucus 37.5 g.
2378. Matzuschita's Peptone Konbu
Preparation
Medium
(1) Dissolve 2, 3 and 4 in 1 by heating
Constituents over a free flame.
1. Water 1000.0 cc. (2) Make up to the original volume by
2. Konbu 100.0 g. addition of water.
3. Peptone, Koch 10.0 g. (3) Press thru a towel, for some of the
4. Glucose 20.0 g. fucus will not dissolve.
Preparation Add Na2C03
(4) or sodium phosphate
(1) Cut 100.0 g. of dry Konbu in small until the reaction is slightly alkaline.
pieces and mix with one liter of water. Pour into a
(5) flask and steam until the
(2) Allow to stay in the water for 24 albuminous material still to be
hours in the cold. removed, is completely separated.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 767
(Usually two hours if sodium phos- (7) Pour into a flask and steam until the
phate is used and longer if Na2C03-) albuminous material still to be
(6) Filter thru cotton, using compressed removed is completely separated.
air to effect a fast filtration. (Usually 2 hours if sodium phosphate
Distribute into small sterile flasks. is used and longer if Na2C03.)
(7)
Finally distribute sterile (7) into (8) Filter thru cotton, using compressed
(8)
sterile test have been
tubes that air to effect a fast filtration.
autoclaved for 2J hours. (The tubes (9) Distribute into smaller sterile flasks.
(10) Finally distribute sterile (9) into
are contained in an enamel container
in the autoclave. The inner tem- sterile tubes that have been
test
(6) Add Na2C03 or sodium phosphate Use: Isolation and cultivation of amoeba.
until the reaction is slightly alkaline. Reference: Celli (1896 p. 537).
GROUP V. INITIALLY LIQUID MEDIA BECOMING PERMA-
NENTLY SOLID. SOLIDIFYING AGENT ORGANIC
Key to the Subgroups of Group V 2383. Matzuschita's Basal Rice Flour
Complete Media .L
Contain- that best growth of mammalian type on
ing Materials of Plant Origin, glycerol medium. Its colonies were
Initially Liquid but Becom- orange yellow. Chicken type gave whit-
ing Permanently Solid ^^^ S!'^^' grayish black or yellowish
colonies.
Ai. Additional materials, if any, of known Added nutrients: The author added 2.0%
chemical composition. glucose or 6.0% glycerol.
Matzuschita's Basal Rice Flour Variants:
Medium 2383 (a) The author used the basal medium
Hefferan's Starch Medium 2384 without any additions.
Lloyd, Clark and McCrae's Gluten (b) Buchanan cultivated Monascus pur-
Medium 2385 purens from silage on a medium
Baginsky's Basal Starch Medium. 2386 . .
prepared from a thick paste prepared
Uschinsky's Asparaginate Starch from rice flour and tap water.
Medium (Smith) 2387 References Matzuschita (1899 p. 128),
:
(9) Allow the starch to stand in the bran with 10.0 g. of water in a 400.0
icebox for about a week in distilled Erlenmeyer flask.
cc.
water (3 liters per jar or beaker). Reference: Kita (1913 p. 446), (1914
p. 354).
Siphon off the water twice a day at
first, and afterwards once a day, 2390. Kita's Rice Medium
stirring up the starch thoroughly Constituents
each time fresh water is added. 1. Water.
(10) Drain the starch very free from 2. Rice.
water, scoop up with sterile spoons Preparation
or spatulas and place in sterile (1) Prepare one of the following com-
petri dishes. binations:
(11) Dry in the blood serum oven at (a) 100.0 g. rice + 100.0 g. water.
56C., the cover being raised on (b) 25.0 g. rice + 10.0 cc. water.
corks an inch to let the moisture (c) 10.0 g. rice + 5.0 cc. water.
out. (d) 100.0 g. rice + 50.0 cc. water.
(b) Smith prepared a similar medium as (2) Place in Erlenmeyer flasks.
follows Sterilization: Not specified.
(1) Dissolve 30.0 g. to 40.0 g. glycerol, Use: Cultivation of Japanese molds, As-
5.0 to 7.0 g. NaCl, pergillus Okazaki, Aspergillus
0.1 g. CaCl., candidus,
0.3 to 0.4 g. MgS04, 2.0 to 2.5 g.
Aspergillus albus, Aspergillus tamarii
K2HPO4, 6.0 to 7.0 g. ammonium Pseudorhizopus Aspergillus glaucus and
lactate, to other molds.
3.0 4.0 g. sodium
asparaginate in 1000.0 cc. of
Variants The author steamed 25.0 of rice
water. :
g.
The glycerol may
be omitted. with 15.0 cc. of water in a 400.0 cc. Erlen-
(2) Add 1.0 g. of potato starch to each meyer flask.
10.0 cc. of (1) in a test tube, and Reference: Kita (1913 p. 446), (1914 p. 353).
proceed as above.
2391. Kita's Soy Bean Medium
References: Smith (1899 p. 102), (1905 p.
196), Tanner (1919 p. 61).
Same as medium
2390 but the author used
fat-free soy bean flour instead of rice.
(2) Distribute as a layer in test tubes (3) Mix (1) and (2) thoroughly (amounts
or flasks. not specified).
Heat over boiling water to solidify (4) Distribute in petri dishes.
(3)
the paste. Sterilization: Sterilize in streaming steam.
Proco, et al.,
Lorrain Smith's Alkali Serum Pergola's Egg Yolk Serum Medium. 2426
(Harvey) 2400 Greenspon's Glucose Citrate Serum
Duval's Trypsinized Serum 2401 Medium 2427
Carrel and Burrows' Plasma Me- Duval's Egg Serum Medium 2428
dium 2402 Eberling's Serum Tissue Plasma
Ball's Coagulated Blood Medium. . . 2403 Medium 2429
(4) Place in a cool place for 24 hours. (3) Close the test tube with a rubber
(5) Transfer the separated serum with a cork.
sterile pipette in quantities of 5.0 (4) Heat one hour at 60C. in a water
cc. to sterile test tubes. bath.
(6) Sterilize 30 minutes at 58C. on (5) Heat, 24 hours later one hour at
each of 8 successive days or 20 70C. in a water bath.
minutes at 100C. three days. (6) Heat, 24 hours later at 70C. until
(7) Test sterility by incubation for 48 the medium becomes syrupy.
hours. (7) Keep in the ice chest till required
(n) Harvey. for use.
(1) Coagulate serum in a slanting (2) Remove a small portion of the lung
position in an inspissator or over of an infected rat under sterile
steam at temperatures varying precautions.
from 65 to 90C. according to the (3) Add 0.5 cc. of sterile calf serum
degree of transparency required. water to the water of condensation.
(2) Add 0.85 sterile salt solution to each (4) Push the piece of tissue down the
test tube, slope to cover the tube and into the liquid at the
medium. bottom.
(3) Sterilize 60 minutes at 115C. (5) Seal the tubes with sealing wax.
(4) Pour off the salt solution at the time (t) Vardon prepared a desiccated
of use. medium as follows: He reported that
Note: The addition of salt solu- the dried serum when dissolved in
tion allows for satisfactory sterili- water and brought to its original
zation and keeps the medium moist volume can be used for all purposes
till required, where serum is used in media.
(p) Harvey cultivated spirochaetes on (1) Measure out a quantity of serum.
the following medium: (2) Pour into shallow trays.
(1) Fill the serum into tall test tubes. (3) Dry at a temperature not exceeding
(2) Heat in the upright position at 50C.
65C. Note: Coagulation of the serum
(3) Remove as soon as the serum begins should not occur in the process
to set. of drying. The drying process may
Note: The heat retained in the be continued overnight by placing
tube will complete the coagulation. the trays in a large incubator.
A soft, almost transparent coagu- To use powder.
lum is formed. (4) Dissolve by shaking the requisite
(q) Harvey prepared a medium as follows amount, calculated from amount
for the cultivation of spirochaetes: of dry material obtained from the
(1) Prepare: horse serum 3; distilled liquid, of (3) in cold water.
water 1. (5) Distribute when dissolved in
(2) Distribute in quantity to nearly amounts of about 7.0 cc. into test
fill test tubes. tubes.
776 CULTURE MEDIA FOR CULTIVATION OF
MICROORGANISMS
(3) Coagulate in the autoclave for 75 Sterilization: Tube and sterilize the m
minutes at 75 to 80 or in a hot air steamer for an hour on three successive
oven at the same temperature and days at a temperature of from 180
time. to
190F.
Sterilization: Method of sterilization of Use: Show presence of B. diphtheriae
glucose, litmus or in
H2SO4 solutions not throats. Author reported that
B. diph-
given.
theriae always gave a characteristic
Use: Diagnosis of diphtheria. red
Author re- colony. He recommended that
ported that diphtheria colonies were red the me-
dium be used in the discharging
Variants: Harvey prepared of
the litmus patients.
tincture as follows:
Reference: Rankin (1912 p. 63).
(1) Grind up litmus in a mortar.
(2) Add 5 volumes 90 per cent alcohol. 2407. Dubois' Glycerol Glucose
Serum
(3) Boil on a water bath.
Constituents:
(4) Decant the supernatant fluid. 1- Serum 100.0 cc.
(5) Add 6 parts distilled water to the 2. Glucose 7.0 g
residue. 3.Glycerol 2.O g^
(6) Boil.
Preparation:
(7) Allow to cool. Tube
(1) non-sterilized serum containing
(8) Divide into two portions.
7 parts glucose and 2 parts glycerol
(9) Render one portion slightly red with per 100 in 2.0 cc. quantities.
dilute sulphuric acid.
(2) Solidification not specified.
(10) Add to this reddened portion the Sterilization Not specified.
:
7. Glycerol 2.0 g
Constituents
1000.0 cc Preparation
1. Blood serum 6 and 7 in 1,
^OS- (1) Dissolve 3, 4, 5,
2. KHoPO^ 80.0 cc. of serum.
20.0 g. (2) Add
3. Glycerol
(3) Solidify (method not given).
Preparation: Method not given.
Sterilization:
(1) Add 0.5% KH2PO4 to blood serum.
Use: Cultivation of tubercle bacilli.
Add 2.0% glycerol to (1).
(2)
Variants: The author omitted the MgS04,
Solidify (method not given).
used 0.2 g. (NH4)2S04 instead of 0.3 g.
(3)
Sterilization: Method not given.
of tubercle bacilli. and added 0.6 g. mannitol.
Use: Cultivation
bacilli Reference: Ficker (1900 p. 510).
Author reported that the tubercle
grew without the glycerol. In concentra- 2411. Proca, et al., Pyrogallic Acid Serum
checked
tion higher than 2.0% glycerol
Constituents
the growth.
1. Distilled water.
Variants
2. Serum.
(a) Thoinot and Masselin prepared a
3. Pyrogallic acid.
similar medium
as follows
Preparation
(1) Autoclave
glycerol at 115C. and
pyrogallic acid
parts by weight of (1) to (1) Dissolve 1.0 g.
(2) Add 6 to 8 NaOH in 100.0 cc. distilled
2.0 g.
100 parts of sterile serum.
water.
(3) Mix well.
serum (beef or horse) add
(2) To 10.0 cc.
(4) Tube.
l.Occ. of (1).
(5) Solidify at a
temperature between
80.
(3) Coagulate at
75 to 86C. serum in the
similar mediiim by Sterilization: Sterilize the
(b) Harvey prepared a
100 steamer using Tyndall's method.
adding 7 parts sterile glycerol to spirochetes.
and Use: Cultivation of syphilus
parts sterile ox serum, tubing
Variants: Harvey specified that the pyro-
coagulating in the steamer or inspis- stored several
gallic acid solution be
sator at 65 to 90C.
weeks before and inoculated the
use,
(c) Harvey gave the
following method of
solidified medium by passing
a pipette
preparation: the
glycerol and 95 parts ox containing a culture material between
(1) Mix 5 parts
medium and the wall of the tubes, seal
serum. incubate.
the tubes hermetically and then
(2) Heat 30 minutes at 56C. in a water (1912
References Proca, Danila and Stroe
bath on each of two successive
:
(9) At the time of the fourth sterilization ^^ ?" '^^^*"'' ^^^ ^'^
l" '''t
solidify
the serum by gradually "' "^^^ Potassium tellurite
g'j^.f
^ ^^"
raising the temperature to 70
C fr,\ d^
You can obtain three different degrees ^^ '"^
^^*" "^'^^^^ ^^^^ bibulous
of hardness of the serum. By heating Paper mside covers.
at a low temperature one obtains ^'^^ Solidify at 85-90C.
a
soft, clear and transparent medium. Sterilization: Sterilize
by repeated heating
By heating at a high temperature one (temperature not given),
obtains a hard solid nearly opaque ^^e: Isolation of diphtheria bacilli,
medium. Then there is a medium Author reported that diphtheria colonies
between the two extremes. were coal black due to the reduction
of
Sterilization: Sterilize (5) by the fractional tellurium.
method in the steamer. Further sterili- Reference: Conradi and Troch (1912
zation
pp
is obtained in step (7) under the 1652-1653).
preparation.
Use: Cultivation of spirochetes. 2414. Hall and Stone's Glycerol Serum
Variants Futaki, et al., studied the motility
:
Medium
of Spirochaetamorsus niuris (cause of rat
bite fever) on a medium Constituents:
prepared as
follows: 1- Water 1000.0 cc.
(1) Shake 0.5 to 0.75 g. of sodium nucleate ^- ^^Ptone (Witte) 10.0 g.
with 100.0 cc. horse serum until the ^- ^^^^ 5.0 g.
nucleate has dissolved. * Glucose lO.o g.
(2) Pass CO2 thru the medium for 2 or 3 ^- ^^y^^rol 5O.0 g.
minutes until the medium is trans- ^' ^^"' blood 3000.0 cc.
parent. Preparation:
(3) Heat on three successive days for ^^^ Dissolve 2, 3, 4 and 5 in 1.
about an hour at 60C. (2) Mix one part of (1) with 3 parts of 6.
(4) On
the 4th day heat to 65C. for 30 ^^^ Filter thru Berkefeld filter,
minutes, when a fluid and coagulated ^^^ ^ube.
portion is formed. (5) Slant.
References: Shmamine (1912 p. 313), Futaki, Sterilization: Sterilize in autoclave at 10
Takaki, Taniguchi and Osumi (1917 pounds pressure for 30 minutes.
p.
^'- Use: Cultivation of diphtheroid bacillus
2413. Conradi P^eisz-Nocard from equine and bovine
and Troch's Extract Broth f
abscesses. Author reported that several
Serum Medium
strains of B. diphtheriae grow on
^ ^. will
Constituents: this medium.
^' ^^^^^'^
1000-0 cc. Reference: Hall and Stone (1916 p. 196).
MICEOOEGANISMS 781
CCLTOEE MEDIA FOB CULTIVATION OF
following coagulation is rec-
Infusion Serum minutes
2415 Greenspon's Veal
(<zr,u\ ommeneded.
^"^ ^ Use: Cultivation of diphtheria
bacilli.
^ ^^^
"
1.0 cc.
2. Sodium citrate (50.0% soln.) .
Blood
2417. Loeffler's Glucose Infusion
3 Serum, pig, sheep or
human.. 75.0 cc.
^ .. Serum
Preparation
(1) Add 1.0 cc. of
a 50.0% sodium citrate Constituents:
solution to 75.0 cc. of clear pig,
sheep 300.0 cc.
^ g^^^^ ^^^^.g^)
human serum and sufficient glucose 100.0 cc.
or ^ Meat infusion
veal infusion broth, or glucose
meat 1-0 S-
^ Peptone
extract broth to bring the
volume to 0.5 g.
^ ^^^^
100.0 cc. 5. Dextrose 10 g.
of prep-
(Dehydrated) gested the following methods
^ i-i *. aration.
Constituents:
hel (1896)
1. Distilled water infusion in the usual
300.0 cc P^^^ m;at
^''\l)'prepare
2. Serum, beef blood. _
(
^^^^^^ ^^^ ^.^^^^^
Glucose infusion broth 100.0 cc.
3.
^.^^^^^^ ^^ ^ ^^^^^^^^ ^^ ^
Preparation: glucose in 100.0
(^^) Dissolve 80 g. of
.
.
Bacto t
Loeffler's m ' NaCl and 10
JNa^^^ anu i.u g.
g. g,
SI!
re\ttl?in'r:,anteci position (4, ^t^ts <3, wit. one pa.
in Koch's apparatus,
(5) Solidify
<4, Sot rilTaTand t.e door of the
autoclave before turning on the
,,, , ^-ff/X
(WO^
steam. Run the pressure to 15 O) Smr^^h ^
Preparation
pounds as quickly as possible and (1)
glucose to (I,,
llZt (2, i'dl"l.O of
Stprilization-
::"sur'e^r20^.::;les"o:to 5
rrrsreforeo minute.
over 15 pounds pressure.
When coagulation
Bo
is
not use
com-
C3, -- -fe
j-^
conditions
^ u^dr alt:
^.
^
Coagulate in the inspissator.
;;:t open lowest'part and replace
le (4)
(4) aqueous)
If bloody, place on ice and
allow
(5) preparation:
the corpuscle to settle out. Method of preparation of bouillon
^^^
(6) Mix 3 parts beef or sheep serum ^^^ gi^gn
and 1 part nutrient broth (neutral ^2) Mix 3 parts good blood serum of the
to litmus at a pH = 6.8 to 7.0) to
^^^^p ^^^ i part (1).
which 1.0% dextrose has been and 5 to (2).
(3) Add 3, 4
added. (4) Tube.
(7) Tube. Sterilization: Coagulate
and sterilize
4. NaCl 10.0 g.
5. Serum (beef or hog) 2423. Picker's Brain Infusion Serum
Preparation Medium
(1) Cut lean beef into small pieces in a
Constituents:
meat cutting machine. Distilled water 500.0 cc.
1.
(2) Add a double weight of distilled
2. Brain 500.0 g.
water to (1).
3. Serum 500.0 cc.
(3) Boil for i hour, stirring continually 30.0 cc.
4. Glycerol
with a glass rod.
Preparation
(4) Make up the loss of water and filter
(1) Pass fresh brain (500.0 g.) thru a meat
thru a filtering cloth.
chopping machine.
(5) Add 1.0% peptone (siccum), and
(2) Add an equal weight of (500.0 g.)
0.5% NaCl. distilled water.
(6) Boil. and heat slowly to a boil.
(3) Stir
(7) Make up the volume of water lost.
Boil slowly for i hour.
(4)
(8) Cool (in a closed container) and
(5) Filter thru a filtering cloth until the
filter.
filtrate assumes a pulpy character.
(9) Distribute in 300 to 500.0 cc. portions
Press the coagulated mass free from
in clean flaskswith patented sealers.
liquid.
(10) Sterilize in streaming steam for one
(6) Mix equal parts of sterile (5) and
hour.
serum.
(11) Bring to boil on a concentrated salt
(7) Add 3.0% glycerol.
solution bath and boil for 45 minutes
(8) Tube.
shaking often.
(9) Solidify in a serum oven.
(12) Take 30.0 cc. of (11), add a drop of
Sterilization: Steam the filtrate from (5)
alcoholic phenolphthalein solution
for two hours. Sterilization of other
and add N/1 sodium solution until a
materials not specified.
red coloration is formed.
Use: Cultivation of tubercle bacilli.
(13) Estimate the amount of (11) and
calculating from (12) add I to f the Author reported that beef lungs, testicles,
(4) Sterilize for two hours (temperature 2426. Pergola's Egg Yolk Serum Medium
not given).
Constituents:
(5) Add an equal volume of sterile solu-
1. Ox serum 50.0 cc.
tion of 3.0% glycerol in serum to (4)
2. NaCl (0.8% soln.) 50.0 cc.
under aseptic conditions.
3. Potassium tellurite (1.0%
(6) Mix thoroly.
soln.) 2.0 cc.
(7) Solidify quickly.
4. Egg yolk 1
References: Ticker (1900 p. 593), Harvey
Preparation: Details of preparation not
(1921-22 p. 98), Klimmer (1923 p. 224).
given in the abstract.
2424. Picker's Potato Juice Serum Medium Sterilization: Not given.
Use: Diagnosis of diphtheria.
Constituents
Reference: Pergola (1918 p. 101 taken
1. Potato juice 100.0 cc.
from 1919 p. 57).
2. Serum (beef) 200.0 cc.
3. Glycerol (3.0%) 6.0 cc.
2427. Greenspon's Glucose Citrate Serum
Preparation
Medium
(1) Exact method of preparation of
potato juice not given. Constituents
(2) Mix one part (1) with two parts beef 1. Glucose veal infusion broth
serum. to 100.0 cc.
(3) Add 3.0% glycerol. 2. Serum, pig, sheep or human . . 75.0 cc.
(4) Reaction may be neutral or slightly 3. Sodium citrate (50.0% soln.) 1.0 cc.
thor reported that best growth was ob- veal infusion broth to bring the
tained on neutral or slightly alkaline volume to 100.0 cc.
medium. (2) Adjust to pH 6.4 by the addition of
Reference: Ficker (1900 p. 509). 3.0% citric acid.
(3) Tube.
2425. Vannod's Nutrose Serum Medium (4) Coagulate in the Koch inspissator.
Constituents Sterilization: Sterilize the fractional
1. Distilled water 40.0 to 50.0 cc. method on each of three successive days.
2. Serum (hog) 15.0 cc. Use: Isolation and cultivation of diph-
3. Glycerol 3.0 cc. theria bacillus.
4. Nutrose 1 .0 cc. Variants: Beef extract broth may be used
Preparation instead of veal infusion.
(1) Obtain hog blood from the abattoir. Reference: Greenspon (1922 p. 32).
(Authors have kept tissue cultures from Corper's, et al.. Gentian Violet Egg
the testicles and brain of guinea pigs in Medium 2447
active growth for four to six generations Twort's Bacteria Infusion Egg
over a period of four to six weeks). Use Medium 2448
guinea pig brain for growing typhus Harvey's Trypsinized Heart Egg
organism and guinea pig testicles for the Medium 2449
cultivation of Rocky Mountain Spotted Rosenthal and Schulz's Meat In-
Fever microorganism. For injection into fusion Egg Medium (Zimmer-
animals the tissue cultures are removed mann) 2450
from the coverslips, placed in Ringer's by egg white.
A2. Solidified
solution and teased into pieces small Thoinot and Masselin's Egg Albumin
enough to pass thru a number 18-gauge Medium 2451
hypodermic needle. Barthel's Egg Albumin Medium 2452
Reference: Wolbach and Schlesinger (1923, Dal Pozzo's Transparent Egg
p. 233). Albumin 2453
Rosenthal and Schulz's Alkaline Egg
SUBGROUP V-C Albumin (Zimmermann) 2454
Barthel's Milk Egg Albumin
Solidifying Agent Egg or Egg Medium 2455
Derivatives, etc. Brown and Orcutt's Veal Infusion
Egg Albumin Medium 2456
Basal or Complete Media Solidified by
A3. Solidified by yolk of egg.
Materials other than Blood or its Deriv-
Pergola's Tellurite Egg Yolk
atives (Egg or Egg Derivatives,
Medium 2457
etc.) Initially Liquid but Becom-
Smith's Egg Yolk Medium 2458
ing Permanently Solid
McCoy and Chapin's Egg Yolk
Ai. Solidified by whole egg. (Francis) 2459
Bi. Additional constituents, if any, of Dorset's Egg Yolk Medium (Heine-
known chemical composition. mann) 2460
Dal Pozzo's Lapwing Egg Medium . 2431 Nastinkoff's Egg Yolk Medium
Ball's Boiled Egg Medium 2432 (Rechtsamer) 2461
Abel's Egg Medium 2433 Dal Pozzo's Egg Yolk Medium 2462
Smith's Egg Medium 2434 Lubenau's Bouillon Egg Yolk
Dorset's Egg Medium (Heinemann) 2435 Medium (Schoenburg) 2463
Modified Dorset's Egg Medium Phisalix's Potato Egg Yolk Medium 2464
(Brown and Smith) 2436 A4. Solidified by other materials.
Dorset's Egg Medium (Abel) 2437 Sputum Medium
Steffen's 2465
Soparkar's Glycerol Egg Medium... 2438 McCann's Ovarian Cyst Fluid
Lubenau's Glycerol Egg Medium Medium 2466
(Abel) 2439
2431. Dal Pozzo's Lapwing Egg Medium
Twort and Ingram's Basal Glycerol
Egg Medium 2440 Constituents
Bj. At least one additional constituent of 1. Water 1000.0 cc.
unknown chemical composition present. 2. Egg (lapwing) 100.0 cc.
Roddy's Glucose Bouillon Egg Preparation
Medium 2441 (1) Wash lapwing's eggs with a 1% sub-
Putnam and Gay's Milk Egg limate solution. (One egg will make
Medium 2442 about 4 or 5 tubes of medium.)
Petroff's Gentian Violet Egg (2) Open the egg under aseptic conditions
Medium 2443 and allow the white of the egg to flow
Harvey's Sperm Egg Medium 2444 into a sterile flask.
Harvey's Olive Oil Egg Medium 2445 (3) Mix the egg albumin with its volume
Lubenau's Glycerol Bouillon Egg of sterile water.
Medium (Park and Krumwiede) 2446
. . .
(4) Distribute into sterile test tubes.
CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 789
(5) Coagulate in the same manner as the (3) Add several drops of sterile water
coagulation of serum, (details of to each piece.
method not given) at a temperature (4) Sterilize (method not given).
of about 70C. References: Ball (1919 p. 84), Bezangon
Sterilization: Method of sterilization of (1920 p. 121), Besson (1920 p. 54), Harvey
water or containers not given. (1921-22 p. 85), Klimmer (1923 p. 223).
Use: Cultivation of saprophytic and para-
sitic forms. 2433. Abel's Egg Medium
Variants The author added a sterile solu-
: