Culture

Media
Manual

A member company of International Diagnostics Group plc

 PROFILE
LAB M has specialised in microbiological
diagnostic products since 1973. The quality of its
dehydrated culture media has an international
reputation, particularly for anaerobic media (FAA,
FAB) and innovative media for improved
detection of Salmonella.
Key services such as SMART QA proficiency
testing scheme and QC Assays media control
cultures are assisting LAB M customers in the
PROFILE
The MALTHUS System enables automated
analysis of microbial activity, by measurement of
changes in the electrical conductance of culture
media. Since metabolic activity, rather than
biomass is being measured, Malthus can provide a
faster and more accurate result than available with
traditional methods.
MALTHUS Systems can reduce analysis time and
labour costs for many routine QC tests in food,
toiletries and other industrial samples. The ability
achievement of laboratory accreditation to
national and international standards. Customers
can also rely upon the broad base of skills and
experience both within LAB M and across the
International Diagnostics Group to find the
appropriate solution for your microbiological
problems
to handle large sample numbers and the flexibility
to add and remove individual samples, without
affecting others, makes it an ideal screening tool.
MALTHUS is recognised as an officially
approved method by the Association of the
Official Analytical Chemists (AOAC), the UK
Ministry of Agriculture Fisheries and Foods
(MAFF), the British Standards Institute (BSI) and
the United States Department of Agriculture
(USDA).
i
 LABORATORIES
PROFILE
Q Laboratories is a leading UKAS accredited
laboratory, providing a range of services to
facilitate the production of microbiologically safe
products.
Routine testing for pathogens and spoilage
organisms is a central part of the business. The
expertise from Q Laboratories and other
companies in the International Diagnostics Group,
enables an extensive range of tests to be offered
across a wide range of food, pharmaceutical and
household products.
RESEARCH
PROFILE
IDG Research services the group’s growing
demand for new product development and this
expertise is extended to external companies
through contract development services.
IDG Research currently produce immunoassay
kits for a leading European producer of food
testing equipment. The kits are based upon
ii
Testing, by itself, does not solve all quality issues.
Q Laboratories specialises in evaluation of
processes, in development of HACCP (Hazard
Analysis Critical Control Point) systems and
advice on all aspects of factory hygiene, which
ultimately contributes to product quality.
Q Laboratories’ customer base includes many
brand leaders. With a testing base across the UK
and worldwide demand for its consultancy,
training and seminar services, Q Laboratories can
provide the specialist support increasingly
required to meet quality standards imposed by
regulations, and more importantly, those expected
by consumers.
magnetic particle technology, which have
applications in traditional microbiology culture,
immunoassay and DNA capture technologies.
The facility has recently established a molecular
biology laboratory to develop technologies to
enhance the specificity of pathogen detection in
food and other industrial applications.
 CONTENTS

Index by product code v Microbiology methods pages 23-27

Index by product name vii Sources of information 23
Index by organism ix TVC 23
Coliforms / enterobacteriaceae 23
Manufacturing process pages 5-8 E. Coli
Flow chart 5 presence or absence 24
Quality criteria 6 enumeration using membranes 24
The application of growth rate analysers 7-8 enumeration without membranes 24
O157:H7 24
Preparing culture media pages 9-13 Yeasts and moulds 25
Dehydrated Media 9 Staph. aureus 25
storage 9 Pseudomonas spp. 25
weighing out 9 Bacillus cereus 25
pH of culture media 9 C. perfringens 26
glassware 10 Listeria
addition of powder to water 10 FDA method 26
heat 10 modified USDA method 26
dispensing prior to sterilisation 10 Salmonella
Autoclaving Culture Media 10 semi-solid method 26
total heat input 10 conventional method 27
measurement points 11 Campylobacter 27
volume of medium 11
heat conductance of agar 11 Culture media information pages 29-95
load distribution / composition 11 Format and abbreviation guide 29
agitation 11 Dehydrated media guide 29
equipment 11 Alphabetical Listing of Products 31
Sterility Indicators 12 amies transport - FAA 31-50
adhesive tape 12 FAB orange serum agar 51-70
Browne’s tubes 12 OGYE - yersinia selective agar 71-96
Spore indicators 12
Molten Media 12 Lyophilised selective agents pages 97-104
re-melting agar 12 Alphabetical listing of products 97
pouring plates 12
drying plates 12 Agars, peptones and
Sterile Supplements 13 media constituents pages 105-109
Storage of Prepared Media 13
Alphabetical listing 105
Troubleshooting Guide 13

Quality control of culture media pages 14-21

The ecometric technique 14
Productivity ratio 15
Liquid media 15
Templates / suggested records 16-19
Preservation of stock cultures 19
Laboratory accreditation 20
HACCP 21
iii
 INDEX by product code
LAB001 Columbia Agar Base 43 LAB061 Eosin Methylene Blue Agar (Levine) 50
LAB002 MacConkey Agar (without salt) 60 LAB062 Tryptose Phosphate Broth 89
LAB003 DCLS Agar 45 LAB063 Tryptone Glucose Extract Agar 88
LAB004 Tryptone Soy Broth USP 15, 89 LAB064 Thioglycollate Medium (Brewer) 85
LAB005 MacConkey Broth Purple 24, 61 LAB065 Desoxycholate Citrate Agar (Hynes) 45
LAB006 CLED Medium (Bevis) 42 LAB066 Anaerobe Identification Medium Base 32
LAB007 Mannitol Salt Agar 62 LAB067 GC Agar Base 53
LAB008 Nutrient Agar 69 LAB068 Nutrient Broth ‘E’ 69
LAB009 Sabouraud Dextrose Agar 78 LAB069 Simmons Citrate Agar 80
LAB010 Plate Count Agar APHA 23, 73 LAB071 Fastidious Anaerobe Broth (FAB) 15, 51
LAB011 Tryptone Soy Agar USP 15, 88 LAB072 Tryptone Bile Agar 24, 87
LAB012 Sensitivity Test Agar (STA) 80 LAB073 Bacillus Cereus Medium (PREP) 25, 33
LAB013A Bismuth Sulphite Agar Base A 34 LAB074 Nusens Agar 68
LAB013B Bismuth Sulphite Agar Base B 34 LAB075 Todd Hewitt Broth 85
LAB014 Nutrient Broth No. 2 BP 69 LAB078 CEMO Agar 40
LAB015 Blood Agar Base No. 2 35 LAB079 WL Nutrient Agar 93
LAB016 Fluorescence Agar 52 LAB080A Minerals Modified Glutamate Medium 24, 64
LAB018 Yeast Extract Agar 23, 95 LAB080B Sodium Glutamate 24, 64
LAB019 Milk Agar 23, 63 LAB082 Membrane Lauryl Sulphate Broth 63
LAB020 Dextrose Tryptone Agar 46 LAB084 Single Step Staph Selective Agar (4S) 81
LAB022 Reinforced Clostridial Medium (Broth) 76 LAB085 Baird Parker Medium 25, 33
LAB023 Reinforced Clostridial Agar 76 LAB086 Rappaport Vassiliadis Medium (RVS) 27, 75
LAB024 Cooked Meat Granules (2 x 250g) 43 LAB087 Sugar Free Agar 83
LAB024 Meat Granules 43 LAB088 Violet Red Bile Glucose Agar (VRBGA) 23, 92
LAB024Z Cooked Meat Medium Tablets 44 LAB089 Oxytetracycline Glucose Yeast Extract Agar 25, 71
LAB025 Fluid Thioglycollate Medium USP 15, 51 LAB090 Fastidious Anaerobe Agar (FAA) 50
LAB027 Hoyle’s Medium 54 LAB091 E.E. Broth 49
LAB028 Blood Agar Base 34 LAB092 M17 Agar 59
LAB029 Desoxycholate Citrate Agar (DCA) 44 LAB093 MRS Agar 65
LAB030 MacConkey Agar (with salt) 59 LAB094 MRS Broth 66
LAB031 Violet Red Bile Agar (VRBA) 23, 92 LAB095 DN’ase Agar 47
LAB032 XLD Agar 26, 27, 94 LAB096 TCBS Cholera Medium 84
LAB033 Sabouraud Liquid Medium USP 78 LAB097 Tetrathionate Broth (APHA) 27, 84
LAB034 Brilliant Green Agar (Modified) 26, 27, 37 LAB098 Potato Dextrose Agar 74
LAB035 TYC Medium 90 LAB099 Wort Broth 94
LAB036 Rose Bengal Chloramphenicol Agar 25, 77 LAB100 Ringers Solution 1/4 Strength 9,77
LAB037 Malt Extract Agar 25, 61 LAB100Z Ringers Solution 1/4 Strength Tablets 9, 77
LAB038 Wort Agar 93 LAB101 Ringers Solution (Calgon) 9, 77
LAB039 Mueller Hinton Agar II 67 LAB102 Ringers Solution (Thiosulphate) 9, 77
LAB041 CLED Medium (single indicator) 42 LAB103 Maximum Recovery Diluent 9, 23-26, 62
LAB042 Mueller Kauffman Tetrathionate Broth 27, 68 LAB104 Peptone Water 72
LAB044A Selenite Broth Base 79 LAB105 China Blue Lactose Agar 41
LAB044B Sodium Biselenite 27, 79 LAB106 Kanamycin Aesculin Azide Agar (complete) 55
LAB045 MacConkey Agar No. 3 60 LAB107 Kanamycin Aesculin Azide Broth (complete) 55
LAB046 Buffered Peptone Water 26, 27, 39 LAB108 Pseudomonas Agar Base 25, 74
LAB048 Brain Heart Infusion Agar 35 LAB109 Perfringens Agar OPSP 26, 72
LAB049 Brain Heart Infusion Broth 14, 19, 36 LAB110 Hektoen Enteric Medium 53
LAB051 Brilliant Green Bile 2% Broth 24, 37 LAB111 Sabouraud Maltose Agar 78
LAB052 SS Agar (Modified) 9, 83 LAB112 Campylobacter Blood Free (CCDA) 14, 39
LAB053 Triple Sugar Iron Agar 86 LAB113Z Salt Meat Broth Tablets 79
LAB055A Selenite Cystine Broth Base 27, 80 LAB114 Mueller Hinton Broth II 67
LAB059 Kligler Iron Agar 56 LAB115 Milk Plate Count Agar 23, 64
LAB060 Endo Agar 49 LAB116 MLCB Agar 65

iv
LAB117 DTM Dermatophyte Test Medium 46 X011 Colistin, Nalidixic Acid 35, 43, 99
LAB119 Yeast Extract Dextrose Chloramphenicol Agar 95 X012 Colistin, Nalidixic Acid 35, 43, 100
LAB120 Yersinia CIN Agar Base 96 X013 Colistin, Oxolinic Acid 35, 43, 104
LAB121 Bromocresol Purple Lactose Agar 38 X015 Neomycin 75 35, 42, 97
LAB122 Listeria Isolation Medium (Oxford) 26, 58 X016 Neomycin 100 35, 42, 98
LAB123 Kirchners T.B. Enrichment Medium 56 X018 Kanamycin 75 98
LAB124 Amies Transport Medium With Charcoal 31 X019 P-INC Supplement 103
LAB125 Amies Transport Medium Without Charcoal 31 X040 VCA 54, 100
LAB126 Lactose Broth 57
X068 VCNT (for Thayer Martin) 102
LAB127 Cooked Meat Medium 44
X069 LCT (for New York medium) 102
LAB129 Tryptone Water 24, 89
X070 LCAT (for New York medium) 53, 101
LAB130 Urea Agar Base 90
X073 Egg Yolk Emulsion 33, 36, 81, 98
LAB131 Urea Broth Base 91
X074 Polymixin B 33, 98
LAB133 Cetrimide Agar 40
X085 Egg Yolk Tellurite 33, 103
LAB135 Campylobacter Enrichment Broth 40
X089 Oxytetracycline (for OGYE) 71, 104
LAB136 Easter - Gibson Pre-enrichment Medium 48
X090 Nalidixic Acid, Vancomycin 50, 97
LAB137 Easter - Gibson Salmonella Medium 48
LAB138 Listeria Enrichment Broth (FDA) 26, 58 X091 Nalidixic Acid 50, 97

LAB139 Buffered Listeria Broth 26, 39 X092 Metronidazole, Nalidixic Acid 50, 97

LAB140 Helicobacter Pylori Agar Base 54 X093 Cycloserine, Cefoxitin 36, 50, 99
LAB144 Palcam Broth 72 X107 CN (for Ps. aeruginosa) 74, 102
LAB147 Orange Serum Agar 70 X108 CFC (for Pseudomonas spp) 25, 74, 102
LAB148 Palcam Agar Base 26, 71 X109 Sulphadiazine 72, 99
LAB149 Plate Count Agar 23, 73 X110 Oleandomycin, Polymixin 72, 99
LAB150 MSRV Medium 26, 66 X112 Cefoperazone, Amphotericin 39, 98
LAB155 UVM Base 26, 91 X120 CIN Selective Supplements 91, 104
LAB157 Aseptic Commissioning Broth 32 X122 CCCAF (for Oxford medium) 58, 100
LAB158 Liquid Baird Parker Medium 57 X131 CVTC 40, 98
LAB159 Malt Extract Broth 62 X137 TMAO Selenite (as X136) 48, 100
LAB160 Brazier’s CCEY Agar 36 X138 NAC (for FDA broth) 39, 58, 101
LAB161 Sorbitol MacConkey Agar 24, 82 X140 Cepacia Selective Supplement 102
LAB162 Tryptone Bile Glucuronide Agar 24, 87
X144 PAC (for Palcam media) 71, 72, 101
LAB163 R2A Medium 75
X150 Novobiocin (for MSRV/Diassalm/
LAB164 Fraser Broth 26, 52 O157 Broth) 47, 66, 70, 99, 103
LAB166 Slanetz & Barley Medium 82 X155 UVM I Supplement 91, 101
LAB165 O157 Broth MTSB 24, 70 X156 UVM II Supplement 91, 101
LAB167 Aeromonas Agar 31 X161 Cefixime Tellurite (for SMAC) 70, 82, 99
LAB537 Diassalm 26, 47 X164 Fraser Supplement (1/2 strength) 52, 101
MC001 Yeast Extract 108 X165 Fraser Supplement (full strength) 52, 101
MC002 Agar No. 1 Bacteriological - High Clarity 105
X207 Methicillin (for MRSA) 103
MC003 Soy Peptone 107
X209 Chloramphenicol 104
MC004 Balanced Peptone No. 1 106
X212 Cefoperazone, Amphotericin 98
MC005 Tryptone 108
X214 Skirrows 98
MC006 Agar No. 2 Bacteriological - Gen. Purpose 24, 105
X215 Neomycin 75 97
MC007 Acid Hydrolysed Casein 105
X219 P-INC Supplement 103
MC008 Tryptose 108
X260 Bacitracin 100
MC009 Mycological Peptone 107
X268 VCNT (for Thayer Martin) 102
MC011 Proteose Peptone A 107
MC019 Beef Extract 106 X269 LCT (for New York medium) 102

MC023 Malt Extract 107 X270 LCAT (for New York medium) 101

MC024 Bacteriological Peptone 106 X271 GC Growth Supplement 102

MC025 Bile Salts No. 3 106 X290 Nalidixic Acid, Vancomycin 97
MC026 Sodium Desoxycholate 107 X291 Nalidixic Acid 97
MC027 Skim Milk Powder (2 x 250g) 107 X546 VCC Supplement 99
MC029 Agar No. 4 - Plant Tissue Culture Grade 106 X555 UVM I Supplement 101
X009 Chloramphenicol 46, 77, 78, 95, 104 X564 Fraser Supplement (1/2 strength) 52, 101
v
 INDEX by product name
Acid Hydrolysed Casein MC007 105 Cycloserine, Cefoxitin X093 36, 50, 99
Aeromonas Agar LAB167 31 DCLS Agar LAB003 45
Agar No. 1 Bacteriological - High Clarity MC002 105 Desoxycholate Citrate Agar (DCA) LAB029 44
Agar No. 2 Bacteriological - Gen. Purpose MC006 105 Desoxycholate Citrate Agar (Hynes) LAB065 45
Agar No. 4 - Plant Tissue Culture Grade MC029 106 Dextrose Tryptone Agar LAB020 46
Amies Transport Medium With Charcoal LAB124 31 Diassalm LAB537 26, 47
Amies Transport Medium Without Charcoal LAB125 31 DN’ase Agar LAB095 47
Anaerobe Identification Medium Base LAB066 31 DTM Dermatophyte Test Medium LAB117 46
Aseptic Commissioning Broth LAB157 32 E.E. Broth LAB091 49
Bacillus Cereus Medium (PREP) LAB073 25, 33 Easter - Gibson Pre-enrichment Medium LAB136 48
Bacitracin X260 100 Easter - Gibson Salmonella Medium LAB137 48
Bacteriological Peptone MC024 106 Egg Yolk Emulsion X073 33, 36, 81, 98
Baird Parker Medium LAB085 25, 33 Egg Yolk Tellurite X085 33, 103
Balanced Peptone No. 1 MC004 106 Endo Agar LAB060 49
Beef Extract MC019 106 Eosin Methylene Blue Agar (Levine) LAB061 50
Bile Salts No. 3 MC025 106 Fastidious Anaerobe Agar (FAA) LAB090 50
Bismuth Sulphite Agar Base A LAB013A 34 Fastidious Anaerobe Broth (FAB) LAB071 15, 51
Bismuth Sulphite Agar Base B LAB013B 34 Fluid Thioglycollate Medium USP LAB025 15, 51
Blood Agar Base LAB028 34 Fluorescence Agar LAB016 52
Blood Agar Base No. 2 LAB015 35 Fraser Broth LAB164 26, 52
Brain Heart Infusion Agar LAB048 35 Fraser Supplement (1/2 strength) X164 52, 101
Brain Heart Infusion Broth LAB049 14, 19, 36 Fraser Supplement (1/2 strength) X564 52, 101
Brazier’s CCEY Agar LAB160 36 Fraser Supplement (full strength) X165 52, 101
Brilliant Green Agar (Modified) LAB034 26, 27, 37 GC Agar Base LAB067 53
Brilliant Green Bile 2% Broth LAB051 24, 38 GC Growth Supplement X271 102
Bromocresol Purple Lactose Agar LAB121 38 Hektoen Enteric Medium LAB110 53
Buffered Listeria Broth LAB139 26, 39 Helicobacter Pylori Agar Base LAB140 54
Buffered Peptone Water LAB046 26, 27, 39 Hoyle’s Medium LAB027 54
Campylobacter Blood Free (CCDA) LAB112 14, 39 Kanamycin 75 X018 98
Campylobacter Enrichment Broth LAB135 40 Kanamycin Aesculin Azide Agar (complete) LAB106 55
CCCAF (for Oxford medium) X122 58, 100 Kanamycin Aesculin Azide Broth (complete) LAB107 55
Cefixime Tellurite (for SMAC) X161 70, 82, 99 Kligler Iron Agar LAB059 56
Cefoperazone, Amphotericin X112 39, 98 Kirchners T.B. Enrichment Medium LAB123 56
Cefoperazone, Amphotericin X212 98 Lactose Broth LAB126 57
CEMO Agar LAB078 40 LCAT (for New York medium) X070 53, 101
Cepacia Selective Supplement X140 102 LCAT (for New York medium) X270 101
Cetrimide Agar LAB133 41 LCT (for New York medium) X069 102
CFC (for Pseudomonas spp) X108 25, 74, 102 LCT (for New York medium) X269 102
China Blue Lactose Agar LAB105 41 Liquid Baird Parker Medium LAB158 57
Chloramphenicol X009 46, 77, Listeria Enrichment Broth (FDA) LAB138 26, 58
78, 95, 104 Listeria Isolation Medium (Oxford) LAB122 26, 58
Chloramphenicol X209 104 M17 Agar LAB092 59
CIN Selective Supplements X120 95, 104 MacConkey Agar (without salt) LAB002 60
CLED Medium (Bevis) LAB006 42 MacConkey Agar (Sorbitol) LAB161 82
CLED Medium (single indicator) LAB041 42 MacConkey Agar (with salt) LAB030 59
CN (for Ps. aeruginosa) X107 74, 102 MacConkey Agar No. 3 LAB045 60
Colistin, Nalidixic Acid X011 35, 43, 99 MacConkey Broth Purple LAB005 24, 61
Colistin, Nalidixic Acid X012 35, 43, 100 Malt Extract MC023 107
Colistin, Oxolinic Acid X013 35, 43, 104 Malt Extract Agar LAB037 25, 61
Columbia Agar Base LAB001 43 Malt Extract Broth LAB159 62
Cooked Meat Granules (2 x 250g) LAB024 43 Mannitol Salt Agar LAB007 62
Cooked Meat Medium LAB127 44 Maximum Recovery Diluent LAB103 9, 23-26, 62
Cooked Meat Medium Tablets LAB024Z 44 Meat Granules LAB024 43
CVTC X131 40, 98 Membrane Lauryl Sulphate Broth LAB082 63
vi
Methicillin (for MRSA) X207 103 Sabouraud Liquid Medium USP LAB033 78
Metronidazole, Nalidixic Acid X092 50, 97 Sabouraud Maltose Agar LAB111 78
Milk Agar LAB019 23, 63 Salt Meat Broth Tablets LAB113Z 79
Milk Plate Count Agar LAB115 23, 64 Selenite Broth Base LAB044A 79
Minerals Modified Glutamate Medium LAB080A 24, 64 Selenite Cystine Broth Base LAB055A 27, 80
MLCB Agar LAB116 65 Sensitivity Test Agar (STA) LAB012 80
MRS Agar LAB093 65 Simmons Citrate Agar LAB069 81
MRS Broth LAB094 66 Single Step Staph Selective Agar (4S) LAB084 81
MSRV medium LAB150 26, 66 Skim Milk Powder MC027 107
Mueller Hinton Agar II LAB039 67 Skirrows X214 98
Mueller Hinton Broth II LAB114 67 Slanetz & Barley Medium LAB166 82
Mueller Kauffman Tetrathionate Broth LAB042 27, 68 Sodium Biselenite LAB044B 27, 79
Mycological Peptone MC009 107 Sodium Desoxycholate MC026 107
NAC (for FDA broth) X138 39, 58, 101 Sodium Glutamate LAB080B 24, 64
Nalidixic Acid X091 50, 97 Sorbitol MacConkey Agar LAB161 24, 82
Nalidixic Acid X291 97 Soy Peptone MC003 107
Nalidixic Acid, Vancomycin X090 50, 97 SS Agar (Modified) LAB052 9, 83
Nalidixic Acid, Vancomycin X290 97 Sugar Free Agar LAB087 83
Neomycin 100 X016 35, 42, 98 Sulphadiazine X109 72, 99
Neomycin 75 X015 35, 42, 97 TCBS Cholera Medium LAB096 84
Neomycin 75 X215 97 Tetrathionate Broth (APHA) LAB097 27, 84
Novobiocin (for MSRV/Diassalm/ 47, 66, Tetrathionate Broth (Mueller Kauffmann) LAB042 27, 68
O157 Broth) X150 70, 99, 103
Thioglycollate Medium (Brewer) LAB064 85
Nusens Agar LAB074 68
Todd Hewitt Broth LAB075 85
Nutrient Agar LAB008 69
TMAO Selenite X137 48, 100
Nutrient Broth ‘E’ LAB068 69
Triple Sugar Iron Agar LAB053 86
Nutrient Broth No. 2 BP LAB014 69
Tryptone MC005 108
O157 Broth MTSB LAB165 24, 70
Tryptone Bile Agar LAB072 24, 87
Oleandomycin, Polymixin X110 72, 99
Tryptone Bile Glucuronide Agar LAB162 24, 87
Orange Serum Agar LAB147 70
Tryptone Glucose Extract Agar LAB063 88
Oxytetracycline (for OGYE) X089 71, 104
Tryptone Soy Agar USP LAB011 15, 88
Oxytetracycline Glucose Yeast Extract Agar LAB089 25, 71
Tryptone Soy Broth USP LAB004 15, 89
P-INC Supplement X019 103
Tryptone Water LAB129 24, 89
P-INC Supplement X219 103
Tryptose MC008 108
PAC (for Palcam media) X144 71, 72, 101
Tryptose Phosphate Broth LAB062 89
Palcam Agar Base LAB148 26, 71
TYC Medium LAB035 90
Palcam Broth LAB144 72
Urea Agar Base LAB130 90
Peptone Water LAB104 72
Urea Broth Base LAB131 91
Perfringens Agar OPSP LAB109 26, 72
UVM Base LAB155 26, 91
Plate Count Agar LAB149 23, 73
UVM I Supplement X155 91, 101
Plate Count Agar APHA LAB010 23, 73
UVM I Supplement X555 101
Polymixin B X074 33, 98
UVM II Supplement X156 91, 101
Potato Dextrose Agar LAB098 74
VCA X040 54, 100
PREP Agar (B.cereus) LAB073 33
VCC Supplement X546 99
Proteose Peptone A MC011 107
VCNT (for Thayer Martin) X068 102
Pseudomonas Agar Base LAB108 25, 74
VCNT (for Thayer Martin) X268 102
R2A Medium LAB163 75
Violet Red Bile Agar (VRBA) LAB031 23, 92
Rappaport Vassiliadis Medium (RVS) LAB086 27, 75
Violet Red Bile Glucose Agar (VRBGA) LAB088 23, 92
Rappaport Vassiliadis Medium, semi-solid LAB150 26, 66
WL Nutrient Agar LAB079 93
Reinforced Clostridial Agar LAB023 76
Wort Agar LAB038 93
Reinforced Clostridial Medium (Broth) LAB022 76
Wort Broth LAB099 94
Ringers Solution (Calgon) LAB101 9, 77
XLD Agar LAB032 26, 27, 94
Ringers Solution (Thiosulphate) LAB102 9, 77
Yeast Extract MC001 108
Ringers Solution 1/4 Strength LAB100 9,77
Yeast Extract Agar LAB018 23, 95
Ringers Solution 1/4 Strength Tablets LAB100Z 9, 77
Yeast Extract Dextrose
Rose Bengal Chloramphenicol Agar LAB036 25, 77 Chloramphenicol Agar LAB119 95
Sabouraud Dextrose Agar LAB009 78 Yersinia CIN Agar Base LAB120 96
vii
 INDEX by organism
Actinomyces 35, 97 Lactobacillus 19, 65, 66, 70, 93
Aeromonas 19, 31 Listeria 26, 39, 52, 58, 71,
Aspergillus niger 20, 60 72, 91, 100, 101

Bacillus cereus 19, 25, 33, 98 Micrococcus 19, 79

Bacillus stearothermophilus 12, 46 Mycobacterium 56

Bacteroides fragilis 19, 31, 97 Neisseria 19, 31, 43, 53, 67, 101, 102
Blastomyces 46 Peptostreptococcus 50
Campylobacter 14, 19, 39, 40, 98 Porphyromonas 50
Candida 19 Propionibacterium 50
Citrobacter 19 Proteus 19, 42, 90
Clostridium difficile 36, 99 Pseudomonas 19, 25, 41, 52, 74, 102
Clostridium perfringens 19, 26, 34, 35, Saccharomyces 20
43, 72, 76, 99, Salmonella 9, 14, 19, 34, 37, 39, 48,
Clostridium sporogenes 19 53, 65, 68, 75, 79,
80, 83, 100, 103
Coccidiodes 46
Salmonella spp 19, 26, 27, 44, 45,
Coliforms 23, 38, 41, 42, 49,
47, 84, 86, 94
50, 57, 61, 63, 64, 89, 92
Corynebacterium 19 Salmonella typhi 49, 65, 68, 84

Dermatophytes 46 Serratia 20

Enterobacteriacieae 23, 38, 49, 60, Shigella 14, 20, 44, 45,
81, 85, 92 53, 83, 94
Enterococcus 55, 82 Staphylococcus aureus 20, 25, 33, 47, 57,
62, 79, 81, 103
Escherichia coli 19, 24, 50, 61, 87
Staphylococcus epidermidis 20
Escherichia coli O157:H7 24, 70, 82, 99
Streptococcus 20, 35, 41, 42,
Eubacterium 50 59, 70, 89, 104
Fusobacterium 50 Taylorella 40
Gardnerella 35, 43, 99 TVC 23, 63, 64, 73, 75, 88, 95
Haemophilus 13, 19, 88, 100 Vibrio 20, 84
Helicobacter 54, 100 Yeasts & Moulds 25, 61, 62, 71, 74,
Histoplasma 36, 46 78, 93, 94, 104
Klebsiella 19 Yersinia 20, 96, 104

viii
 LAB M Culture Media:
The Process Outline

RAW MATERIALS
Agars, Peptones, Extracts, Dyes, Chemicals etc.
Each component is individually tested for suitability.
Growth promoting components are selected with the help of
an automated growth rate analyser (Malthus Instruments)

PRODUCTION
Weighing, Milling, Blending
A production batch is made from raw materials of specified
batch number which have been pre-tested for compatibility.
The components are individually milled to ensure uniform
particle size. Weighings are double checked before the
components are blended.

QUALITY CONTROL
Physical, Biological parameters. Comparison with previous
batch and competition
Quality control first checks that the batch is completely
blended, then a series of physical and biological tests are
performed to ensure the product meets the exacting
standards required by our customers. Comparisons with
previous and competitor’s batches are made. Results are
recorded and a reference sample stored.

BOTTLING
Into 500g sealed containers, or bulk containers at request
Automated equipment delivers pre-weighed amounts
into containers which are hermetically sealed.
Each container is immediately labelled with product details,
code and batch number.

CUSTOMERS
LAB M products are dispatched all over the world to
microbiologists in all types of laboratory. Strict batch
traceability in accordance with ISO9001 ensures we can recall
all products if necessary, safeguarding your products/process.

5
This section intentionally starts at page 5
 LAB M Culture Media – Production
All components from specified batch numbers. All components
The quality criteria weighed accurately and checked.
Components milled to uniform particle size. Components blended for
specified time, multiple samples taken to ensure thorough blending.

Raw materials Quality control

Peptones and Extracts – Clarity, pH, moisture, growth promoting
properties with Gram positive and Gram negative organisms Physical – pH, clarity, gel strength, colour, heat stability, viscosity,
aerobically and anaerobically, freedom from toxicity. Compatibility redox.
with other components, haemolysis patterns, antibiotic antagonists. Biological – Growth characteristics, productivity ratio, chemical
Agar – Clarity, pH, gel strength, melting point, setting point, heavy reactions and colour changes, comparison with previous batch and
metal content (particularly Ca++, Mg++) compatibility with other competition.
components. Clarity on re-melt.
Bile Salts – Clarity, pH, thin layer chromatography, compatibility
with other components.
Dyes & Chemicals – pH, chemical parameters, growth promotion
/inhibition, properties after incorporation into culture media.

Selection of ingredients
LAB M pioneered the use of impedance technology for the selection
of Culture Media ingredients.
We were the first Company to utilise automated growth rate analysers
to ensure only the most suitable ingredients available are chosen. For
example with the use of growth rate analysis, we can select those
peptones that trigger the exponential growth of inoculated micro-
organisms in the shortest possible time; this technology is also useful
in minimising batch to batch variation.
Further information on this technique is on page 5 and 6.

Choosing peptones for enrichment media - peptone A is chosen because of a faster response time.
The survival time of the test organisms is also taken into consideration.
6
 The application of growth rate
analysers in the manufacture of
bacteriological culture media
by W.A. Hyde and K. Denton
Introduction
One of the fundamental performance parameters of a bacteriological
culture medium is its ability to promote the early and rapid growth of
micro-organisms. There have been many methods devised to assess
parameters, most of which rely on the viable count.
The viable count gives information on the state of the culture at a
single moment in time but gives no indication of the growth rate,
unless serial viable counts are performed. Methods based on the
classical method of Lister are used for estimating growth rate. Using
this method he was able to plot the classic growth rate curve of a
bacterial culture.
Unfortunately this technique is both laborious and inaccurate.
Halverston and Ziegler in 19331 showed the technique to be subject
to very large experimental errors. For example, with five tubes
counted from each dilution, variation can be from -70 to +260 per
cent!
Other variations on the Lister technique for assessing substrate
performance are:
a) Agar dilution techniques
b) Surface inoculation techniques such as the drop method of Miles
and Misra2 and the ecometric technique of Mossel et al3
c) The novel spiral plater technique4
d) Photometric and nephelometric techniques
Techniques a-c still suffer from giving a point source of information
in what is a dynamic analysis.
The nephelometric/photometric techniques are relatively insensitive
and give little information about the log phase, and they cannot be
used with materials which produce turbidity.
The introduction of electrical methods into the field of culture media
performance analysis has provided microbiologists with the means to
accurately record development of a culture over a large section of the
growth curve. The main electrical parameters measured, are
conductance and impedance. There are advantages in measuring
either of these parameters, and these are outlined in the excellent
book by Eden, R. and G5.
Impedance/conductance techniques
In 1898, Stewart showed that bacterial growth in fluid could be
detected by changes in the electrical properties of that fluid. Since
this time several workers have made use of conductance and
impedance measurements in the analysis of bacterial growth, finally
crystallising in the 1970’s with papers by Ur and Brown (1975)6 and
Cady (1975)7 describing the use of continuous impedance monitoring
as a tool with wide potential in microbiology.
In 1977, workers at the Torry Research Station in Aberdeen produced
equipment capable of accurately plotting the growth curve of bacteria
in various fluid culture media using impedance techniques8.
The curves produced on the equipment could be directly related to the
total viable count.
Multi-channel growth rate analysers are commercially available, eg
the Malthus system9.
Peptones
The peptones, infusions and extracts that are used by microbiologists
are manufactured from biological raw materials and these are subject
to variations and inconsistency (e.g. meat, milk, plants). These are
manufactured by acid or enzyme digestion processes which are
cumbersome and difficult to control.
It is not surprising, therefore, that products described as ‘meat
peptone’, for example, are much more variable in performance than
reagent grade chemicals. (Figure 1) Because of this variation it is
standard practice for culture media manufacturers to test samples of
these raw materials from many sources in order to maintain a supply
of products with suitable performance characteristics.
Normally pre-shipment samples will be evaluated, both alone and
made up into typical formulations, and both performance and
comparison with ‘stock’ material will be taken into account before a
decision to purchase is made. Full quality control of the bulk material
is then also carried out.
LAB M introduced an eight channel Malthus Growth Rate Analyser
into its raw material selection and quality control procedures in 1982.
This has enabled closer control of the performance characteristics of
the individual peptones and culture media formulations available
from the company.
Method of use
The eight channel machine is used to compare the performance of
two substrates inoculated with three to four organisms or when
testing finished product to compare the new batch of product
against a stock batch, and against a competitor’s product.
The organisms are inoculated into 2ml or 10ml volumes of the
sterile substrate in tubes which contain the electrodes. The tubes
are then placed in an accurately controlled water bath and
connections are made between the electrodes and the analyser. The
base lines for each channel are set on the chart recorder and the
growth curves for each test are recorded.
The speed of the chart and the sensitivity of the instrument can be
adjusted in order to accommodate the growth characteristics of
various organisms. Using this technique it is possible with some
organisms and substrates to obtain a result in less than half a
working day.
Figure 1
7
 Growth curve information
There are several parameters which can be assessed from the growth
curve produced by the Malthus system. These include:
Lag time – The time that elapses before a change in electrical
properties can be detected. Rapid metabolism causing changes in
electrical properties may be detected before cell division begins.
Detection time or take off – When the curve shifts from the baseline
lag phase into the log phase, this information can be used as a
measure of the number of organisms present in a sample with the aid
of suitable calibration graphs (Figure 2).
Log phase - The log phase gradient is a measure of the rate of a
metabolism in the multiplying bacteria.
Maximum peak of the curve - This is related to the maximum
‘number’ of bacteria achieved in the culture. The information can be
used in a number of ways, for example:
a) The peptone with the shortest lag time can be chosen for those
media that are required to give rapid growth from a small
inoculum, e.g. sterility test media, blood culture media, and
fermentation media.
b) For culture media that demand standardisation of performance
from batch to batch, it is possible to choose peptones with a
specified lag time growth rate.
c) The maximum peak is of interest when choosing a raw material
for production of large numbers of bacteria such as for vaccines.
As equipment based on impedance methods became established in
the microbiological quality control procedures of the food industry, a
new aspect of culture media quality control is opened up, and needs
to be tested for, these are the electrical properties of the product.
Only by the use of impedance/conductance measuring equipment in
quality control, can performance of the culture media on similar
equipment in customers’ laboratories be assured.
Figure 2

Conclusion
Electrical methods of monitoring bacterial growth have been known
since the latter part of the 19th Century, however, only in the last few
years with the development of micro-processors and sophisticated
electronics are these methods beginning to reach their full potential in
Growth Rate Analysers.
In culture media manufacture the Growth Rate Analyser provides
information that makes a significant contribution to improving both
the performance and standardisation of products manufactured from
variable raw materials. (Figure 3)

References
1. Halvorson, H.O., Ziegler, N.R. (1933a) J. Bact. 25, 101, (1933b) I.
Bid., 26, 331, 559
2. Miles, A.A., Misra, S.S. (1938) J. hyg., Camb., 38, 732
3. Mossel, D.A.A., Van Rossem, F., Koopmans, M., Hendriks, M.,
Verdouden M., Eelderink, I. (1980) J. Appl. Bact., 49, 439-454
4. Gilchrist, J.E., Donelly, C.B., Peeler, J.T., Delany, J.M. (1973)
Appl. Microbiol. 25, 244-252
5. Eden, R. Firstenberg, Eden, G., Impedance Microbiology (1984)
Research Studies Press, John Wiley and Sons, New York
6. Ur, A., Brown, D.F. J., (1975) New Approaches to the
Identification of Microorganisms (edited by C.G. Heden and T. Illeni)
P.61-71 John Wiley and Sons, New York.
7. Cady, P. (1975) (edited by C.G. Heden and T.Illeni, New York).
P.73-99, John Wiley and Sons, New York
8. Hobbs, G., Gibson, D. M. (1977) J. Appl. Biol. 43, 3
9. Malthus Instruments Ltd., William Clowes St., Burslem, Stoke on
Trent.
First published in International Labmate 1987 Vol.12 Issue 2 pp 36-
37

Figure 3
8
 Preparing Culture Media
Quality Assured
Before each batch of LAB M Culture media is passed for sale it
undergoes a rigorous quality control procedure to ensure it gives
maximum recovery and reproducibility. Reconstitution of media in
the users laboratory must be done with care to ensure the same high
standards of performance.
The following section outlines the correct procedures which will
ensure high quality reconstituted products, and suggests simple
quality control techniques that can be used to check the performance
of prepared media.
Dehydrated Culture Media
Storage
Dehydrated media stored unopened under optimal conditions have a
shelf life of 3-5 years but once the container is opened the contents
should be used within six months. In-house quality control by the
user will help determine the condition of product in opened
containers. The best conditions for storing dehydrated media are in a
cool, even temperature away from any sources of moisture such as
washing up areas or laboratory autoclaves and away from strong
light. Storage in a refrigerator is generally not recommended as there
is the risk of condensation on the container when it is brought out of
the refrigerator.
TABLE 1 – Deterioration of SS Agar stored in various conditions
for 6 months.
Storage conditions moisture gain %
Unopened bottle stored in cool,
dark, dry conditions 0 by pH
Loose cap, stored in light on bench 1.1
Water
Purification by distillation or deionisation is advisable. It is important
that the equipment is properly maintained; the output of ion resins
need to be electronically monitored and microbial colonisation of the
resin and tubing must be avoided. Storage vessels for purified water
must also be monitored for microbial colonisation. It is advisable to
use only fresh purified water with a conductivity of less than 10
microsiemens. Stored water tends to become acidic because it
absorbs atmospheric CO2. Tap water is not recommended because of
the potential presence of heavy metal ions which can cause inhibition
and precipitation problems.
pH of culture media
Meter Sensitive to one decimal place.
Electrodes Calomel, gel filled unbreakable, combination
electrodes shielded with detachable shield for
cleaning. Flat electrodes may be used, but
some problems have been found by users.
Short-term storage pH 4.0 buffer
of electrode
Electrode faults Indicated by slow or erratic readings and
inability to obtain two points on the meter
without adjustment.
Cleaning of 0.5% pepsin in N HCl for 1-2 hours.
electrode Wash in deionised water, soak in pH 7.0
buffer for 2 hours.
Daily checks Use pH 4.0 and pH 7.0 buffer and, if possible,
pH 10.0.
Testing meter pH calibrating or checking attachment.
(Available from BDH.).
Water Deionised or double-distilled water is only
weakly dissociated. Measure conductivity
< 10 micro Siemens. To check pH add 0.3ml.
saturated KCl to 100ml water.
Culture media Acidity: Bile salts precipitated; H2S reactions
reactions affected depressed; sugar fermentation; antibiotics less
active (aminoglycocides, cephalosporins,
macrolides).

Loose cap, stored in light in autoclave room 4.4 Alkalinity: Potentiates aminoglycocides;
sugar fermentation; antibiotics less active
(fusidin, tetracycline, penicillins).
The effect of the moisture gain on the performance of the agar can be Media difficult to Ringers, Maximal Recovery Diluent
quite dramatic. A 1.1% gain in moisture on storage will lead to a 53% pH because of
reduction in the numbers of Salmonella isolated. Similarly a 4.4% low ionic strength
gain in moisture will result in a 78% reduction in isolation rate. This Effect of Autoclaving or irradiation will lower the pH
demonstrates the importance of ensuring the container lid is tightly autoclaving or about 0.2 - 0.5 units, but not predictable.
closed and the pot stored in cool, dry, dark conditions. Barry, A. L. irradiation
and Fay, G. D. A review of some common sources of error in the
Preparation of Agar Media. 1972. Am. J. Med. Tech. Vol. 38 No. 7. Effect of volume Mixed volume loads should be avoided. Time
of media on pH allowance must be made for various volume
When a container is opened for the first time the date should be noted sizes of media to ensure that overheating
on the container. Dehydrated media should not be used if it shows any leading to acid formation and caramelisation
sign of moisture gain i.e. become lumpy or discoloured. The lid on does not occur. Further details are given under
the container should be replaced quickly after media has been taken Autoclaving Culture Media.
out and closed tightly.
Temperature These adjust for changes within the electrode
compensator only. They DO NOT compensate for changes
in ionisation of solution at high temperatures.
Optimum 20 - 25˚C. or according to media
Weighing Out temperature to manufacturer’s instructions.
pH culture media
Using a top-pan balance with an accuracy of ±0.1 gram the powder
should be spooned onto a weighing boat or clean beaker. Do not tip Acceptable ± 0.2 is usual. This assumes 1 litre volume
the media out of the container as this will cause excess dust which pH variations produced strictly according to media
may be irritant and will certainly need cleaning up. The components manufacturer’s instructions.
of some formulations can be irritant so the wearing of a face mask at Adjustment of pH Should not be necessary if all systems correct,
this stage is advisable. of media e.g. water quality, balance, volume etc., and
sterilisation is carried out precisely to
manufacturer’s instructions.

9
 pH out of If pH needs to be adjusted because
tolerance manufacturer’s instructions cannot be
followed, titration of small additions of N/10
HCl. or N/10 NaOH into aliquots of media
should be made, media processed and pH
measured when cooled and appropriate
calculations carried out.
QUALITY CONTROL USING MICROBIOLOGICAL
PERFORMANCE CRITERIA ARE ESSENTIAL
Glassware
All glassware must be undamaged and clean having been rinsed
with purified water before storage. Borosilicate glass is preferable to
soda-glass because the latter may leach alkali into any solution
contained in it.
Addition of Powder to Water
There are a number of ways to do this, attempting to mix the water
and powder too quickly can cause the formation of lumps which are
difficult to disperse.
1. Pour approximately 1/3 of the volume of water required into a flask,
add the powder slowly whilst constantly swirling, then add
remaining water.
2. Pour the full amount of powder onto the full amount of water in a
flask. Allow to stand for 10 minutes before swirling to mix.
3. Add the powder to the flask and slowly pour on the water with
frequent swirling.
NOTE: The water should always be measured before adding to the
flask. Never heat water before adding to the medium.
Heat
Remember that heat denatures the nutrients and the agar in culture
media. Heat is necessary for sterilisation and for dissolving the agar
if the medium is to be distributed into tubes or bottles prior to
sterilisation. However these processes must be carefully controlled to
ensure the minimum possible heat input is used. Heat input is less in
equipment that allows rapid heating and rapid cooling, e.g. media
preparators. Media should never be left at high temperatures for
prolonged periods for example holding in a 56˚C water bath
overnight will noticeably reduce a culture medium’s performance and
gel strength.
Dispensing prior to Sterilisation
Most broth media are readily soluble at room temperature or with the
aid of gentle heat. Ensure a clear, well mixed, solution is obtained
before dispensing into final containers and sterilising. Agar
containing media need to be brought to the boil to take the agar into
solution. This boiling should be done with frequent agitation to
ensure even heat distribution. As soon as the medium begins to boil
it should be removed from the source of heat.
CAUTION: Agar media, particularly those with low agar content,
may boil unexpectedly and overflow out of the flask. To prevent this
agitate frequently and gently as the medium approaches boiling and
the agar begins to dissolve. Allow the medium to cool to 47˚C before
dispensing with constant mixing into final containers for sterilisation.
10
Autoclaving culture media
To achieve optimum performance from reconstituted culture media it
is important to ensure sufficient heat input to kill all spores whilst
protecting the medium from excessive heat input that would damage
the nutrient and gelling properties of the medium. In our experience
the commonest cause of problems with culture media is the heat
sterilisation, a dynamic process which has many variables that must
be considered. These are:
Total Heat Input
Figure 4 Schematic diagram of heat input during sterilisation
The total heat input of a sterilisation cycle can be shown graphically.
The total time above 50˚C is important because both nutrients and
agar will be undergoing a denaturation process. The higher the
temperature the faster the denaturation, i.e. 30 minutes at 121˚C will
noticeably affect the gel properties of most agars whilst the medium
could be held at 50˚C for several hours before denaturation can be
detected. Total heat input should be controlled to protect the
medium from denaturation. Prolonged heating and cooling can
lead to excessive heat input.
The total time spent above 100˚C is important, because not only is
this the temperature at which spores are being killed, but it is the
temperature with the potential to cause most damage to the medium’s
performance. The aim of effective sterilisation is to ensure all spores
are destroyed with the minimum necessary heat input in order to
ensure no ill effects are caused to the medium. This can be done by
using the lethal rate equation or tables (Stumbo 1973). From a chart
recording of the sterilisation process (the recording must be from a
thermocouple placed inside the medium during processing), the
portion of the graph between 100˚C and 121˚C for both the heating
and cooling stages is divided into 1 minute intervals, and using the
equation or table, (shown below) can be converted into an equivalent
time at 121˚C. The time is added together and the total time removed
from the holding time. This ensures that the heating process is
sufficient to kill the spores, but keeps damaging heat input to a
minimum. Thus the instruction to ‘autoclave at 121˚C for 15 minutes’
should be taken to mean ‘sterilise by an equivalent heat process to
121˚C for 15 minutes’. Once calculated the new holding time can be
used for future cycles, but only for the same volume of medium.
Different types of media differ in heat penetration i.e. agars and
broths so the reduction in holding time must be calculated for
different volumes of both.
Lethal Rate Equation:
L = Log-1 (T - 121.1)
Z
Where L = Equivalent time at 121˚C
T = Actual temperature for 1 minute
Z = Temperature coefficient (= 10˚C for spores)
Log-1 = antilog of the number. Using this equation,
a lethal rate table can be produced: (see facing page)
TABLE 2 – Lethal Rate Table:
Heat Conductance of Agar
Temp Time Temp Time
Agar is a very poor conductor of heat and heat penetration into agar
100 0.008 111 0.098 containing media is significantly slower than into non-agar
101 0.010 112 0.123 containing media. Brecker & Bridson showed that 500mls of agar in
a thin-walled bottle took 6 minutes longer to reach 121˚C than did
102 0.012 113 0.155 500mls of water in an identical bottle. If a thermocouple is used in a
103 0.015 114 0.195 500ml bottle of water to control the sterilisation of 500mls of agar
medium, the difference in heat penetration must be taken into
104 0.019 115 0.245 account. The poor heat conductivity of agar has significant effects
105 0.025 116 0.309 when trying to sterilise large volumes of medium (e.g. 2 litres or
more). Pre-heating of the medium to get the agar into solution before
106 0.031 117 0.389 autoclaving is recommended. Brecker & Bridson found that: “In 4
107 0.039 118 0.490 litres of medium, with the agar settling undissolved to the bottom, the
temperature at the centre of the agar mass had not reached 121˚C
108 0.049 119 0.617 1 hour after the autoclave chamber had.”
109 0.062 120 0.776
110 0.078 121 1.000
Temp = Temperature for 1 minute interval
Load Distribution / Composition
Time = The equivalent time at 121˚C
Heat penetration in an overloaded autoclave will be hampered as will
evacuation of air. Ensure sufficient space is left between items in a
load to allow free passage of steam. As covered in point 3, the
Measurement Points volume of medium in the bottles is directly related to the heat input.
It is therefore impossible to properly control a cycle if mixed volumes
Some autoclaves will measure chamber temperature, some will (e.g. 1 litre flasks and 20ml bottles) are put together in the same load.
measure the temperature of the medium (by use of a thermocouple), Mixed volume loads should therefore be avoided if at all possible.
some will measure both. It is important to remember that the
temperature of the medium will lag behind that of the chamber both
in heating up and cooling down.

Agitation
The continuous agitation of a medium during sterilisation greatly
shortens the heating and cooling times. Modern media preparators
have this ability which gives them a significant advantage over
general purpose autoclaves.

Figure 5 Schematic diagram showing the lag of medium
temperature compared to chamber temperature.
Volume of Medium
The heating and cooling periods of large volumes of media are longer
than for small volumes of media. Therefore, the total heat treatment
in cycles, based on holding time alone, can be significantly greater
for large volumes than for small volumes. Adjustments have to be
made to the fluid heat penetration time in the sterilisation cycle
shown below. Brecker & Bridson gave the following guide times for
heat penetration at 121˚C in glass bottles:
TABLE 3 – Heat Penetration Times
Volume Time to reach 121˚C
500ml 18 minutes
1 litre 22 minutes
2 litre 27 minutes
5 litre 37 minutes
The thickness of the glass and the shape of the container were not
specified; both these factors affect the rate of heat penetration. These
times seem to be based on both autoclave and medium being heated
from cold to 121˚C without the effective ‘holding’ at 100˚C that
occurs with modern pulsing autoclaves or with traditional autoclaves
before the outlet valve is closed.
In our experience, with a 5 minute hold at 100˚C, a 1 litre Pyrex flask
of medium will attain 121˚C within 5 minutes of the chamber
reaching that temperature. Be aware that a sterilisation cycle that
is optimum for 1 litre of medium will be excessive for l00ml
of medium.
Equipment
The equipment used for autoclaving varies from the very simple to
very complex, although they all operate by using steam under
pressure to attain temperatures above 100˚C. The crudest autoclave is
the domestic pressure cooker. We consider the lack of proper controls
make it unsuitable for routine use. Simple bench top autoclaves are
larger versions of pressure cookers but with the ability to measure the
temperature and pressure of the chamber. This equipment is suitable
for the sterilisation of small to medium sized volumes and the heating
and cooling periods are short. It is important to allow this type of
equipment to free steam before closing the outlet valve, otherwise,
not all the air will be flushed out of the chamber, reducing the
effectiveness of the equipment.
Larger laboratory autoclaves now have separate steam generation
facilities allowing rapid heating and some models evacuate the air by
vacuum and then inject the steam under pressure ensuring there are
no air pockets causing cold spots. This type of equipment will have
built-in thermocouples and chart recorders capable of monitoring
both chamber and load temperatures throughout the cycle. This type
of equipment also incorporates a cooling jacket through which cold
water is run after the completion of the holding time. For safety
reasons the door will not open until the chamber has cooled to 80˚C
- this can prolong cooling times.
Media preparators are dedicated to sterilising media. The main
advantage of this equipment is that they agitate the medium speeding
up heating and cooling and ensuring thorough mixing. They are well
equipped with both time and temperature controls. The cooling is
brought about by use of a water jacket. The only disadvantage of
media preparators is that they can only handle one medium at a time
with a cycle time of just under 1 hour. Corry et al made the following
comment. “General purpose lab autoclaves are difficult to standardise
and more consistent results are obtained by purpose-built media
sterilisers.”
It is important that whatever equipment is used is properly
maintained to ensure accuracy of gauges.

11
 References:
Stumbo C.R. (1973) Thermobacteriology in Food Processing. 2nd
Edition. Academic Press, New York.
Bridson E.Y. and Brecker A. (1970) Design and Formulation of
Microbial Culture Media. Methods in Microbiology Volume 39
edited by Norris S.R. and Ribbon D.W. Academic Press, New York.
Gardner J.F. and Peel M.M. (1986) Introduction to Sterilisation and
Disinfection. Churchill Livingstone.
Sterility indicators
Sterility indicators can be a useful tool to highlight deficiencies in
sterilising equipment, but only if they are used correctly and their
limitations are fully understood. Use of an inappropriate indicator for
the product or equipment can lead to the mistaken belief that the
process is being properly monitored. The major drawback of sterility
indicators is that even when used carefully for the correct application,
they will show that the minimum conditions have been achieved, but
give no information about the total heat input. Consequently they
give no indication as to whether the medium has been subjected to
excessive heat resulting in loss of performance of the sterilised
product. Wherever possible an appropriate equivalent heat process
should be calculated for the load to be sterilised.
Three types of indicator are commonly used, physical, chemical and
biological, examples of which are given below:
Adhesive Tape
Bowie-Dick tape is classed as a physical indicator even though it
relies upon a chemical change to produce the black diagonal stripes
which indicate the tape has been subjected to heat. More commonly
referred to as autoclave tape, it was designed for use in the
demonstration of adequate steam penetration. A cross of the tape was
placed on a sheet of steam permeable paper across a towel at the
mid-point of a stack of towels, and this was examined after
sterilisation to make sure the diagonal stripes changed to black
uniformly along the tape cross. It is therefore only an indicator of
steam penetration, as the stripes will change colour before the full
cycle is complete. In most of the applications to which Bowie-Dick
tape is put in the microbiology laboratory, it will be a very
unsatisfactory method for demonstrating the achievement of sterile
conditions.
Browne’s Tubes
The Browne’s tube is a chemical indicator comprising a heat sensitive
solution within a glass tube, which changes colour from red to green
when the tube is subjected to a high temperature for the required
length of time. In this respect it is a more reliable indicator of sterility
than Bowie-Dick tape, as it shows both temperature and time are
sufficient for sterility, whereas Bowie-Dick tape merely indicates a
heating process has occurred. However the tube only indicates that
the correct conditions have been achieved at that particular site in the
steriliser. The use of multiple tubes throughout the load is more
satisfactory, but again no information is given about the total heat
input. Furthermore, the tubes can deteriorate on storage which may
lead to premature colour change and false information as to the
performance of the sterilising equipment.
Spore Indicators
The spores of Bacillus stearothermophilus are extremely resistant to
heat and can thus be used as a biological indicator of sterilisation.
Spore strips prepared in the laboratory or commercially produced
strips, are placed in various parts of the load. After the cycle is
complete they are removed aseptically and placed in a bottle of
thioglycollate medium or cooked meat medium, and incubated at
55˚C for 7 days, during which they are examined for growth. Again
12
this type of indicator shows if the time and temperature requirements
have been achieved but not if excessive heat has been used. Like
Brown’s tubes, spore strips only show ‘spot’ conditions, and may
deteriorate on storage giving false results. However, the length of
time needed to obtain a result means they are inconvenient for routine
use.
All the above indicators are a compromise in the absence of
accurate steriliser control, and none can demonstrate
overheating of the medium. The only way to ensure sterilisation
without overheating, is to use thermocouples introduced into the
medium being processed, linked to chart recorders, and in
accordance with a previously determined equivalent heat process
for the equipment and volume of media being sterilised.
Molten Media
Holding molten media in water baths at 47 ˚C or above for more than
a few hours should be avoided. The extra heat input will damage both
the nutrient and gelling properties of the medium. Holding at 47˚C
overnight will result in noticeable denaturation of agar gel . Ideally,
media should be poured as soon as possible; holding at 47˚C for 4
hours should be the maximum holding time any medium is subjected
to. The addition of extra agar to compensate for the reduction in
gelling caused by overheating is not recommended as the overheating
will have also damaged the nutrient properties.
Re-melting Agar
Most media will stand re-melting once in a boiling water bath. Again,
the total heat input into the medium has to be considered. If a medium
is likely to be allowed to set then re-melted, it is doubly important
that holding at 47˚C in a water bath is minimised. Never re-melt an
agar more than once.
Pouring Plates
Media should be cooled, in a water bath, to 47˚C before pouring.
Immediately before pouring the medium should be swirled gently to
ensure thorough mixing. Plates should contain at least 5mm. of
medium if they are to be stored before use to minimise the effects of
the drying that will occur during storage. Single vent petri dishes lose
moisture more slowly than triple vent dishes but they are also more
prone to condensation problems if not adequately dried. The amount
of medium specified for poured plate techniques is generally less
because the medium is not stored in vented petri dishes. When
layered plates are used the total depth should not be less than 5 mm.
NB: -It is important to note that the tubing through which the medium
is dispensed can retain inhibitory substances on the inner surfaces.
This means it is necessary to use separate tubing for non-selective
media and for inhibitory/antibiotic supplemented media. Failure to do
this can result in traces of inhibitory substances being incorporated
into non-selective agars, thereby reducing their ability to support the
growth of fastidious organisms.
Drying Plates
In order to achieve well isolated colonies on agar plates streaked for
single colonies, it is necessary to make sure the agar is free from
surface moisture by drying, but caution must be taken to ensure over
drying does not occur as this can be detrimental to the performance
of the agar. It is worth noting that numerous procedures in the stages
of preparation of agar plates can result in loss of moisture, and the
sum of these can impair performance if not considered and kept to a
minimum. Boiling of the medium to dissolve agar before autoclaving,
autoclaving, and pouring at temperatures above 50˚C can all result in
moisture loss, and so media should only be boiled before autoclaving
if absolutely necessary, the autoclaving carefully controlled in an
equivalent heat process, and cooled to 47˚C before pouring.
There are 2 commonly used methods of drying plates:

agar
Lid
Fig. 6 Rapid drying of plates
1. Carefully place the plate in the incubator with the medium
containing side up. Lift the base of the dish up and rest it on the
lid as in the diagram. This allows excess moisture to evaporate
whilst minimising the possibility of contaminating the agar.
2. Plates are allowed to stand (with lids on) overnight on the bench.
The effectiveness of this technique will depend on the venting of
the petri dishes, the humidity of the surroundings and the amount
of excess moisture present. The practice of incubating plates
overnight could lead to excessive moisture loss and reduced
medium performance. To check for sterility, incubate a
representative sample at the temperature and time parameters used
for performing the test for which the medium is employed.
Sterile Supplements
The addition of sterile supplements is performed after the medium
has been sterilised and cooled to approximately 47˚C. Most sterile
supplements such as blood, serum and antibiotics are denatured at
higher temperatures. The supplement itself should be warmed up to
room temperature before adding to the medium and it should be
added whilst mixing to prevent the formation of ‘cold spots’ and
premature gelling of the agar. Care must be taken to observe strict
aseptic precautions whilst adding the supplement. The supplemented
medium should be thoroughly mixed by swirling before any plates
are poured. Any antibiotic supplement reconstituted but not used
should be thrown away. Antibiotics vary widely in their stability once
reconstituted and can deteriorate rapidly - even deep frozen. Serum
products are best stored frozen then thawed. It is wise to aliquot
serum on arrival into suitable volumes to be frozen until required. To
produce ‘chocolate’ or heated blood plates add the sterile blood to the
medium at 80˚C or add at a lower temperature and gently re-heat with
frequent swirling until the medium ‘chocolates’. This chocolating of
the blood destroys enzymes which would otherwise inactivate the
nicotinamide adenosinedinucleotide (NAD or V factor) required for
growth by Haemophilus spp.
Storage of prepared media
The shelf life of prepared media is dependent upon the composition
of the medium, the form in which it is stored and the conditions of
storage. All media should be stored in the dark to prevent the
formation of bacteriostatic and bactericidal substances (e.g.
peroxides).
Each time a batch of medium is prepared some form of performance
quality control should be carried out. It is unwise to use media
beyond their minimum shelf life without repeating the quality control
and comparing it with the initial result.
Plates
Most plates stored medium side up at 4˚C in the dark will have a
minimum life of 7 days. This can be extended up to 3-4 weeks for
simple nutrient media by using some form of airtight packing. Plates
containing antibiotics have a shelf life governed by the stability of the
antibiotics. Generally speaking it is unwise to extend the shelf life of
an antibiotic containing medium beyond 7 days. As a medium loses
moisture the ingredients of the medium will be concentrated making
selective media progressively more selective. A plate with an original
medium depth of 5mm will have its ingredients concentrated 20% by
the time its gel has shrunk to a depth of 4 mm. Plates showing visible
signs of shrinkage (drying) should not be used. Plates should be
brought up to room temperature before use to avoid any ‘thermal’
shock to the bacteria. Any plates left on the bench for more than 8
hours should be discarded as unsuitable for use.
Bottled Media
Any medium in an airtight capped container will have a longer shelf
life than in a plate. Many simple nutrient media can be stored at 15-
20˚C for 3 months in the dark. Indeed, many can be stored for longer
but we advise repeat of a simple Q.C. procedure after 3 months. If a
medium is stored as a gel and then is re-heated before use, repeat of
a simple Q.C. procedure is recommended. Bottled media containing
antibiotics will have shelf lives governed by the activity of the
antibiotic.
Troubleshooting Guide
The laboratory that routinely quality controls the media it produces
will occasionally find it has produced a batch that is not up to
standard. The following is a list of potential problems and their
possible causes:
FAULT POSSIBLE CAUSE
Soft Gel Excess heat, pH too low causing acid
hydrolysis, inaccurate weighing, inadequate
mixing, agar not dissolved.
pH incorrect Alkaline glassware, impure water, overheating,
chemical contamination, pH taken at wrong
temperature, pH equipment faulty or poorly
standardised, deterioration of dehydrated
medium. (See specific section on pH of culture
media)
Abnormal colour Impure water, dirty glassware, deterioration of
dehydrated medium, excess heat, pH wrong.
Darkening Excess heat, deterioration of dehydrated
medium.
Precipitation Excess heat, deterioration of dehydrated
medium, impure water or glassware.
Toxicity Excess heat (scorching or burning),
deterioration of dehydrated medium.
Poor growth Contaminated water or glassware, deterioration
of dehydrated medium, incorrect weighing and
mixing. Excess heat.
Poor selective or Contaminated water or glassware, incorrect
differential weighing and mixing, deterioration of
properties dehydrated medium. Excess heat.
13
 5. Note the last point at which growth occurs, on the test and control
Quality Control of plates, and record the segment and line, e.g. C4 or D5 etc. This is
the end point and can be used to calculate the absolute growth
Culture Media index (AGI) and relative growth index (RGI) of a medium. The
AGI is obtained by noting the end point in Table 4. below and this
gives the AGI. e.g. if the end point is C4 then the AGI is 75.
The routine quality control of culture media is an essential ‘good
laboratory practice’ necessary to maintain the standards and
performance of any bacteriological culture technique. More recently
it is a requirement of many laboratory accreditation schemes such as
NAMAS, and CLAS etc.
We recommend the following: A full quality control when a new
batch of dehydrated medium is introduced into the laboratory. This
full Q.C. to be repeated every 3-4 months on opened containers. A
short-form quality control when a new batch of medium is
reconstituted from dehydrated media previously quality controlled.
A short-form quality control whenever prepared media are used
beyond their minimum shelf life or are re-heated more than once.
Keeping of records - both full and short-form quality controls - so
that trends, e.g. fall off in performance can be detected. Each medium
used in the laboratory should have its own quality control protocol
and the necessary organisms should be maintained.

Techniques
The parameters of growth on culture media are:
● Lag time
● Organisms grown from known inoculum Figure 7 Schematic diagram of the ecometric method
● Organisms inhibited from known inoculum
● Comparative growth with standard inoculum TABLE 4 – Absolute Growth Index (AGI)
● Comparative inhibition with standard inoculum
Al = 5 Bl = 10 Cl = 15 Dl = 20
● Colony size
A2 = 25 B2 = 30 C2 = 35 D2 = 40
● Colonial appearance
A3 = 45 B3 = 50 C3 = 55 D3 = 60
In practice absolute measurements of growth are time-consuming or
A4 = 65 B4 = 70 C4 = 75 D4 = 80
require sophisticated equipment whilst colonial appearance is
subjective and difficult to record. Colony size is easily measured but A5 = 85 B5 = 90 C5 = 95 D5 = 100
is an insensitive indicator of performance. Comparative methods are
the most suitable ones for routine quality control of culture media The RGI is a comparison of the AGI of the test plate and that of the
they can be used for comparisons of growth and inhibition. The control plate. For example:
ecometric technique of Mossel is simple and gives numerical
readings that can form the basis of records suitable for trend analysis. Plate A—non-selective control—end point A5, AGI = 85
Both absolute growth index (AGI) and relative growth index (RGI) Plate B—selective agar test —end point C4, AGI = 75
can be obtained by this method.
Using the formula RGI = AGI Test x 100
AGI Control
RGI = 75 x 100
85
The Ecometric Technique = 88.24 %
This plating technique is simple enough to use in both full and short- Thus, for this particular organism the test plate was 88.24% as
form quality controls. It is based on streaking an inoculum to efficient as the non-selective control plate. The performance of a
extinction. The results obtained can be compared with previous selective agar can be thoroughly tested using an organism which it
batches of the same medium or with batches of the same medium has been designed to isolate and one which it is designed to inhibit.
from different manufacturers. The results can also be compared with For the former the RGI should be as close to 100 as possible and the
results obtained using the same organisms on non-selective media. latter as close to 0 as possible. The very nature of selective agars and
the bacteria which must be selected or repressed means that
effectiveness of different media will vary quite considerably. For
example, Campylobacter Selective Agar (LAB 112) is a very good
selective medium as it will give high RGI’s for Campylobacter spp.
Method and achieve suppression of bacteria without inhibiting the organism
1. Inoculate 5mls. of Brain Heart Infusion Broth (LAB 49) with a which is to be isolated. Examples of less effective selection occurs
loopful of the chosen test organism (see individual products) and with Salmonella media, where there is a close relationship between
incubate for 4 hours. pathogens (Salmonella and Shigella) and the other enteric organisms,
making it difficult to inhibit one without reducing the isolation rate of
2. Divide the plate to be tested into quarters designated A, B, C and the other. The onus is therefore on the end user to decide which
D as shown below: medium is best for their use. Having found a medium which is
3. Charge a 1 microlitre loop with the incubated 4-hour culture and suitable, use that as the yardstick to measure the performance of new
spread the test plate, going from Al - Bl - Cl - Dl - A2 - B2 etc. batches and to check that the efficiency of all media-making
without flaming or recharging the loop and finish at D5. processes are being maintained. It is important to note that the
ecometric method is a simple technique by which laboratories can
4. Repeat this process with a control plate. For a batch of dehydrated check the media they are producing. However, it has to be performed
media new to the laboratory, use a suitable non-inhibitory plate. with care, as simple errors such as holding the inoculation loop at a
For a new batch of plates from the laboratory stock of dehydrated different angle may introduce errors. This can be overcome by using
medium, use a plate of the same medium from the last batch to a spiral plater to produce the plates to ensure the plating method is
check consistency. Incubate both plates for 18 hours. identical for test and control agars.
14
Reference: Liquid Media
Mossel D.A.A. et al (1983) Quality Assurance of Selective Culture Another simple technique can be used to quality control fluid media
Media for Bacteria, Moulds and Yeasts: An Attempt at Standardisation such as Fastidious Anaerobe Broth (LAB 71) and Fluid
at the International Level. J. Appl. Bacteriol. 54 313-327 Thioglycollate Medium (LAB 25).
1. Prepare tenfold dilutions (to 10-12) of an overnight culture of the
test organism in Tryptone Soy Broth (LAB 4).
2. Add 1ml. of each dilution to 9mls. of test and control broths.
Productivity Ratio (P.R.) Incubate at 37˚C for 18 hours.
3. Examine the broths and note the highest dilution showing growth
Determining the productivity ratio of a medium is another way to
(turbidity of the broth).
check its performance related to a control medium, which should be
a nutritious agar such as Tryptone Soy Agar (LAB 11). The inoculum This method can be used in conjunction with the Miles-Misra
used must be the same for both media and the P.R. is calculated by technique to demonstrate recovery of known levels of CFU’s in broth
counting the colonies on the test and control media: media.
P.R. = No. of colonies on test x dilution factor .
No. of colonies on control x dilution factor
A simple method to obtain the P.R. of a medium is using the Modified
Miles-Misra technique:
1. Prepare tenfold dilutions of an overnight culture of the test
organism in Buffered Peptone Water (LAB 46).
2. Divide the test plates into quadrants and mark each quadrant with the
dilution to be used as shown below. Repeat with the control plates.
3. Starting with the highest dilution (10-8 in example below) place
one drop of the dilutions on the relevant quadrant. Repeat for
control plate.
4. Spread each drop over the quadrant and incubate the plates at 37˚C
for 18 hours.
5. Count colonies at the lowest dilution they can be easily counted,
for both test and control. Calculate the P.R.

Figure 8 Template for Miles-Misra plates

For example: Test = 20 colonies at 10 -3 dilution

Control = 25 colonies at 10 -3 dilution
P.R. = 20 x 103
25 x 103
P.R. = 20
25
P.R. = 0.8
Thus in this example the test medium is 80% as efficient as the
control medium.
This technique is not as simple as the ecometric method and is
probably more usefully used to quality control broth media.
However, it is less prone to error than the ecometric technique and its
accuracy can be further increased by using duplicate plates.

15
 Templates for the
Ecometric Method
Mark the bottom of the plate as shown below,
using a permanent marker pen.

Once the quadrants are marked, place the plate face-up on

the template below. Charge a lµl loop with a 4-hour broth
culture and spread the plate, going from
Al-Bl-Cl-Dl-A2-B2-etc. without flaming or recharging the
loop. Replace on template after incubation to read result.

16
 Suggested Quality Control
Record Sheet

Quality Control Record (A)

DATE: VOLUME OF WATER ADDED:

MEDIUM: CONDUCTIVITY OF WATER ADDED:

LAB CODE: METHOD OF STERILISATION:

BATCH NO: SUPPLEMENTS ADDED:

WEIGHT OF AGAR: EXPIRY DATE OF SUPPLEMENTS:

CONTROL MEDIUM USED:

End Point RG1

Q.C. Organism RG1
Test Control Test Control
1

End Point RG1 Mean

Q.C. Organism RG1
Test Control Test Control RG1
1

N.B. The same inoculum should be used for all plates. All plates must be spread at the same time, and incubated under identical conditions.

17
 Suggested Quality Control
Record Sheet

Quality Control Record (B)

MEDIUM: DATE RECEIVED:

BATCH NO: DATE OPENED:

APPEARANCE OF POWDER WHEN OPENED:

Date RGI Appearance Comments

1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4

N.B. Comparisons of relative growth index are only useful to show a trend in the condition of the medium. Variation between individual figures will occur due to
inevitable inaccuracies of the technique.
18
 Organism/Bacteria ATCC NCTC Other
Preservation of Escherichia coli 11775 9001
Stock Cultures Escherichia coli 10090
Escherichia coli NCIMB 9483
The following method has been used in our laboratory for several Escherichia coli O:111 9111
years. It does not require ultra low temperature equipment, a
domestic freezer at -20˚C will suffice. Haemophilus influenzae 10479
Hafnia alvei 13337 8109
Preservation of Bacteria using a
Modified Glass Bead Technique Klebsiella oxytoca CMCC 2703
1. Grow the organism on an appropriate solid medium until a heavy Lactobacillus acidophilus 19992
growth is obtained. Use several plates with organisms forming
Lactobacillus brevis 14869
small colonies.
Lactobacillus bulgaricus 11842
2. Remove growth from plate using a sterile cotton swab and
emulsify in 10% w/v glycerol in Brain Heart Infusion Broth. Lactobacillus casei 7469
3. Carefully pipette the suspension on to sterile glass beads with a Lactobacillus plantarium 14917
hole for threading, then place them in a plastic freezer tube.
Lactobacillus sake 15521
Replace the cap and tap the bottle to remove the bubbles from
bead centres. Lactobacillus viridescens 12706
4. Pipette off the excess fluid into phenolic disinfectant in a discard Leuconostoc mesenteroides DSM 20241
jar. Cap the tube, label and place in freezer.
Microbacterium flavum NCIMB 8707
5. To recover the organisms, remove the beads using forceps, roll on
Microbacterium lacticum NCIMB 8540
an appropriate medium and streak out to obtain single colonies.
Micrococcus luteus 15307 2665
6. There are potential problems with this technique which can be
avoided: Moraxella sp. NCIMB 10762
(i) The tubes should be duplicated and then stored in separate pots Morganella morganii 8076h 235 NCIMB 235
- one for routine access, the other as a back-up in a separate
freezer. Neisseria gonorrhoeae 19242 8375
(ii) The frozen beads will soon defrost on the bench. A block of Pediococcus damnosus 29258
aluminium or copper drilled out to take the plastic tubes will
prevent thawing. The block is kept in the freezer along with the Proteus mirabilis 25933
beads, and removed wearing a glove. Pseudomonas aeruginosa 25668 10662
A commercially available form of the above system called Protect Pseudomonas fluorescens 10038
Beads is available from LAB M, please call for further details.
Details of recommended QC strains of organisms are contained in the Pseudomonas fragi 10689
individual entries for media. QC Assays, which combines certified Pseudomonas putida 10936
media control cultures and Protect storage beads, is also available as
a convenient kit. Salmonella dublin 9676

TABLE – 5 Culture collection strains for the quality control of Salmonella enteritidis 5188
culture media Salmonella gallinarum 9240
Salmonella saint paul 6022
Organism/Bacteria ATCC NCTC Other Salmonella senftenberg 10384
Acinetobacter calcoaceticus 15309 5866 Salmonella typhi 8394
Aerococcus viridans 11563 8251 Salmonella typhimurium 13311 74
Aeromonas hydrophila 7966 8049 NCIMB 9240 Salmonella virchow 5742
Bacillus cereus 11778 Serratia liquefaciens NCIMB 9321
Bacillus coagulans 7050 10334 NCIMB 9365 Serratia marcescens 13880 10211
Bacillus licheniformis 14580 10341 NCIMB 9375 Shigella flexneri 29903
Bacillus megaterium 14581 10342 NCIMB 9376 Shigella sonnei 29930
Bacillus subtilis 5398 Staphylococcus aureus 25923 637
Bacteroides fragilis 25285 9343 Staphylococcus aureus 6538
Brocothrix thermosphacta 11509 10822 NCIMB 10018 Staphylococcus aureus CMCC 2656
Campylobacter coli 11366 CIP 7080 Staphylococcus epidermidis 19990 1466
Campylobacter jejuni 11168 Streptococcus bovis Kiel 27421
Campylobacter laridis 11352 Streptococcus lactis 19435 6681 NCIMB 6681
Citrobacter freundii 6272 Streptococcus pneumoniae 10319
Clostridium bifermentans 506 Streptococcus pyogenes 8198
Clostridium perfringens 8237 Streptococcus thermophilus 19258
Clostridium perfringens 8238 Vibrio cholerae 11348
Corynebacterium diptheriae 19409 3984 Vibrio fluvialis 11327
Enterobacter aerogenes 13048 10006 Vibrio parahaemolyticus 11344
Enterobacter cloacae 13047 10005 Yersinia enterocolitica 9610 CCUG 11291
Enterococcus faecalis 8043 775 Yersinia enterocolitica CCUG 4588
Enterococcus faecium DSM 2918 Yersinia enterocolitica CCUG 4586
19
 Yeasts & Moulds ATTC NCYC CMI
Alternaria alternata 89343
Laboratory Accreditation
Aspergillus amstelodami 17455
The accreditation of a microbial testing facility must be the goal of all
Aspergillus flavus 91856
involved in the production of goods or provision of services to
Aspergillus niger NCIMB 50097 consumers. Accreditation is the means by which external assessment
ensures that the facility, personnel, and methods of a testing
Aurobasidium cladosporoides 45534
laboratory are appropriate, monitored and challenged within a
Candida albicans 18804 continuously improving quality system.
Cladosporum cladosporoides 45534 The impetus for laboratory accreditation has, in the past, differed by
industry sector; for example pharmaceutical laboratories have for
Debaryomyces kloeckeri 10620
many years been subjected to Good Laboratory Practice and regular
Fusarium moniliforme 61274 inspection by bodies such as the FDA and MCA. However, more
recently, the benefits of accreditation are being applied in diagnostic
Hansenula anomala 432
and food testing laboratories, and probably the greatest area of
Mucor racemosus 17364 growth is the food industry.
Penicillium cyclopium 19795 The impetus for this growth is essentially the Food Safety Act of
1990, which laid down the requirements for food manufacturers with
Pichia burtonii 439
regard to the provision of safe food, and introducing the ‘Due
Rhizopus stolonifer 61269 Diligence Defence’ whereby the defendant must show that all
reasonable precautions which could have been taken to avoid an
Saccharomyces cerevisiae 79
incident were indeed in place. It follows from this that ensuring the
Zygosaccharomyces rouxii 1522 testing laboratory is operating to agreed standards could be
interpreted as a reasonable precaution which should be taken.
Abbreviation key: There are a number of schemes which provide assessment and
ATCC American Type Culture Collection, 12301 Parklawn Drive certification of laboratory performance; NAMAS, CLAS, MAFF, GLP,
Rockville, Maryland, USA. ISO9000, CPA. The choice of scheme is bewildering, but narrowed
depending upon the nature of testing carried out, e.g. a food laboratory
CCUG Culture Collection, University of Goteborg, Guldhedsgaten would not use CPA (Clinical Pathology Accreditation scheme) and
lOA, 5-413 46, Goteborg, Sweden. may also consider GLP (Good Laboratory Practice) not targeted to its
CMCC Colworth Microbiological Culture Collection, Colworth specific needs. In addition, other schemes are available and the choice
House, Sharnbrook, Bedford MK44 1 LQ, U. K. [strains may continue to expand. The choice of scheme should not be taken
from this source will be deposited with NCIMB]. lightly, and should take into consideration the demands of the
CMI Commonwealth Mycological Institute, Ferry Lane, Kew, laboratory customers (internal and external), and industry practice.
Surrey, UK Further consideration might be given to possible future legislation, as
CIP Collection de l’Institut Pasteur, Paris, France. changes between systems will almost certainly involve extra cost if it
becomes necessary. One driving force for any future regulations is
DSM Deutsche Sammlung von Mikroorganismen, (German
likely to be The Official Control of Foodstuffs Directive (89/397)
Collection of Micro-organisms), Grisebachstrasse 8, D-
which is an EC Directive concerned with the establishment of the
3400, Gottingen, FRG.
Single Market in Foodstuffs, and sets out the requirements for official
KIEL Federal Dairy Research Institute, Hermann Weigmanstrasse food testing laboratories. Part of this sets out the requirements for
1, Kiel, FRG. accreditation, stating that this must be to the EN 45000 Series of
NCIMB National Collections of Industrial and Marine Bacteria Ltd., standards. Currently only NAMAS in the UK meet the criteria for
AURIS Business Centre, 23 St. Machar Drive, Aberdeen, accreditation bodies laid down in European Standard EN 45003.
AB2 1RY. Scotland. Whilst these standards relate specifically to official food testing
laboratories, i.e. those laboratories who will be monitoring food with
NCTC National Collection of Type Cultures, Central Public Health
regard to enforcement of the law, it will reflect the best practice to
Laboratory, Colindale Avenue, London NW9 5HT, U. K.
which all testing facilities should aspire.
NCYC National Collection of Yeast Cultures, Food Research
Institute,Colney Lane, Norwich NR4 7UA, U. K.
The above table is adapted from the original published by the IUMS
- ICFMH Working party on Culture media. Int. J. Food Microbiol.
1987 5 297 -299.

20
 Hazard Analysis Critical
Control Points (HACCP)
In the same way that European legislation is driving the issue of
laboratory accreditation, it also affects other areas where the expertise
of the food microbiologist is essential, and a prime example of this is
in the use of HACCP in the production of food.
The European Directive on the Hygiene of Foodstuffs 93/43/EEC
includes the requirement that all businesses shall develop a system in
accordance with the HACCP principles to identify and control food
hazards.
Developed in the USA to ensure the absolute safety of food used in
the NASA space programme (can you imagine suffering food
poisoning in zero gravity?), it is a system which recognises the
limitations of end-product testing and identifies the points in the
production process critical to producing a safe product. It does not
replace end product testing; it puts end product testing into the correct
context as a validation that the control system is functioning
correctly.
The hazards inherent in any food production process are specific to
that process in that company at that time, and so the HACCP system
used to control it also has to be designed specifically and reviewed
regularly.
Like Quality Systems HACCP can only work with the backing of the
entire organisation starting from the top and requires a team approach
to implement. The team must be multi-disciplinary and typically will
include personnel from production, engineering, microbiology, and
hygiene.
The team will then follow the principles of HACCP to implement a
system which will ensure the safety of the product:
Hazard Analysis
This is the identification of the hazards associated with the
production of the product and the ranking of the risk involved with
each and involves the input of relevant members of the team with the
necessary technical background. Once hazards are agreed then the
CCP’s can be identified.
Critical Control Points
This is any process, location, practice, or raw material which, if not
controlled, will allow the product to pose a threat to consumer of the
product. For example, if a product must reach 70˚C on cooking,
failure to do so would pose a threat to the consumer. This is a critical
control point.
Establish Control Criteria
The control criteria must be a parameter or practice which can be
monitored, adjusted and verified in a time scale relevant to the
production of the food. For example, oven temperature can be set
accurately determined limits, monitored by probes and adjusted in
real time. With the advent of rapid technology the hygienic status of
surfaces can be measured in real time and re-cleaning performed if
necessary.
Establish Monitoring Procedures
The parameters which ensure that the critical control point is actually
in control of the safety of the product, which allow positive feedback
and rapid corrective action. These are generally physical (time,
temperature) chemical (pH, biocide levels, aw, ATP) or visual
(handling procedures, equipment). Documentation of all monitoring
results is essential.
Microbiology is seldom an effective monitoring tool due to the time
involved. However it may be useful for monitoring contamination
levels of raw materials prior to use.
Establish Corrective Action Plans
Agreed actions necessary to regain control if monitoring of a CCP
indicates that it is not controlling the safety of the product.
Documentation is again essential to show the ‘quality loop’ -
monitoring > non-conformance > action > monitoring > control.
Verification Procedures
Further actions taken to verify the monitoring of CCP’s, to ensure that
the control is in place, and the monitoring parameters are correct. If a
product fails verification when the monitoring process shows no
problem, it indicates a failure in the system and a review is necessary
to identify a missing CCP or other source of the problem.
End product testing falls into this category, as could measuring the
level of microbial contamination of the manufacturing environment.
Auditing and Review
The system must be reviewed and audited at regular intervals to
ensure that nothing has changed which would affect the efficacy of
the control process, or omission of important detail. This must
include audit of the documentation and monitoring records, as well as
physical examination of the whole procedure. As with verification,
any areas of concern must be subject to the corrective action and
demonstrate the ‘quality loop’.
Like all systems, HACCP is only effective if implemented properly
with the co-operation of all employees involved. A poorly operated
system could make things appear under control, when the reality is
that potentially dangerous products may be produced.
Developing a HACCP system can be a time consuming and complex
business, and the above representation is vastly over-simplified. It is
therefore advisable to seek professional help via research associations
or expert consultants with many years experience of developing
HACCP systems.
21
22 This page left intentionally blank
 Microbiology Methods Total Viable Count
(Total Aerobic Count)
We have set out some of the more common methods in use, plus one Prepare an appropriate 1:10 dilution of the sample
or two newer methods, indicating the LAB M products required to e.g. 10g* sample + 90ml MRD LAB103
|
perform them. This is not intended to be a comprehensive list, nor |
replace other sources of information, but as a simple guide only. Homogenise for 2 mins
There are numerous sources of information regarding microbiology |
|
methods, just some of which are listed below:
Prepare serial dilutions by adding 1ml of homogenate to 9ml
of MRD LAB103
|
British Standards Institute, Customer Services, Linford Wood, Milton |
Keynes, MK14 6LE. Numerous microbiological standards e.g. Inoculate 1ml of appropriate dilutions into a sterile petri dish and
BS5763 Methods for Microbiological examination of food and add aseptically 15ml of molten, cooled:
animal feeding stuffs. Part 11. Enumeration of Bacillus cereus. Plate Count Agar APHA LAB010 or
Plate Count Agar LAB149 or
Milk Plate Count Agar LAB115 or
Compendium of Methods for the Microbiological Examination of Milk Agar LAB019 or
Foods(1992) Third edition. Edited by Vanderzant, C. and Yeast Extract Agar LAB018
Splittstoesser D.F. American Public Health Association. ISBN 0- |
|
87553-173-3 Incubate at the appropriate temperature to count the required
bacterial population:
30˚C for 48hrs (aerobic mesotroph count)
Food and Drug Administration. Bacteriological Analytical Manual 21˚C for 5 days or 6.5˚C for 10 days (aerobic psycrotroph count)
7th edition (1992) AOAC International. ISBN 0-935584-49-8 55˚C for 48hrs (aerobic thermotroph count)
|
|
Foodborne Pathogens An Illustrated Text (1991) Varnam, A.H. and Select plates with between 30 and 300 colonies for enumeration.
Evans, M.G. Wolfe Publishing (now Times Mirror International). If the highest dilution has more than this use 300 as the figure to
calculate the original count and express as ‘greater than…’
ISBN 0-7234-1521-8) |
|
Calculate the original count in the sample expressed as CFU’s
Manual of Microbiological Methods for the Food and Drink Industry. per gram or ml (The number should be given in standard scientific
Technical Manual No. 43 2nd edition. (1995) Campden & notation e.g. 2.4 x 103)
Chorleywood Food Research Association, Chipping Campden, This method can be adapted to perform an anaerobic count by
Goucestershire, GL55 6LD. incubating in anaerobic conditions. However incubation times may
vary to those above and specific texts should be consulted.
Remember that facultative organisms will give a count for both the
Micro organisms in foods - Their significance and methods of aerobic and anaerobic counts.
enumeration 2nd edition (1978) ICMSF edited by Thatcher F.S. and *For liquid samples use 10ml to 90ml.
Clark D.S. ISBN 0-8020-2293-6 No homogenisation is required prior to preparing serial dilutions.

Practical Food Microbiology. Methods for the Examination of Food

for Micro organisms of Public Health Significance 2nd edition (1995)
Edited by Roberts D. Hooper W. and Greenwood M. Public Health Coliform/Enterobacteriaceae
Laboratory Service ISBN 0-901144-36-3
(Enumeration)
Prepare an appropriate 1:10 dilution of the sample
Reports on Public Health and Medical Subjects No.71. The e.g. 10g* sample + 90ml MRD LAB103
Bacteriological Examination of Drinking Water Supplies 1994. |
|
Methods for the Examination of Waters and Associated Materials.
HMSO. ISBN 0-11-751675-9 Homogenise for 2 mins
|
|
Prepare serial dilutions by adding 1ml of homogenate to 9ml
of MRD LAB103
|
|
Inoculate 1ml of appropriate dilutions into a sterile petri dish and
add aseptically 15ml of molten, cooled:
VRBA LAB031 (Coliform count)
or VRBGA LAB088 (Enterobacteriaceae count)
When set, overlay with more of the same agar to cover surface
|
|
Incubate at 37˚C for 24hr
(some USA methods 35˚C, coliforms for dairy purposes 30˚C)
|
|
Count all colonies >0.5mm diameter present on the plate. Select
plates with between 15 and 150 colonies for enumeration. If the
highest dilution has more than this use 150 as the figure to calculate
the original count and express as ‘greater than…’
|
|
Calculate the original count in the sample expressed as CFU’s per
gram or ml (The number should be given in standard scientific
notation e.g. 2.4 x 103)
*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.

23
 Escherichia coli E.coli
(Presence or Absence) (Enumeration Without Membranes)
Prepare an appropriate 1:10 dilution of the sample Prepare an appropriate 1:10 dilution of the sample
e.g. 10g* sample + 90ml MRD LAB103 e.g. 10g* sample + 90ml MRD LAB103
| |
| |
Homogenise for 2 mins Homogenise for 2 mins
| |
| |
To detect presence in 0.1g of product, inoculate 1ml of homogenate Prepare serial dilutions by adding 1ml of homogenate to 9ml of
into 10ml of single strength MacConkey Broth Purple LAB005 or MRD LAB103
Brilliant Green Bile Broth LAB051 (with inverted Durham tubes). |
|
To detect presence in 1g of product, inoculate 10ml of double
strength medium with 10ml of homogenate Inoculate the surface of Tryptone Bile Glucuronide Agar (LAB162)
| |
| |
Incubate at 37˚C for 24hr and examine for acid and gas Incubate at 30˚C for 4hrs then transfer to 44 ± 0.5˚C for 18-24hrs
|
(LAB005) or turbidity and gas (LAB051) |
(30˚C is used in the dairy industry) *For liquid samples use 10ml to 90ml.
| No homogenisation is required prior to preparing serial dilutions.
|
If negative incubate for a further 24hrs, and examine again
|
|
Subculture 0.1ml of positive broth into 10ml single strength
Brilliant Green Bile Broth (LAB051), and
Tryptone Water (LAB129)
E.coli O157:H7
| (Presence or Absence)
|
Incubate at 44 ± 0.5˚C for 24hrs and examine for turbidity Add 25g sample to 225ml of Modified Tryptone Soy Broth
and gas production (MTSB) LAB165
| |
| |
If negative incubate for a further 24hrs and examine again Homogenise for 2 mins
| |
| |
For all positive tubes add 2-4 drops of indole reagent to the Incubate at 42˚C for 6 and 24hrs
matching Tryptone Water. Wait one minute and examine for a red |
|
colour in the alcohol layer at the top of the broth, indicating a
positive indole reaction Perform Captivate™
| immunomagnetic separation
| at 6 and 24hrs
Record result as presumptive presence of E.coli in |
|
0.1g or 1g of product as appropriate
Inoculate 50µl of Captivate™ beads onto SMAC
*For liquid samples use 10ml to 90ml. (LAB161) and CT SMAC (LAB161 + X161).
No homogenisation is required prior to preparing serial dilutions. Incubate plates at 37˚C for 24hrs.
|
|
Examine plates for sorbitol negative colonies
|
|
Confirm as E.coli O157 by serology and biochemistry
E.coli (commercial ID kits)
(Enumeration Using Membranes)
It should be noted that not all O157 isolates produce the
Prepare an appropriate 1:10 dilution of the sample verocytotxin which is the mode of pathogenesis of the organism.
e.g. 10g* sample + 90ml MRD LAB103 Furthermore, other serovars of E.coli can produce the verocytotxin
| and therefore be pathogenic for humans.
|
Homogenise for 2 mins
|
|
Prepare serial dilutions by adding 1ml of homogenate to 9ml of
MRD LAB103
|
|
Inoculate membrane on the surface of Minerals
Modified Glutamate Agar (LAB080A + LAB080B
+ 12g/litre of Agar No.2 MC006)
|
|
Incubate at 37˚C for 4hrs then transfer membrane
to a Tryptone Bile Agar plate (LAB072)
|
|
Incubate with membrane uppermost at 44 ± 0.5˚C for 18-24hrs
|
|
Put 2ml of Vracko-Sherris reagent into lid of dish, and place
membrane on top. Allow to soak in reagent for 5 min.
|
|
Dry membrane in natural daylight or expose to UV for 5-30 min.
|
|
Indole positive colonies will turn pink. Count all pink colonies as
presumptive E.coli and calculate the original count in the sample,
expressed as CFU’s per gram or ml. (The number should be given
in standard scientific notation e.g. 2.4 x 103
*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.
24
Yeasts and Moulds Pseudomonas species
(Enumeration) (Enumeration)
Prepare an appropriate 1:10 dilution of the sample Prepare an appropriate 1:10 dilution of the sample
e.g. 10g* sample + 90ml MRD LAB103 e.g. 10g* sample + 90ml MRD LAB103
| |
| |
Homogenise for 2 mins Homogenise for 2 mins
| |
| |
Prepare serial dilutions by adding 1ml of homogenate to 9ml of Prepare serial dilutions by adding 1ml of homogenate to 9ml of
MRD LAB103 MRD LAB103
| |
| |
Inoculate 1ml of appropriate dilutions onto the surface of : Inoculate 0.1ml or 0.5ml by spreading over the surface of
Rose Bengal Chloramphenicol Agar (LAB036), or Pseudomonas Agar (LAB108) supplemented with CFC (X108)
Malt Extract Agar (LAB037), or |
|
Oxytetracycline Glucose Yeast Extract Agar (LAB089)
| Incubate at 25˚C for 48hrs
| |
|
Incubate at 25˚C for 5 days
| Select plates with between 15 and 150 colonies for enumeration.
| If the highest dilution has more than this use 150 as the figure to
Select plates with between 15 and 150 colonies for enumeration. calculate the original count and express as ‘greater than…’
If the highest dilution has more than this use 150 as the figure to |
|
calculate the original count and express as ‘greater than…’
| Calculate the original count in the sample expressed as CFU’s per
| gram or ml (The number should be given in standard scientific
Calculate the original count in the sample expressed as CFU’s notation e.g. 2.4 x 103)
per gram or ml (The number should be given in standard It is advisable to confirm growth on the plate as Pseudomonas spp
scientific notation e.g. 2.49 x 103) by performing an oxidase test. Yeasts and moulds may grow on the
It is always prudent to perform a simple wet preparation of suspect medium, and these may be differentiated by performing a wet
yeast colonies by emulsifying a colony in a drop of saline on a slide preparation and examining under the microscope for typical cells.
and placing a coverslip on top. Examine microscopically to confirm *For liquid samples use 10ml to 90ml.
large, rounded, usually budding yeast cells as opposed to the much No homogenisation is required prior to preparing serial dilutions.
smaller bacterial rods and cocci).
*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.

Bacillus cereus
(Enumeration)
Staphylococcus aureus Prepare an appropriate 1:10 dilution of the sample
(Enumeration) e.g. 10g* sample + 90ml MRD LAB103
|
|
Prepare an appropriate 1:10 dilution of the sample
e.g. 10g* sample + 90ml MRD LAB103 Homogenise for 2 mins
| |
| |
Homogenise for 2 mins Prepare serial dilutions by adding 1ml of homogenate to 9ml of
| MRD LAB103
| |
|
Prepare serial dilutions by adding 1ml of homogenate to 9ml of
MRD LAB103 Inoculate 0.1ml or 0.5ml by spreading over the surface of Bacillus
| Cereus Medium (PREP, LAB073)
| |
|
Inoculate 0.1ml or 0.5ml of appropriate dilutions by spreading over
the entire surface of Baird Parker medium (LAB085) Incubate at 30˚C for 24hrs
| |
| |
Incubate at 37˚C for 48hr Examine the plate for colonies. If not clearly visible incubate for a
| further 24hrs.
| |
|
Select plates with between 15 and 150 black colonies for
enumeration. If the highest dilution has more than this use Count typical colonies (large, 2-4mm, rough, pink in colour,
150 as the figure to calculate the original count and express with or without precipitate in surrounding medium).
as ‘greater than…’ Due to the large size of the colonies, count the dilution with less
| than 15 colonies on the plate if possible.
| |
|
Confirm at least 5 colonies of each colony type present using slide
or tube coagulase tests, or a proprietary kit Calculate the original count in the sample expressed as CFU’s per
| gram or ml (The number should be given in standard scientific
| notation e.g. 2.4 x 103)
Calculate the original count in the sample expressed as CFU’s per This is only a presumptive count, as other Bacillus spp. will grow
gram or ml (The number should be given in standard scientific on PREP agar (also known as MYP agar). Confirmation can be
notation e.g. 2.4 x 103), taking into account the ratio of coagulase done by biochemical methods, although this is not straight forward.
positive and negative colonies. Use reference schemes such as Bergey’s Determinative
The advice to count all black colonies is a reflection of the variation Bacteriology Volume 2 or Cowan and Steele. Alternatively a
of appearance of S.aureus (and other staphylococci) on Baird Parker proprietary kit for detection of specific enterotoxin may be used,
Medium. The production of the ‘typical’ lecithinase and lipase and strains of other Bacillus spp. have been reported to produce
reactions is not a stable characteristic, and in particular damaged enterotoxin.
organisms may lose the ability to produce them. Moreover, other *For liquid samples use 10ml to 90ml.
staphylococci can mimic these reactions and so confirmation of all No homogenisation is required prior to preparing serial dilutions.
colony types is necessary to reduce the risk of reporting false counts.
*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.

25
 Clostridium perfringens Isolation of Listeria species
(Enumeration) (the ‘modified USDA’ method)
Prepare an appropriate 1:10 dilution of the sample Add 25g sample to 225ml of UVM I (LAB155)
e.g. 10g* sample + 90ml MRD LAB103 |
| |
| Homogenise for 2 mins
Homogenise for 2 mins |
| |
| Incubate at 30˚C for 24hrs
Prepare serial dilutions by adding 1ml of homogenate |
|
to 9ml of MRD LAB103
| Subculture 0.1ml of enriched UVM I into 10ml Fraser Broth
| (LAB164) and incubate at 35˚C for 24 and 48hrs
Inoculate 1ml of appropriate dilutions into a sterile petri dish and |
|
add aseptically 15ml of molten, cooled, Perfringens Agar (OPSP,
LAB109).When set, overlay with more of the same agar to cover Streak a loopful of Fraser Broth (10 µl) onto Oxford Agar
surface (LAB122) or Palcam Agar (LAB148).
| Incubate plates at 30˚C for 24 and 48hrs.
| |
|
Incubate anaerobically at 37˚C for 24hrs
| Examine plates for typical colonies
| |
|
Select plates with between 15 and 150 colonies for enumeration.
If the highest dilution has more than this use 150 as the figure to Confirm colonies as Listeria spp. and speciate using
calculate the original count and express as ‘greater than…’ biochemical tests
| Colonies can be speciated using Listeriazym (T500),
| and a blood agar plate to determine haemolytic activity.
Calculate the original count in the sample expressed as CFU’s per Some workers have reported improved isolation by using both
gram or ml (The number should be given in standard scientific Oxford and Palcam agars together, similar to the use of different
notation e.g. 2.4 x 103) plating media for the isolation of Salmonella.
*For liquid samples use 10ml to 90ml.
No homogenisation is required prior to preparing serial dilutions.

Isolation of Salmonella species

Isolation of Listeria species (Semi-solid technology)
(the ‘FDA’ method) Add 25g sample to 225ml of Buffered Peptone Water (LAB046)
|
Add 25g sample to 225ml of Listeria Enrichment Broth |
(LEB, LAB138) or Buffered LEB (LAB139) Homogenise for 2 minutes and incubate at 37˚C for 24hrs
| |
| |
Homogenise for 2 mins Subculture three drops of enriched BPW centrally onto the surface
| of MSRV (LAB150) or Diassalm (LAB537) plates in duplicate.
| |
Incubate at 30˚C for 24 and 48hrs |
| Incubate with the lid uppermost at 37˚C or 42˚C for 24hrs
| |
Streak a loopful (10 µl) onto Oxford Agar (LAB122) or Palcam |
Agar (LAB148). Incubate plates at 30˚C for 24 and 48hrs. Examine for a spreading zone of growth. If no growth incubate for
| a further 24hrs
| |
Examine plates for typical colonies |
| Subculture spreading growth to XLD (LAB032) and Brilliant
| Green Agar (LAB034) to obtain pure growth for confirmation
Confirm colonies as Listeria spp. and speciate using by biochemical and serological methods
biochemical tests Use of a filter paper disc soaked in polyvalent H Salmonella
Colonies can be speciated using Listeriazym (T500), antiserum can increase the specificity of the media. Place the disc
and a blood agar plate to determine haemolytic activity. mid way between the inoculation point and the edge of the dish
Some workers have reported improved isolation by using both before incubation. The motility of salmonellas will be inhibited
Oxford and Palcam agars together, similar to the use of different around the disc, whereas non-salmonellas will grow up to the disc.
plating media for the isolation of Salmonella. Use of the disc with Diassalm enhances the H2S reaction on this
medium. The area where the motility is inhibited by the disc has a
high concentration of Salmonella and gives a good H2S reaction
indicated by blackening of the growth around the disc.

26
Isolation of Salmonella species
(conventional method)
Add 25g sample to 225ml of Buffered Peptone Water (LAB046)
|
|
Homogenise for 2 minutes and incubate at 37˚C for 24hrs
| |
| |
Subculture 1ml of enriched BPW into 9ml of Selenite Cystine Broth Subculture 0.1ml of enriched BPW into 10ml of Rappaport
(LAB055A + LAB044B). Incubate at 37˚C for 24 and 48hrs Vassiliadis (Soy) Broth (LAB086).
| Incubate at 41.5 ± 0.5˚C* for 24hrs
| |
Inoculate selective agars: Brilliant Green Agar (LAB034) |
and XLD Agar (LAB032) Inoculate selective agars: Brilliant Green Agar (LAB034) and
| XLD Agar (LAB032)
| |
Incubate at 37˚C for 24 hrs and examine for typical colonies |
| Incubate at 37˚C for 24 hrs and examine for typical colonies
| |
Confirm suspect colonies by serological and biochemical methods |
Confirm suspect colonies by serological and biochemical methods

If Selenite cystine broth is not acceptable because of the use of sodium biselenite, then an alternative medium may be substituted, such as
Mueller Kauffman Tetrathionate Broth (LAB042) or Tetrathionate Broth (LAB097). Both these formulations require the addition of
chemicals not supplied by LAB M.

Typical colony descriptions for Brilliant Green Agar and XLD agar can be found under the individual listings in the main body of the catalogue,
and are also available as colony cards which show typical colonies in full colour.

*It is critical that the incubation temperature does not exceed 43˚C as this can inhibit some salmonellas. To ensure this lower temperatures are
recommended: 41.5 ± 0.5˚C for incubators, 42 ± 0.1˚C for water baths. (Reference: Peterz M., Wiberg C., and Norberg P. 1989. The effect of
incubation temperature and magnesium chloride concentration on growth of Salmonella in home-made and in commercially available dehydrated
Rappaport-Vassiliadis broths. J. Appl. Bact. 66 523-528).

Isolation of Campylobacter species

Add 25g sample to 75ml (or 1:4 w/v) of
Campylobacter Enrichment Broth (LAB135)
|
|
Homogenise for 2 mins
|
|
Incubate at 37˚C for 2-4 hrs followed by a further 14-44 hrs at 42˚C
|
|
Streak a loopful (10µl) onto Campylobacter Blood Free Medium
(LAB112) and incubate at 37˚C for 40-48 hrs
|
|
Examine plates for typical colonies
|
|
Confirm colonies as Campylobacter spp. using biochemical tests

27
28 This page left intentionally blank
 Format and Dehydrated culture media
abbreviation guide selection guide
Agars, Peptones and other culture media ingredients
Product Name MC7 Acid Hydrolysed Casein
(Alternative name or commonly used abbreviation) MC2 Agar No. 1 Bacteriological-High Clarity
MC6 Agar No. 2 Bacteriological-General Purpose
PRODUCT CODE MC29 Agar 4-Plant Tissue Culture
MC24 Bacteriological Peptone
Description: MC4 Balanced Peptone
MC19 Beef Extract
A brief outline which may include any of the following information
MC25 Bile Salts No. 3
on the medium:
MC23 Malt Extract Powder
● HISTORY MC9 Mycological Peptone
●
MC11 Proteose Peptone A
MECHANISMS
MC27 Skim Milk Powder
● APPLICATIONS MC26 Sodium Desoxycholate
●
MC3 Soy Peptone
RECOGNITION BY REGULATORY/ADVISORY BODIES
MC5 Tryptone
● ADVANTAGES MC8 Tryptose
MC1 Yeast Extract Powder
Formula: The product composition in grams per litre; minor
adjustments to the published formula may be made to meet
performance criteria. Anaerobes
LAB 160 Brazier’s CCEY Agar
Method for reconstitution LAB 24 Cooked Meat Granules
Distilled water can be substituted for deionised water. LAB 90 Fastidious Anaerobe Agar (F.A.A.)
“Allow to soak times” are not critical. If agar media are to LAB 71 Fastidious Anaerobe Broth (F.A.B.)
be dispensed prior to sterilising, first bring to the boil to LAB 109 Perfingens Agar (O.P.S.P.)
dissolve the agar. LAB 22 Reinforced Clostridial Medium (Broth
Appearance: – of the finished cooled medium. LAB 23 Reinforced Clostridial Agar
LAB 64 Thioglycollate Medium (Brewer)
pH: at 20˚C. For agars, pour a small quantity into a universal bottle, LAB 25 Thioglycollate Medium (Fluid)
allow to set and plunge the probe into the medium.
Minimum Q.C. organisms – for use every time a new batch of
prepared medium is reconstituted. This short form check should not Blood Agar Bases
be confused with a full Q.C. of the medium. Where an organism LAB 1 Columbia Agar Base
should show inhibition this could be complete or partial. Records LAB 28 Blood Agar Base
should be kept of these results to help recognise changes in LAB 15 Blood Agar Base No. 2
performance over a period of time. LAB 90 Fastidious Anaerobe Agar
Storage of Prepared Media – All prepared media should be stored
in the dark. If a medium is to be used beyond the suggested shelf life,
appropriate quality control should be performed to demonstrate that Blood Culture Media
there has been no detectable fall off in performance.
LAB 49 Brain Heart Infusion Broth
LAB 71 Fastidious Anaerobe Broth (F.A.B.)
Growth characteristics LAB 4 Tryptone Soy Broth
Abbreviation key for colonial descriptions:
CV = convex CR = crenated Coliform Media
F = flat Rz = rhizoid LAB 51 Brilliant Green Bile 2% Broth
LAB 91 E.E. Broth
E = entire G = glossy LAB 61 Eosin Methylene Blue Agar
P.P. = pinpoint D = dull LAB 60 Endo Agar Base
LAB 126 Lactose Broth
( ) brackets are used to denote occasional variations. LAB 5 MacConkey Broth (Purple)
LAB 72 Tryptone Bile Agar
LAB 162 Tryptone Bile Glucuronide Agar (TBGA)
References LAB 31 Violet Red Bile Agar
A list of related publications and sources of information. LAB 88 Violet Red Bile Glucose Agar
N.B. The formulae in this manual and on the product label are
adhered to wherever possible. However it is occasionally necessary Diagnostic Medical Microbiology
to make minor adjustments to meet performance criteria.
LAB 121 Bromocresol Purple Lactose Agar
LAB 41 C.L.E.D. (Mackey & Sandys)-single indicator
LAB 6 C.L.E.D. (Bevis) – double indicator
LAB 67 G.C. Agar Base
LAB 123 Kirschners-T.B. enrichment
LAB 35 T.Y.C Medium-investigation of dental caries

29
 Enteric Pathogens Sensitivity Test Media
LAB 13 Bismuth Sulphite Agar LAB 39 Mueller Hinton Agar (II)
LAB 34 Brilliant Green Agar LAB 114 Mueller Hinton Broth (II)
LAB 46 Buffered Peptone Water – pre enrichment broth LAB 74 Nu-Sens Agar
LAB 112 Campylobacter Agar (Blood Free – Improved) LAB 12 Sensitivity Test Agar
LAB 135 Campylobacter Enrichment Broth
LAB 161 CT-SMAC
LAB 29 Desoxycholate Citrate Agar (DCA) Staphylococci Media
LAB 65 Desoxycholate Citrate Agar (Hynes) LAB 85 Baird Parker Medium
LAB 3 D.C.L.S. LAB 95 DN’ase Test Agar
LAB 537 Diassalm LAB 158 Liquid Baird Parker Medium
LAB 16 Fluorescence Agar – Pseudomonas spp. LAB 7 Mannitol Salt Agar
LAB 110 Hektoen Enteric Agar – Shigella spp. LAB 84 Single Step Staph Selective Agar
LAB 2 MacConkey Agar (without salt) (4S)-rapid method for S. aureus
LAB 30 MacConkey Agar No 3 (crystal violet) LAB 113Z Salt Meat Broth (tablets)
LAB 116 M.L.C.B. Agar
LAB 150 M.S.R.V. Streptococci Media
LAB 42 Muller Kauffman Tetrathionate Broth
LAB 165 0157 Broth (MTSB) LAB 106 Kanamycin Aesculin Azide Agar
LAB 86 Rappaport Vassiliadis Medium (broth) LAB 107 Kanamycin Aesculin Azide Broth
LAB 44 Selenite Broth Sterility Test Media
LAB 55 Selenite Cystine Broth
LAB 161 Sorbitol MacConkey Agar. (SMAC) LAB 25 Fluid Thioglycollate U.S.P.
LAB 97 Tetrathionate Broth Base LAB 14 Nutrient Broth No. 2 B.P.
LAB 96 T.C.B.S. Cholera Medium LAB 33 Sabouraud Liquid Medium U.S.P.
LAB 32 XLD Agar LAB 11 Tryptone Soy Agar
LAB 120 Yersinia CIN Agar LAB 4 Tryptone Soy Broth U.S.P.

Food Microbiology Transport Media

LAB 85 Baird Parker Medium LAB 124 Amies with charcoal
LAB 34 Brilliant Green Agar LAB 125 Amies without charcoal
LAB 105 China Blue Lactose Agar
LAB 135 Campylobacter Enrichment Broth
LAB 537 Diassalm Total Viable Counts
LAB 136 Easter and Gibson Pre-enrichment
LAB 137 Easter and Gibson Salmonella LAB 19 Milk Agar
LAB 164 Fraser Broth LAB 115 Milk Plate Count Agar
LAB 122 Listeria Isolation Medium LAB 10 Plate Count Agar A.P.H.A.-standard methods
LAB 138 Listeria Enrichment Broth LAB 149 Plate Count Agar-for spiral platers or pour plates
LAB 139 Listeria Enrichment Broth (Buffered) LAB 63 Tryptone Glucose Extract Agar A.P.H.A.
LAB 158 Liquid Baird Parker Medium LAB 11 Typtone Soy Agar
LAB 93 M.R.S. Agar
LAB 94 M.R.S. Broth
LAB 150 M.S.R.V. Veterinary
LAB 109 Perfringens Agar (O.P.S.P.)
LAB 78 C.E.M.O. Agar Base
LAB 149 Plate Count Agar
LAB 10 Plate Count Agar A.P.H.A.
LAB 73 P.R.E.P. Agar-cultivation of Bacillus cereus
LAB 108 Pseudomonas Agar Water Testing
LAB 23 Reinforced Clostridial Agar-enumeration of anaerobes LAB 5 MacConkey Broth Purple
LAB 87 Sugar Free Agar-enumeration of organisms in LAB 82 Membrane Lauryl Sulphate Broth
butter and similar products LAB 80 Minerals Modified Glutamate Broth
LAB 162 T.B.G.A. LAB 126 Lactose Broth
LAB 155 U.V.M. Broth LAB 163 R2A Medium
LAB 31 V.R.B.A.
LAB 88 V.R.B.G.A.
LAB 79 W.L. Agar-examination of worts, Yeasts and Moulds
beers and yeast cultures
LAB 38 Wort Agar-yeasts in dairy and sugar products LAB 117 Dermatophyte Test Medium
LAB 99 Wort Broth LAB 37 Malt Extract Agar
LAB 89 O.G.Y.E. Agar
LAB 98 Potato Dextrose Agar
Identification Media LAB 36 Rose Bengal Chloramphenicol Agar
LAB 104 Peptone Water LAB 9 Sabouraud Dextrose Agar
LAB 69 Simmons Citrate Agar LAB 33 Sabouraud Liquid Medium
LAB 53 Triple Sugar Iron Agar LAB 111 Sabouraud Maltose Agar
LAB 129 Tryptone Water LAB 38 Wort Agar
LAB 130 Urea Agar LAB 99 Wort Broth
LAB 131 Urea Broth LAB 119 Yeast Extract Dextrose Chloramphenicol Agar

Nutrient Media for general use

LAB 48 Brain Heart Infusion Agar
LAB 49 Brain Heart Infusion Broth
LAB 8 Nutrient Agar
LAB 68 Nutrient Broth ‘E’
LAB 62 Tryptose Phosphate Broth
LAB 18 Yeast Extract Agar

30
Aeromonas Agar Amies Transport Medium
Bile Salt Irgasan Brilliant Green Agar without Charcoal
LAB 167 LAB 125
Description
Aeromonas Agar is a highly selective medium for the isolation of Description
Aeromonas spp. from food, clinical and environmental samples. This medium is as LAB 124 without the charcoal. It is used when
Based on the selective agents, brilliant green and irgasan, this microscopic examination of a film is an important part of the
medium will not inhibit those strains of Aeromonas sensitive to procedure and the charcoal may interfere with interpretation.
ampicillin used in other media.
Formula g/litre
Formulation g/litre
Sodium chloride 3.0
Beef Extract 5.0
Potassium chloride 0.2
Meat Peptone 5.0
Disodium hydrogen phosphate 1.15
Xylose 10.0
Sodium thioglycollate 1.0
Bile Salts No.3 8.5
Calcium chloride 0.1
Sodium thiosulphate 5.44
Magnesium chloride 0.1
Irgasan 0.005
Potassium phosphate 0.2
Brilliant green 0.005
Agar No. 1 4.0
Neutral red 0.025
Agar 11.5 Method for reconstitution
Weigh 9.75 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to boil to
Appearance: Purple, Clear gel
dissolve agar. Distribute into bijou bottles filling to shoulder.
pH: 7.0 ± 0.2 Constantly mix whilst distributing. Sterilise by autoclaving at
121˚C for 15 minutes. Screw caps down after autoclaving.
Method for reconstitution Appearance: Soft translucent gel.
Weigh 45.5 grams of powder and disperse in 1 litre of deionised
water. Allow to soak for 10 minutes, swirl to mix and sterilise by pH: 7.1 ± 0.2
bringing to the boil. Cool to 47˚C, mix well and dispense into petri
dishes. Minimum Q.C. organisms: N. gonorrhoeae
S. pyogenes
Inoculation: Faecal specimens: Inoculate surface of medium
directly, spreading for single colonies. Samples requiring enrichment: Storage of Prepared Medium: Capped containers up to 6 months
Inoculate alkaline peptone water and incubate at 37˚C for 18-24 hr. at 15-20˚C dark.
Subculture onto Aeromonas Agar, surface spreading for single
colonies. Inoculation: As LAB 124.
Incubation: Incubate plates aerobically at 37˚C for 18-24 hr.
Examine for typical colonies and confirm as Aeromonas spp.
Storage: Poured plates: 7 days at 2-8˚C in the dark (may be extended
if moisture tight packaging used). Amies Transport Medium
Minimum Q.C. organisms: Aeromonas hydrophilia with Charcoal
NCIMB 9240
E.coli LAB 124
NCIMB 50034 (inhibited) Description
Confirmation Amies introduced his modification of Stuarts transport medium to
overcome a number of problems. Stuarts transport medium suffered
Typical colonies (translucent pink colonies 0.5-3.0mm diameter) from overgrowth by coliforms that were capable of utilising sodium
should be confirmed as presumptive Aeromonas spp. by performing glycerophosphate. Amies replaced the problem component with an
an oxidase test and inoculating into Hugh & Leifsons O/F medium. inorganic phosphate buffer system. Calcium and magnesium salts are
Aeromonas spp. will give a positive oxidase reaction and demonstrate added to control the permeability of the bacterial cell wall and
both oxidative and fermentative metabolism. Pseudomonas spp. will
thus prolong their survival. The addition of charcoal to the
also be oxidase positive, but do not possess fermentative metabolism.
medium extended the survival time of Neisseria gonorrhoeae from
An alternative method is to inoculate triple sugar iron tubes.
24 to 72 hours.
Aeromonas will typically produce an acid butt (yellow) and an
alkaline or unchanged slant (red) whilst Pseudomonas spp. will
remain unchanged in both the butt and slant. To fully identify Formula g/litre
colonies as Aeromonas spp. the above tests should be supported using
Charcoal-activated 10.0
a proprietary kit such as API 2ONE or Microbact 24E (other products
may be available). Sodium chloride 3.0
Potassium chloride 0.2
Interpretation
Organism Size Shape Colour Disodium hydrogen phosphate 1.15
Aeromonas spp.* 0.5-3.0 CV.E.G Translucent pink Sodium thioglycollate 1.0
Pseudomonas spp. 0.5-1.0 CV.E.G Translucent pink Calcium chloride 0.1
S.aureus No growth Magnesium chloride 0.1
E.coli No growth
Potassium phosphate 0.2
*The selective nature of the medium may mean occasional strains do not grow, Agar No. 1 4.0
or grow poorly.
31
 Method for reconstitution
Weigh 19.75 grams of powder, disperse in 1 litre of deionised water.
Aseptic Commissioning Broth
Allow to soak for 10 minutes, swirl to mix then bring to boil to LAB157
dissolve agar. Distribute into bijou bottles filling to shoulder.
Constantly mix whilst distributing. Sterilise by autoclaving at 121˚C Description
for 15 minutes. Screw caps down after autoclaving.
A general growth medium specifically designed for the
Appearance: Soft gel with heavy concentration of evenly suspended commissioning of aseptic filling machines in the pharmaceutical and
charcoal. allied industries. It is a simple formulation incorporating peptone,
yeast extract and sucrose as energy sources, and phenol red to
Minimum Q.C. organisms: N. gonorrhoeae indicate growth of organisms producing acid. Gas production and
S. pyogenes turbidity also indicate growth and are indicated by bubbles in
Durham’s tubes or distortion of plastic packaging. It is suitable for
Storage of Prepared Medium: Capped containers – up to 6 months sterilisation by filtration, passing quickly through the filters, and
at 15-20˚C dark. reducing blockage.
pH: 7.2 ± 0.2
Formula g/litre
Inoculation: Push the swab containing the sample into the gel to
approximately one third of the gels depth. Cut off the unwanted swab Tryptone 5.0
stick then screw on the cap pushing the swab further down into the
gel. Cap tightly and keep cool during transport to the laboratory. Yeast Extract 2.5
Sucrose 5.0
References
Sodium chloride 5.0
Amies, C.R. 1967. Can. J. Pub. Health. 58: 296-300. A modified
formula for the preparation of Stuarts Transport Medium. Phenol red 0.005

Method for reconstitution

Anaerobe Identification Medium Base Weigh 17.5 grams of powder and disperse in 1 litre of deionised
water. Allow to soak for 10 minutes, swirl to mix and heat gently to
LAB 66 dissolve. Sterilise at 121˚C for 15 minutes, or alternatively, by
filtration.
Description
Appearance: Pale brown/straw, clear broth.
A medium introduced by Phillips in 1976 for testing the fermentation
capabilities of non-sporing anaerobes. The base is carbohydrate free pH: 7.2 + 0.2
and nutritious.
Minimum Q.C. organisms: Escherichia coli (acid and gas
Formula g/litre production) NCIMB 50034
Proteus spp. (turbidity, no acid or gas)
Beef Extract 4.0
Peptone mixture 16.0 Inoculation: Dispense medium through filling line into the final
Sodium chloride 5.0 packaging, or suitable container, as appropriate.

Agar No. 2 15.0 Incubation: As per routine sterility testing laid down in
Pharmacopoeias or in house methodology. Typically 20-25˚C for
Method for reconstitution up to 14 days.
Weigh 40 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 48˚C and aseptically
add 50-70mls of sterile defibrinated horse blood. Mix well and pour.
Before use, flood the surface with 1ml of a sterile solution of the
substrate under test.
Appearance: Blood agar plate.
pH: 7.2 ± 0.2

Minimum Q.C. organisms: B. fragilis

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark. Capped Container – up to 3 months at 15-20˚C in the dark.
Inoculation: Heavily inoculate a small area of the plate with a
loopful of a fresh culture of the test organism. Up to 4 organisms per
plate can be tested.
Incubation: 37˚C anaerobically for 24-48 hours. Recognition of
fermentation: Remove a small plug of agar from below the growth.
Cover the plug with bromothymol blue indicator (0.04%). Colour
changes due to production of acidity will develop in a few seconds
and should be viewed against a white background. Comparison with
controls is useful, a plug taken from an area well away from any
growth can be used as a negative control.

References
Phillips K.D. 1976. A Simple and sensitive technique for determining
the fermentation reactions of non-sporing anaerobes. J. Appl. Bact.
41: 325-328.

32
Bacillus Cereus Medium
Phenol Red Egg Yolk Polymixin Agar (P.R.E.P.)
Mannitol Egg Yolk Polymixin Agar
LAB 73
Description
Introduced by Mossel and his co-workers in 1967 for the enumeration
of Bacillus cereus in foods, this formula was shown to be the most
effective for this purpose by Inal in 1972. Two reactions on this
medium differentiate B. cereus from other members of the Bacillus
group, these are mannitol fermentation and lecithinase production.
Mannitol fermentation on this medium produces a yellow colour,
B. cereus is mannitol negative and produces red colonies.
The lecithinase production of B. cereus is indicated by a white
precipitate around the colonies. Polymixin is added to suppress
coliforms but some Proteus spp and Gram positive cocci may
grow through.
Baird-Parker Medium Base
LAB 85
Description
Baird-Parker introduced this complex medium in 1962 to overcome
the problems of recovering damaged Staphylococcus aureus from
foodstuffs. The medium is highly selective due to potassium tellurite
and lithium chloride. The tellurite inhibits most coliforms and is also
reduced by S. aureus to telluride giving typical black colonies.
Glycine and sodium pyruvate are both utilised by staphylococci as
growth factors, pyruvate also neutralises toxic peroxides which may
be formed in the medium. Sulphamethazine may be added to inhibit
Proteus spp. Two reactions typical of S. aureus can be detected by the
egg yolk. (1) lecithinase production – an opaque zone round the
colony; (2) lipase production – a zone of clearing outside the opaque
zone.
Colonies suspected of being S. aureus should be confirmed by the
coagulase test or by a latex agglutination kit.

Formula g/litre Formula g/litre

Beef Extract 1.0 Tryptone 10.0
Balanced Peptone No. 1 10.0 Beef Extract 7.5
D-Mannitol 10.0 Yeast Extract 1.0
Sodium chloride 10.0 Lithium chloride 5.0
Phenol red 0.025 Glycine 12.0
Agar No. 1 15.0 Sodium pyruvate 10.0

Method for reconstitution Agar No. 2 20.0

Weigh 46 grams of powder, disperse in 900ml of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by Method for reconstitution
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and aseptically Weigh 65.5 grams of powder, disperse in 1 litre of deionised water.
add 100mls of X073 egg yolk emulsion and 2 vials of X074 Allow to soak for 10 minutes, swirl to mix then sterilise by
Polymixin. autoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 50mls of
Appearance: Pink, opaque gel. X085 sterile egg yolk tellurite emulsion. Mix well and dispense into
petri dishes.
pH: 7.2 ± 0.2
Appearance: Cream/pale fawn, opaque.
Minimum Q.C. organisms: B. cereus NCIMB 50014 pH: 6.8 ± 0.2
E. coli (inhibition) NCIMB 50034
Minimum Q.C. organisms: S. aureus NCIMB 50080
Storage of Prepared Medium: – up to 7 days at 2-8˚C in the dark. S. epidermidis NCIMB 50082
E. coli (inhibition) NCIMB 50034
Inoculation: Surface, spreading or streaking for single colonies.
Incubation: 30˚C aerobically for 24-48 hours. Storage of Prepared Medium: Plates – up to 3 days at 2-8˚C in
the dark.
Growth Characteristics Inoculation: Surface spread.
colony size shape & Incubation: 37˚C aerobically for 48 hours.
organism (mm) surface colour
B. cereus 3.0-4.0 F.CR.D. Pink, white halo Growth characteristics
B. subtilis 2.0-3.0 F.CR.D. Yellow colony size shape &
B. coagulans 2.0 F.CR.D. Yellow organism (mm) surface colour other
B. licheniformis 2.0 F.Rz.D. Yellow S. aureus 1.0-3.0 CV.E.G. Black Narrow opaque
margin surrounded
Proteus spp. 1.0 CV.E.G. Pink (swarms) by a 2-5 mm zone
E. faecalis 0.5 CV.E.G. Yellow of clearing
E. coli no growth S. saprophyticus 0.5-2.0 CV.E.G. Black (poor growth)
S. aureus 1.0 CV.E.G. Yellow (white halo) Other
coagulase 0.5-1.0 CV.E.G. Black (no growth)
negative
References staphylococci
Inal, T.: Vergleictiende Untersuchungen über die Selektivmedien zum
Proteus spp. 0.5-2.0 F.Rz.G Brown (no growth)
qualitativen und quantitativen Nachweis von Vacillus cereus in
Black
Lebensmitteln.
Bacillus spp. 0.5-1.0 F.Rz.D. Brown (no growth)
I. Mitteilung: Fleischwritsch, 51: 1629-1632 (1971). IV. Mitteilung:
Fleischwritsch, 52: 1160-1162 (1972). Entero- no growth
bacteriaceae
Mossel, D. A. A., Koopman, M. J. and Jongerius, E. 1967.
Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15: 650-
653. Thatcher, F. S., Clarke, D. S. 1978 Micro-organisms in foods.
Volume 1 second edition. University of Toronto.
BS5763 Part 1L:1994. ISO7932:1993 3/100

33
 References Growth characteristics
Baird-Parker, A. C. 1962. An improved diagnostic and selective
medium for isolating coagulase positive staphylococci. J. Appl. Bact. colony size shape &
25(1): 12-19. organism (mm) surface colour other
Baird-Parker, A. C. and Davenport, E. 1965. The effect of Recovery S. typhi 1.5-2.0 CV.E.G. Black Metallic sheen
medium on the isolation of Staph. aureus after black deposit in
heat treatment and after storage of frozen or dried cells. J. Appl. Bact medium. (H2S-ve
28: 390-402. strains green)
Ten Broeke, R. 1976. The Staphylococcus medium of Baird-Parker in Other
practical use. The occurrence of coagulase-positive, egg yolk non- Salmonella spp. 1.0-2.5 CV.E.G. Black/ Metallic sheen
clearing staphylococci. Antonie van Leeuwenhoek 33: 220-236. Green especially in
heavy growth,
Smith, B. A. and Baird-Parker, A. C. 1964. The use of single colonies may
sulphamethazine for inhibiting Proteus spp. on Baird-Parker’s give rabbit eye
isolation medium for Staphylococcus aureus. J. Appl. Bact 27(1): appearance
78-82.
E. coli P.P.-1.0 CV.E.G. Green
Klebsiella spp. P.P-2.0 CV.E.G. Green
Citrobacter spp. 1.0-2.5 CV.E.G. Green (black centre)
Bismuth Sulphite Agar Proteus spp. 1.0-2.5 CV.E.G. Green/ (black centre)
Brown
(Wilson and Blair Medium)
LAB 13A + LAB 13B References
Wilson, W. J. and Blair, E. M. M’V 1926. A combination of bismuth
Description and sodium sulphites affording an enrichment and selective medium
A modification of Wilson and Blair’s original medium for the for the typhoid-paratyphoid groups of bacteria. J. Pathol. Bacteriol.,
isolation of Salmonella typhi and other Salmonella from clinical 29: 310-311.
samples, sewage and other materials. The presence of bismuth
International Journal of Food Microbiology 1987 5:3:200-202.
sulphite and brilliant green make this medium highly selective.
As the medium contains neither lactose nor sucrose it can be used to I.C.M.S.F. 1978 Micro organisms in Foods I. Their significance and
detect lactose and sucrose fermenting Salmonella. enumeration. 2nd edition Univ of Toronto Press. Speck M. L. 1984.
Compendium of methods for microbiological examination of foods.
Formula g/litre 2nd edition. American Public Health Association, Washington. 3/102

Bismuth Sulphite Agar Base ‘A’ LAB 13a

Beef Extract 6.0
Balanced Peptone No. 1 10.0 Blood Agar base
Ferric citrate BPC 0.4 LAB 28
Brilliant Green 0.01
Description
Agar No. 2 20.0 An inexpensive general purpose agar base which, with the addition of
Bismuth Chemical Mixture ‘B’ LAB 13b 5% sterile blood, can be used to cultivate a wide range of micro
organisms of clinical significance. Typical haemolysis patterns are
Bismuth ammonium citrate 3.0 obtained with this medium.
Sodium sulphite 5.0
Formula g/litre
Disodium phosphate 5.0
Beef Extract 10.0
Glucose 5.0
Balanced Peptone No. 1 10.0
Method for reconstitution Sodium chloride 5.0
Agar Base ‘A’: Weigh 36.4 grams of powder and mix with 1 litre of Agar No. 2 12.0
deionised water. Sterilise for 15 minutes at 121˚C. Cool to 50˚C
approx. and add 100ml of Chemical Mixture ‘B’. Mix well and pour
thin plates. Store at 4˚C for 3 days to mature, before use. Method for reconstitution
Weigh 37 grams of powder, disperse in 1 litre of deionised water.
Chemical Mixture ‘B’: Suspend 18 grams of powder in 100ml. Allow to soak for 10 minutes, swirl to mix then sterilise by
of deionised water. Bring to boil over a tripod and gauze, and cool autoclaving for 15 minutes at 121˚C. Cool to 47˚C and add 5-7%
quickly in cold water. Add to 1 litre of Agar Base ‘A’ prepared sterile defibrinated blood. Mix by swirling the flask and pour into
as above. petri dishes.
Appearance: Pale green, opaque gel. Appearance: Dependent upon blood additive.
pH: 7.6 ± 0.2 pH: 7.4 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076 Minimum Q.C. organisms: S. aureus. NCIMB 50080
E. coli (inhibition) NCIMB 50034 S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – store 3 days before use. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Use within 7 days. Store at 2-8˚C in the dark. the dark.
Inoculation: Surface, streak out to single colonies. Inoculation: Surface, streaking to single colonies.
Incubation: 37˚C for 24 hours aerobically. Incubation: 37˚C aerobically, anaerobically or microaerobically
for 24 hours.

34
 Growth characteristics Growth characteristics
colony size shape & colony size shape &
organism (mm) surface colour other organism (mm) surface colour other
S. aureus 0.5-1.5 CV.E.G. White- haemolytic S. aureus 1.5-2.0 CV.E.G. White/ (haemolytic)
Golden Golden
S. pyogenes P.P.-1.0 CV.E.G. Grey beta haemolytic S. pyogenes 1.0-1.5 CV.E.G. Grey beta haemolytic)
alpha haemolytic (alpha or non
non-haemolytic haemolytic)
S. pneumoniae P.P.-1.0 F.E.G. Grey alpha haemolytic S. pneumoniae 0.5-1.0 F.E.G. Grey (draughtsman
draughtsman (alpha haemolytic)
(mucoid)
N. meningitidis P.P.-1.5 CV.E.G. Grey mucoid
(require CO2)
E. coli 1.5-2.5 CV.E.G. Grey haemolytic
N. meningitidis 0.5-1.0 CV.E.G. Grey (May require
Ps. aeruginosa 0.5-3.0 F.CR.D. Grey many colonial CO2)
forms
E. coli 2.0-3.0 CV.E.G. Grey (haemolytic)
green pigment
Ps. aeruginosa 1.0-3.0 F.CR.D. Grey (green pigment
C. perfringens 0.5-1.5 CV.CR.G. Grey Target haemolysis
(haemolytic)
non-haemolytic
C. perfringens 1.0-2.5 CV.CR.-(E)G Grey “Target”-
B. fragilis 0.5-1.5 CV.E.G. Grey mucoid
haemolysis
P. anaerobius P.P.-0.5 CV.E.G. Grey- (non-haemolytic)
White
B. fragilis 1.0-1.5 CV.E.G. Grey non haemolytic
F. necrophorum P.P. CV.E.G. Trans- haemolytic
P. anaerobius 0.5-1.0 CV.E.G. White non haemolytic
parent

References
Cruikshank, R. 1972. Medical Microbiology. 11th edn. Livingstone,
London Brain Heart Infusion Agar
LAB 48
Description
Blood Agar Base No. 2 A general purpose nutritious agar base. This medium was first used
for the isolation of dental pathogens. The mixture of brain and heart
LAB 15 infusions is particularly useful in the isolation of Actinomyces israeli
and Histoplasma capsulatum. With the addition of 7% defibrinated
Description blood the medium will support the growth of a wide range of
A very rich agar base which, with the addition of blood, is capable of fastidious organisms, the phosphate buffer will help neutralise the
growing delicate clinical pathogens. The medium gives colonial acids produced from the utilisation of glucose and thus maintain
appearances, haemolysis patterns and pigment production of viability. The medium is not recommended for the determination of
diagnostic value. When the blood is ‘chocolated’ the medium gives haemolytic reactions because of the glucose content.
good recovery of Haemophilus spp. The medium can be made The use of porcine material in this product ensures there are no
selective for various groups by the addition of appropriate antibiotic Specified Risk Materials (SRM’S) with respect to Transmissible
mixtures eg: Spongeform Encephalopathies (TSE’S).
Streptococci – Colistin/Oxolinic acid (XO13)
Gardnerella spp. – Colistin/Oxolinic acid (XO11) Formula g/litre
C. perfringens – Neomycin (XO15) (XO16)
Staphylococci/streptococci – Colistin/Naladixic acid (XO12) Brain-Heart Infusion Solids (porcine) 17.5
Tryptose 10.0
Formula g/litre
Glucose 2.0
Tryptose 15.0
Sodium chloride 5.0
Soy Peptone 2.5
Disodium phosphate 2.5
Yeast Extract 5.0
Agar No. 2 12.0
Sodium chloride 5.0
Agar No. 2 12.0 Method for reconstitution
Weigh 49 grams of powder, disperse in 1 litre of deionised water.
Method for reconstitution Allow to stand for 10 minutes then swirl to mix. Sterilise by
Weigh 39.5 grams of powder, disperse in 1 litre of deionised water. autoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour into
Soak for 10 minutes, swirl to mix then sterilise for 15 minutes at petri dishes.
121˚C. Cool to 47˚C then aseptically add 5-7% sterile, defibrinated Appearance: Pale Straw colour, clear gel.
horse or sheep blood. Mix well before pouring.
pH: 7.4 ± 0.2
Appearance: Dependent upon blood additive.
pH: 7.4 ± 0.2 Minimum Q.C. organisms: S. aureus NCIMB 50080
E. coli NCIMB 50034
Minimum Q.C. organisms: S. aureus NCIMB 50080
S. pyogenes ATCC 19615 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in Inoculation: Surface, streaking out to single colonies.
the dark.
Incubation: Time and temperature to suit specimen/organisms.
Inoculation: Surface, streaking out to single colonies.
Incubation: 37˚C aerobically or microaerobically for 24 hours,
anaerobically for 24 and 48 hours.
35
 Growth characteristics (with horse blood) Brain Heart Infusion Broth
colony size shape &
organism (mm) surface colour LAB 49
S. aureus 1.0-1.5 CV.E.G. White/ Description
Golden A rich isotonic infusion medium with tryptose (a mixture of meat and
other staphylococci 0.5-1.5 CV.E.G. White/ milk peptones) providing a wide range of substrates. A low
Yellow concentration of glucose is used to stimulate early growth. The
medium is lightly buffered to prevent the early death of some species
S. pyogenes 0.5-1.0 CV.E.G. White due to acid production. Organisms which produce significant
S. milleri P.P.-0.1 CV.E.G. Transp. amounts of acid may well overwhelm the buffering system and auto-
(White) sterilise. The medium is suitable for use as a blood culture medium or
as an enrichment broth for fastidious organisms.
E. faecalis 1.0-1.25 CV.E.G. Grey/
Green The use of porcine material in this product ensures there are no
Specified Risk Materials (SRM’S) with respect to Transmissible
S. pneumoniae 0.5-1.0 F.E.G. Grey/ Spongeform Encephalopathies (TSE’S).
Green
E. coli 2.0-3.0 CV.E.G. Grey Formula g/litre
Pseudomonas 2.0-4.0 F.CR.D. Grey Brain-Heart Infusion solids (porcine) 17.5
aeruginosa
Tryptose 10.0
References Glucose 2.0
Roseburg. T., Epps, L. J. and Clarke, A. R. 1944. A study of the
isolation, cultivation and pathogenicity of Actinomyces israeli Sodium chloride 5.0
recovered from the human mouth and from actinomycosis in man. Disodium hydrogen phosphate 2.5
J. inf. Dis., 74: 131-149.
Howell, E. 1948 Efficiency of methods of isolation of Histoplasma Method for reconstitution
capsulatum. Pbl. Hlth. Rep. 63: 173-178. 3/108 Weigh 37 grams of powder then disperse in 1 litre of deionised water.
Allow to stand for 10 minutes then dissolve with gentle heat before
dispensing into tubes or bottles. Sterilise at 121˚C for 15 minutes.
Overheating will cause caramelisation and darkening of the medium.
Appearance: Straw colour, clear liquid.
pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080

E. coli NCIMB 50034

Storage of Prepared Medium: Capped container – up to 3 months

at 15-20˚C in the dark.
Inoculation: (as a blood culture medium). Using a minimum volume
of 50mls of medium add the blood to a dilution of from 1:10 to 1:20.
Use in conjunction with an anaerobic culture medium eg Fastidious
Anaerobe Broth LAB 71.
Incubation: 37˚C aerobically for 7 to 15 days.
Interpretation: Observe daily, subculture after 1, 2, 3, 7 and 15 days
or immediately on showing signs of growth.

References
Rosenow. E.C. 1919. Studies on selective localisation; focal infection
with special reference to oral sepsis. J. Dent. Res. 1:205-267.

Brazier’s CCEY Agar

LAB 160
Description
Brazier’s CCEY agar is the formulation currently used by the
Anaerobe Reference Unit for the isolation of C.difficile, resulting
from work initiated by Ken Phillips and Paul Levett, and completed
by Jon Brazier.
Based upon the market leading anaerobe medium, Fastidious
Anaerobe Agar, it incorporates additional ingredients to improve the
isolation and differentiation of C.difficile from clinical specimens.
Cholic acid is present to promote spore germination following
alcohol shock treatment, and p-hydroxyphenylacetic acid to enhance
the production of p-cresol, a distinctive metabolite of C.difficile
Selectivity is achieved by addition of supplement X093 (cefoxitin
cycloserine) and egg yolk emulsion X073 is added to help
differentiate C.difficile from lecithinase positive clostridia. Finally
the addition of lysed horse blood optimises the recognition of colony
fluorescence when cultures are examined using UV light.
36
 and Shigella spp. Less inhibitory media such as X.L.D. and Hektoen
Formula g/litre Enteric will be useful in detecting salmonellae and shigellae inhibited
Peptone Mix 23.0 by Brilliant Green Agar.

Sodium chloride 5.0 Formula g/litre

Soluble Starch 1.0 Beef Extract 5.0
Agar 12.0 Balanced Peptone No. 1 10.0
Sodium bicarbonate 0.4 Yeast Extract 3.0
Glucose 1.0 Disodium hydrogen phosphate 1.0
Sodium pyruvate 1.0 Sodium dihydrogen phosphate 0.6
Cysteine HCl 0.5 Lactose 10.0
Haemin 0.01 Sucrose 10.0
Vitamin K 0.001 Phenol red 0.09
L-arginine 1.0 Brilliant green 0.0047
Soluble pyrophosphate 0.25 Agar No. 2 12.0
Sodium succinate 0.5
Method for reconstitution
Cholic acid 1.0
Weigh 52 grams of powder and disperse in 1 litre of deionised water.
p-Hydroxyphenylacetic acid 1.0 Allow to soak for ten minutes and then bring to the boil with frequent
swirling to dissolve the solids and cool to 47˚C in a water bath. Pour
plates and dry the surface before inoculation. DO NOT remelt or
Method for reconstitution autoclave: overheating causes precipitation of the medium. Store
Weigh 48 grams of powder and add to 1 litre of deionised water. plates away from light.
Allow to soak for 10 minutes, swirl to mix, and sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and aseptically Appearance: Tan, clear gel.
add the following: 2 vials of X093, 40ml of Egg Yolk Emulsion X073 pH: 6.9 ± 0.2
and 10ml lysed horse blood. Mix well and pour into petri dishes.
Appearance: Tan opaque gel. Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli (inhibition) NCIMB 50034
pH: 7.0 + 0.2
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Minimum Q.C. organisms: C.difficile
the dark.
E.coli (inhibition) NCIMB 50034
Inoculation: Surface streaking for single colonies, a heavy inoculum
Storage of prepared medium: Plates – up to 7 days at 2-8˚C in can be used.
the dark. Incubation: 37˚C for 18-24 hours aerobically.
Inoculation: Surface streak untreated or alcohol shocked specimens
for single colonies. Growth characteristics
Incubation: 37˚C for 24-48hrs under anaerobic conditions colony size shape &
Characteristics of C.difficile: Gray opaque flat colonies, raised organism (mm) surface colour other
elevation, 2-3mm diameter, generally circular but tending to elongate Salmonella spp. 1-1.5 CV.E.G. Pink (red zone in
in the direction of spreading, ground glass appearance and a rough, colonies medium)
fimbriate edge. Lecithinase negative. Incubation longer than 48hrs
may result in a lighter gray or white centre to the colony. Phenolic S. typhi 1.0 CV.E.G. Pink/Red (may not grow)
odour due to the production of p-cresol. Colonies fluoresce yellow- E. coli no growth (0.5-1.0 yellow
green under UV light. Confirm by latex agglutination. colony)

References Proteus spp no growth

Brazier J S (1993) Role of the Laboratory in Investigations of Ps aeruginosa no growth (crenated small red
Clostridium difficile Diarrhoea. Clinical Infectious Diseases 16 (4) colonies)
S228-33. Klebsiella spp. 1-1.5 CV.E.G. Green (yellow) (NG)
colonies
Enterococcus
spp. no growth
Brilliant Green Agar (modified) S. sonnei no growth (0.5 mm red)
Phenol Red Brilliant Green Agar
References
LAB 34 Edel, W. and Kamplemacher, E.H. 1968. Comparative studies on
Salmonella isolation in eight European laboratories. Bull. Wld. Hlth.
Description Org. 39: 487-491.
First introduced by Kristensen et al in 1925 as a selective medium for Edel, W. and Kamplemacher, E.H. 1969. Salmonella infections in
the isolation of salmonellae (except S. typhi). The medium was nine European laboratories using a standard technique. Bull Wld.
modified by the Netherlands Institute for Public Health, Utrecht. The Hlth. Org. 41: 297-306.
modification was to increase the selectivity of the medium by
increasing the dye concentration. This formulation is quoted by the American Public Health Association 1966. Recommended Methods
International Standards Organisation, standard European Community for the Microbiological Examination of Foods, 2nd end. (ed. J.M.
Methods, the American Public Health Association and the Sharf) A.P.H.A. Washington.
Association of Official Analytical Chemists. The medium is suitable Association of Official Analytical Chemists (AOAC) 1978
for subcultures from selective enrichment media. However because Bacteriological Analytical Manual, 5th edn., Washington D.C.
this medium is highly selective, small numbers of salmonellae may
be missed. This medium is definitely not recommended for S. typhi Pharmacopoeia of culture media for food microbiology. 1987. Int. J.
Food Microbiol. 513: 245-247. 3/112
37
 Brilliant Green Bile 2% Broth Bromocresol Purple Lactose Agar
LAB 51 (Drigalski agar)
Description LAB 121
A modification of MacConkey’s medium, formulated in 1926 by
Dunham and Schoenlein, for the recovery of coliform bacteria in Description
foodstuffs and water. The brilliant green and bile inhibit most Gram A non selective differential medium for the isolation and enumeration
positive organisms thus overcoming the problem of some of Enterobacteriaceae from urine, water and food products. Lactose
Clostridium spp. fermenting lactose and giving false positive results. fermenting organisms produce yellow colonies, non lactose
fermenters produce purple colonies.
Formula g/litre
Formula g/litre
Balanced Peptone No. 1 10.0
Peptone mixture 7.4
Lactose 10.0
Lactose 8.5
Ox Bile 20.0
Bromocresol purple 0.025
Brilliant green 0.0133
Agar No. 1 12.0
Method for reconstitution
Weigh 40 grams of powder, disperse in 1 litre of deionised water. Method for reconstitution
Allow to soak for 10 minutes, swirl to mix then warm to dissolve. Weigh 28 grams of powder and disperse in 1 litre of deionised water.
Dispense into tubes or bottles with inverted Durham tubes. Sterilise Allow to soak for 10 minutes, swirl to mix then sterilise by
by autoclaving at 115˚C for 15 minutes. autoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pour
into petri dishes.
Appearance: Green, clear.
Appearance: Purple, clear agar.
pH: 7.4 ± 0.2
pH: 6.8 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli NCIMB 50034 Minimum Q.C. organisms: E. coli. NCIMB 50034
S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 1 month
at 2-8˚C in the dark. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation: Serial 1:10 dilutions of homogenised sample are
inoculated into the broth in the proportion of 1ml sample to 9mls Inoculation: Surface, plating either over entire surface for colony
broth. Ensure the Durham tube is free from gas bubbles before count or streak out to single colonies.
commencing inoculation. B.G.B. broth can be used at double strength Incubation: 37˚C aerobically for 18-24 hours.
if required but cannot be sterilised by autoclaving, pasteurisation
must be used instead.
Growth Characteristics
Incubation: E. coli and thermotrophs 44˚C for 18 hours aerobically.
Mesopholic coliforms 32˚C for 24-48 hours aerobically. colony size shape &
Psychrotrophic coliforms 4˚C for 10 days aerobically. organism (mm) surface colour other
Interpretation: Turbidity, colour changes (to yellow or yellowish E. coli 1.5-2.0 CV.E.G. Yellow (N.L.F. – purple)
green) and production of gas are all presumptive evidence of the Klebsiella spp. 4.0-6.0 CV.E.G. Yellow (mucoid)
growth of organisms of the coli-aerogenes group. Confirmation by
indole production in Tryptone Water LAB 129 (44˚C for E. coli). Citrobacter spp. 2.0-2.5 CV.E.G. Yellow
Proteus spp. 1.0-1.5 CV.E.G. Purple
References
Salmonella spp. 1.0-2.0 CV.E.G. Purple
Pharmacopoeia of Culture Media for Food Microbiology 1987. Int. J.
Food Microbiol. 5:3:206-207. S. aureus 0.5 CV.E.G. Cream (purple if N.L.F.)
American Public Health Association, American Water Works E. faecalis 0.5 CV.E.G. Yellow
Association and Water Pollution Control Federation, 1975, Standard
Methods for the Examination of Water and Wastewater, 14th ed., References
Washington D.C.
Drigalski, Conrad. 1902. Uber ein Verfahren zum Nachweis der
Association of Official Analytical Chemists (AOAC). Bacteriological Typhusbacillen. Z. Hyg. Infekt. 39:283-300.
Analytical Manual, 5th ed., Washington, D.C. Association of Official
Analytical Chemists. 1978.
Hausler, W. J. (ED) 1972. Standard Methods for the Examination of
Dairy Products. 13th ed., Washington. D.C. American Public Health
Association.
Shane, M.S. 1947. Studies on false confirmed test using B.G.B. and
comparison studies on Lauryl Sulfate Tryptose Broth as presumptive
medium. J. Am. Water Works Assoc., 39: (4), 337.

38
Buffered Listeria Enrichment Broth Minimum Q.C. organisms: E. coli NCIMB 50034

LAB 139 Storage of Prepared Medium: Capped containers – up to 3 months

at 15-20˚C in the dark.
Description
Inoculation: Add 25 grams of sample to 225ml of Buffered Peptone
A medium for the selective enrichment of food and environmental
Water and homogenise.
samples for Listeria spp, LAB 139 is a buffered version of the ‘FDA’
broth LAB 138. The extra buffering capacity maintains the pH of the Incubation: Aerobically at 37˚C for 18-24 hours.
enrichment culture during incubation, ensuring optimum conditions Subculture: 10ml aliquots in 100mls of Selenite Cystine Broth
for the recovery of Listeria spp. LAB 55 and 0.1ml into 10ml Rappaport Vassiliadis Medium LAB 86.
Formula g/litre References
Tryptone 17.0 Edel W. and Kampelmacher E. H. 1973. Bull. Wld Hlth Org. 48: 167-
174.
Soy peptone 3.0
Poemla P. K. and Silliker J. H. 1976 Salmonella in Compendium of
Sodium chloride 5.0 Methods for microbiological examination of foods. Am. Pub. Health
Ass., Washington.
Dipotassium hydrogen phosphate 2.5
Glucose 2.5
Yeast Extract 6.0
Potassium dihydrogen phosphate 1.35 Campylobacter Blood Free
Disodium hydrogen phosphate 9.6 Selective Medium
(Modified CCDA-Improved)
Method for reconstitution
Weigh 47 grams of powder and add to 1 litre of deionised water. LAB 112
Allow to soak for 10 minutes then swirl to mix and sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials of Description
X138 reconstituted in 50% alcohol. Aseptically dispense into sterile A blood free medium which will support the growth of most enteric
tubes or bottles. campylobacters. The selective cocktail X112 makes the medium
Appearance: Yellow, clear. selective for C. jejuni. C. coli and C. laridis when incubated at 37˚C.
With this product incubation at 42˚C is no longer necessary and
pH: 7.2 ± 0.2 higher recovery rates have been reported at 37˚C than at 42˚C.
The antibiotic sensitive strains of campylobacter e.g. C. cinaedi and
Minimum Q.C. organisms: L. monocytogenes NCIMB C. fennelliae can be isolated on this medium by the membrane filter
50007 method, but it is essential the antibiotic supplement is omitted to
E. coli (inhibition) NCIMB 50034 allow these organisms to grow. The supplement X112 consists of
cefoperazone and amphoteracin and is superior to the selective
Storage of Prepared Medium: Capped containers – up to 14 days at cocktails of Skirrow, Butzler and Blazer-Wang all of which contain
2-8˚C in the dark. antibiotics shown to be inhibitors to C. coli. The colonial
morphologies of Campylobacter spp. on this medium are distinctive.
Inoculation: Homogenised samples of food, 25 grams of
homogenate to 225mls of broth.
Formula g/litre
Incubation: 30˚C aerobically for up to 48 hours.
Peptone blend 25.0
Subculture: After 24 and 48 hours onto Listeria Isolation Medium –
LAB 122. Bacteriological Charcoal 4.0
Sodium chloride 3.0
Sodium desoxycholate 1.0

Buffered Peptone Water Ferrous sulphate 0.25

Sodium pyruvate 0.25
LAB 46
Agar No. 2 12.0
Description
A pre-enrichment medium design to help sublethally damaged Method for reconstitution
salmonellae recover before introducing them into a selective Weigh 45.5 grams of powder, disperse in 1 litre of deionised water
medium. This nutrient medium is free from inhibitors and is well and allow to soak for 10 minutes. Swirl to mix, then sterilise by
buffered to maintain the pH at 7.2 for the incubation period. Sublethal autoclaving at 121˚C for 15 minutes. Cool to 47˚C then add 2 vials of
injury to salmonellae occurs in many food processes and this pre- X112 supplement, mix well and pour into petri dishes. Continuously
enrichment step greatly increases recovery of these organisms. mix whilst pouring to prevent the charcoal settling.
Appearance: Black agar.
Formula g/litre
pH: 7.4 ± 0.2
Peptone 10.0
Minimum Q.C. organisms: C. jejuni
Sodium chloride 5.0 E. coli (inhibition) NCIMB 50034
Disodium hydrogen phosphate 3.7 Candida albicans (inhibition)
NCIMB 50010
Potassium dihydrogen phosphate 1.5
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
Method for reconstitution dark.
Weigh 20 grams of powder and disperse in 1 litre of deionised water. Inoculation: C. jejuni, C. coli. C. laridis surface streaking to single
Mix to dissolve then distribute into tubes or bottles. Sterilise by colonies. C. cinaedi, C. fennelliae use the membrane filtration
autoclaving at 121˚C for 15 minutes. method of Goosens et al.
Appearance: Pale straw, clear liquid. Incubation: 37˚C for 48 hours in an atmosphere of 5% oxygen, 10%
pH: 7.2 ± 0.2 carbon dioxide and 85% nitrogen. C. cinaedi and C. fennelliae
require up to 7 days. 39
 Growth Characteristics Method for reconstitution
Weigh 27.6 grams of powder, disperse in 1 litre of deionised water
colony size shape & and allow to soak for 10 minutes. Swirl to mix and autoclave at 121˚C
organism (mm) surface colour other for 15 minutes. Cool to 47˚C, add 2 vials of selective supplement
C. jejuni 2.0-3.0 F.E.G. Grey/ X131 (reconstituted with 5mls of 50% alcohol) and 50mls of saponin
White Efflorescent lysed horse blood, mix well and dispense into sterile containers.
(spreading moist) Appearance: Translucent, wine-red with a fine black suspension.
C. coli 1.0-2.5 CV.E.G. Creamy Moist pH: 7.4 ± 0.2
Grey
C. laridis 1.5-3.0 F.E.G. Grey (C.V. moist) Minimum Q.C. organisms: Campylobacter jejuni
E. coli (inhibition) NCIMB 50034
C. cinaedi 2.0-3.0 F.E.G. Pale Grey requires
7 days in Storage of Prepared Medium: Capped containers: 7 days at 2-8˚C
absence of X112
in the dark.
C. fenneliae 2.0-3.0 F.E.G. Pale Grey requires
Inoculation: Food homogenate is added to broth in a ratio of 1:4
7 days in
(w/v) in screw cap containers leaving a head space of 1.5 cm. For
absence of X112
faeces 1ml of a 10% suspension in Buffered Peptone Water LAB 46
Other Enterobacteriacae – No growth if sensitive to cefoperazone. is added to 5ml of broth.
Incubation: Aerobically at 37˚C for 2-4 hours, followed by a further
References 16-44 hours at 42˚C.
Bolton F. J. Hutchinson D.N., Parker G. Reassessment of Selective Subculture: Onto Campylobacter Blood Free Selective Medium
Agars and Filtration Techniques for Isolation of Campylobacter LAB 112.
Species from Feces. Eur.J. Clin. Microbiol. Infects. Dis. 1988 7 p
155-160.
References
Bolton F. J. 1988 Personal Communication. Bolton, F. J. Personal Communication.
Bolton F. J. Hutchinson D. N., Parker G. Isolation of Campylobacter: Hunt J. M., Abeyta C., and Tran T. (1998) Chapter 7 Campylobacter
What are we missing? J.Clin.Path. 1987 40 p 702-703. in FDA Bacteriological Analytical Manual 8th Edition.
Goosens H., De. Boeck M., Coignau H.. Vlaes L., Van Den Borre C.,
Butzler J. P. Modified Selective Medium for Isolation of
Campylobacter spp from Feces : Comparison with Preston Medium,
a Blood Free Medium, and a Filtration System. J.Clin. Micro. 1986
24 p 840-843. C.E.M.O. Agar Base
Gun-Munro J., Rennie R. P., Thornley J. H. Richardson H. L., Hodge (Contagious Equine Metritis Organism)
D., Lynch J. Laboratory and Clinical Evaluation of Isolation Media
for Campylobacter jejuni J. Clin Micro. 1987. 25 p 2274-2277. LAB 78
Herbert G. A., Hollis D. G., Weaver R. E., Karmali M. A., Simor A.
E., Roscoe M., Fleming P. C., Smith, S. S. Lane J. Evaluation of a Description
Blood-Free, Charcoal-Based, Selective Medium for the Isolation of This medium is a selective isolation medium for Taylorella
Campylobacter organisms from Faeces. J. Clin. Micro. 1986 23 p equigenitalis the causative organism of contagious equine metritis.
456-459. The medium is a sugar free base with a mixture of high grade casein
and soy peptones as nutrients and with L-cystine and sodium sulphite
as supplements and reducing agents. The medium is made selective
with the addition of amphoteracin (5 mg/L) and trimethoprim (10
mg/L). Streptomycin (200 mg/L) can also be used but sensitive
Campylobacter Enrichment Broth variants of T. equigenitalis have been described.

LAB 135 Formula g/litre

Description Tryptone 15.0

A selective enrichment broth for the isolation of Campylobacter spp. Soy Peptone 5.0
from food, environmental samples and faeces. The use of a selective
enrichment broth enhances the recovery of sub-lethally damaged Sodium chloride 5.0
organisms due to processing of foods, or if small numbers of Agar No. 2 12.0
campylobacters are present in heavily contaminated specimens.
This broth has been shown to give appreciably better results than L-Cystine 0.3
Preston Broth.
Sodium sulphite 0.2
Formula g/litre
Method for reconstitution
Meat Peptone 10.0 Weigh 37.5 grams of powder, disperse in 1 litre of deionised water.
Lactalbumin Hydrolsates 5.0 Allow to soak for 10 minutes, swirl to mix then sterilise by autoclaving
at 121˚C for 15 minutes. Allow to cool to 80˚C, add 50ml of sterile
Yeast Extract 5.0 horse blood and allow to ‘chocolate’. Further cool to 47˚C before
adding antibiotic selective agents. Mix well and pour into petri dishes.
Sodium chloride 5.0
Appearance: Chocolated Blood Agar.
Haemin 10.0mg
pH: 7.3 ± 0.2
Sodium pyruvate 0.5
Minimum Q.C. organisms: T. equigenitalis
α – ketoglutaric acid 1.0 E. coli (inhibition) NCIMB 50034
Sodium metabisulphite 0.5
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Sodium carbonate 0.6 the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C in 10% CO2 for 2-3 days.

40
 Growth Characteristics References
United States Pharmacopoeia XXI. 1985.
colony size shape &
organism (mm) surface colour other Brown V. I., Lowbury E. J. L. 1965. Use of an improved Cetrimide
Agar Medium and other culture methods for Pseudomonas
T. equigenitalis 0.1-1.0 CV.E.G. Cream colony size aeruginosa. J. Clin. Pathol. 18, 752-756.
variation is
common

References
Atherton, J. G. 1978. Inhibition of the C.E.M. organism in mixed
cultures. Vet. Rec. 432.
Mackintosh, M. E. 1981. Bacteriological techniques in the diagnosis
of equine genital infections. Vet. Rec. 108, 52-55. Atherton, J. G.
Personal Communication.
Fleming, M. P. Tribe. G. W. 1977. Vet. Rec. 101, 1470.
Cetrimide Agar
China Blue Lactose Agar
LAB 105
Description
China Blue Lactose Agar is a non-inhibitory medium for the
differentiation and enumeration of bacteria in milk. The formulation
is the same as that described by the Methodenkomission für
Milchivirtschaft. Lactose fermenters in milk form blue colonies. The
medium is non-inhibitory to the growth of cocci, making it a useful
medium for the detection of streptococci and staphylococci as well as
coliform organisms.

U.S.P. Formula g/litre

LAB 133 Balanced Peptone No. 1 5.0
Beef Extract 3.0
Description
A medium recommended by the United States Pharmacopoeia for the Lactose 10.0
isolation of Pseudomonas aeruginosa from pharmacological
preparations. Subculture is carried out onto the medium after Sodium chloride 5.0
enrichment in LAB 4 Tryptone Soy Broth. Cetrimide inhibits the Aniline blue 0.325
growth of many micro organisms whilst allowing Ps. aeruginosa to
develop typical colonies which will fluoresce in ultraviolet light and Agar No. 2 12.0
produce green pigment.
Method for reconstitution
Formula g/litre Weigh 35 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
Pancreatic Digest of Gelatin 20.0
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into petri
Magnesium chloride 1.4 dishes.
Potassium sulphate 10.0 Appearance: Blue clear agar.

Cetyl trimethylammonium bromide (cetrimide) 0.3 pH: 7.0 ± 0.2

Agar 13.6 Minimum Q.C. organisms: E. coli NCIMB 50034

Method for reconstitution
Weigh 45.3 grams of powder, disperse in 1 litre of deionised water.
Add 10mls of glycerol, allow to soak for 10 minutes then swirl to
mix. Sterilise at 121˚C for 10 minutes.
Appearance: Opalescent, pale yellow agar.
pH: 7.2 ± 0.2
Staph. aureus NCIMB 50080
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation: Surface, spread inoculum evenly over entire surface.
Incubation: 37˚C aerobically for 18-24 hours.
Interpretation: Count all colonies

Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067

E. coli (inhibition) NCIMB 50034
Growth Characteristics
colony size shape &
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the organism (mm) surface colour other
dark. E. coli 1.5-2.0 CV.E.G. Blue (colourless if NLF)
Inoculation: Subculture from enrichment broth, streak out for single Klebsiella spp. 2.0-2.5 CV.E.G. Blue (mucoid)
colonies.
Proteus spp. 0.5-2.5 F.Rz.G. colourless (spreading)
Incubation: 30-35˚C aerobically for 24-48 hours.
Salmonella spp. 2.0 CV.E.G. colourless
Growth Characteristics Shigella spp. 1.0 CV.E.G. colourless
colony size shape & Pseudomonas
organism (mm) surface colour spp. 2.5-3.0 F.CR.D. colourless
Ps. aeruginosa 0.5-1.0 F.CR.D. green pigment S. aureus 0.5-1.0 CV.E.G. Pale Blue
(non pigment) S. epidermidis 0.5-1.0 CV.E.G. Blue
green/yellow
fluorescence
References
Ps. fluorescens 0.5 CV.R.E.G. green/yellow Methodenbuch Band VI. Vergand Deutscher Landwirtschaftlicher
fluorescence Untersuchungs – und Forschugsanstallen.
E. coli N.G.
S. aureus N.G.
Proteus spp. N.G.

41
 C.L.E.D. (Bevis modification) References
Bevis, T. D. 1968. A modified electrolyte-deficient culture medium.
(Cystine Lactose Electrolyte Deficient – Double Indicator) J. Med. Lab. Tech., 25: 38-41.
Mackey, J. P. and Sandys, G. H. 1966. Diagnosis of urinary
LAB 6 infections, Brit.Med. J., 1: 1173.
Description Sandys, G.H. 1960. A new medium for preventing swarming of
Bevis modified Mackey and Sandys original medium by introducing Proteus spp. with a description of a new medium suitable for use in
a double indicator to improve the differentiation of lactose and non routine laboratory practice. J. Med.Lab. Tech., 17: 224-233.
lactose fermenting coliforms, staphylococci and streptococci. The
swarming of Proteus spp. is inhibited. LAB M C.L.E.D. will grow
many of the more demanding streptococci of Lancefield groups A, B,
C, G and F. This medium may not grow Pasteurella spp. or halophilic
organisms. C.L.E.D. Medium
(Mackey and Sandys)
Formula g/litre
(Cystine Lactose Electrolyte Deficient-Single Indicator)
Balanced Peptone No. 1 4.0
LAB 41
Beef Extract 3.0
Tryptone 4.0
Description
A medium for urine culture first described by Mackey and Sandys in
Lactose 10.0 1960. The absence of electrolytes inhibits the swarming of Proteus
spp. Cystine is added for the benefit of those organisms which have
L-Cystine 0.128 a specific cystine requirement. Differentiation of lactose and non
Bromothymol blue indicator 0.02 lactose fermenters is achieved using bromothymol blue as pH
indicator. This medium supports the growth of Streptococcus
Andrade’s indicator 0.08 pyogenes and most other fastidious organisms that do not require
blood.
Agar No. 1 15.0
Formula g/litre
Method for reconstitution
Weigh 36 grams of powder, disperse in 1 litre of deionised water. Balanced Peptone No. 1 4.0
Allow to soak for 10 minutes, swirl to mix then sterilise by
Beef Extract 3.0
autoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix before
pouring. Tryptone 4.0
Appearance: Green/blue, clear gel. Lactose 10.0
pH: 7.5 ± 0.2
L-Cystine 0.128
Minimum Q.C. organisms: E. coli NCIMB 50034 Bromothymol blue indicator 0.02
S. aureus NCIMB 50080
Agar No. 1 15.0
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Method for reconstitution
Inoculation method: Surface inoculation, either streaking for single Weigh 36 grams of powder, disperse in 1 litre of deionised water.
colonies or spread evenly over entire surface for colony counts. Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving for 15 minutes at 121˚C. Cool to 47˚C mix and distribute
Incubation: 37˚C for 24 hours aerobically. into petri dishes.
Appearance: Green/blue clear gel.
Growth Characteristics
pH: 7.3 ± 0.2
colony size shape &
organism (mm) surface colour other Minimum Q.C. organisms: E. coli NCIMB 50034
E. coli 2.0-3.0 CV.E.G. Yellow/ (Blue if S. aureus NCIMB 50080
Orange non-lactose
fermenter) Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
K. aerogenes 3.0-4.0 CV.E.G. Yellow/ mucoid
Orange Inoculation: Surface inoculation either spreading for single colonies
or spread evenly over entire surface for colony counts.
Proteus spp. 2.0-3.0 CV.E.G. Blue
Incubation: 37˚C aerobically for 18-24 hours.
Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (Green pigment
& odour)
Shigella spp. 1.5-2.5 CV.E.G. Blue
Salmonella spp. 2.0-3.0 CV.E.G. Blue (Yellow-orange
if lactose +ve)
S. aureus 1.0-1.5 CV.E.G. Yellow/ (Blue if
Orange non-lactose
fermenting)
Other (Yellow if
Staphyloccocci 0.5-1.5 CV.E.G. Blue-White lactose
fermenting)
Enterococcus spp. 0.5 CV.E.G. Yellow-
Orange

42
 Growth Characteristics colony size shape &
organism (mm) surface colour other
colony size shape &
S. aureus 1.5-2.0 CV.E.G. White-
organism (mm) surface colour other
Yellow Haemolytic
E. coli 2.0-3.0 CV.E.G. Yellow (blue if non
S. pyogenes 0.5-1.0 CV.E.G.(D) White α, β-haemolytic
lactose fermenters)
dependent on
K. aerogenes 3.0-4.0 CV.E.G. Yellow (mucoid) strain
Proteus spp. 2.0-3.0 CV.E.G. Blue S. pneumoniae 0.5-1.5 F.E.G. Grey greenish
discolouration
Ps. aeruginosa 1.0-4.0 F.CR.D. Blue (green pigment
in medium, mucoid
& odour)
in H2/C02
Shigella spp. 1.5-2.5 CV.E.G. Blue
Neisseria
Salmonella spp. 2.0-3.0 CV.E.G. Blue (yellow if meningitidis 1.0-2.0 CV.E.G. trans/ (mucoid)
lactose +ve) Grey
Staph. aureus 1.0-1.5 CV.E.G. Yellow (blue if non- E. coli 2.0-3.0 CV.E.G. Opaque/ (haemolytic)
lactose Grey
fermenting)
Ps. aeruginosa 0.5-4.0 F.CR.D. Opaque many colonial
other Staphylococcus (yellow if Grey forms
spp. 0.5-1.5 CV.E.G. Blue-white lactose (green pigment)
fermenting) (haemolytic)
Enterococcus (mucoid)
spp. 0.5 CV.E.G. Yellow C. perfringens 1.5-2.0 CV.CR.G. Grey usually target
haemolysis
(non haemolytic)
References
Mackey, J. P. and Sandys, G. H. 1966. Diagnosis of urinary B. fragilis 1.0-1.5 CV.E.G. Grey (mucoid)
infections. Brit.Med.J. 1: 1173. P. anaerobius P.P.-0.5 CV.E.G. White/
Guttman, D and Naylor, G.R.E. 1967. Dip-slide: an aid to Grey
quantitative urine culture in general practice. Brit.Med. J. 3: 343-345.
References
Ellner, P. D., Stoessel, C. J., Drakeford, E and Vasi, F. 1966. A new
culture medium for medical bacteriology. Amer. J. Clin Pathol.,
45:502-504.
Columbia Agar Base Goldberg, R. L., and Washington, J. A., 1976. Comparison of
LAB 1 isolation of Haemophilus vaginalis (Corynebacterium vaginale) from
Peptone-Starch-Dextrose Agar and Columbia Colistin-Nalidixic Acid
Description Agar. J. Clin. Microbiol., 4:245-247.
A general purpose nutritious agar base formulated by Ellner et al. Thayer, D. D. and Martin, H. E. 1966. An improved medium for the
When further enriched by the addition of sterile blood, Columbia cultivation of N. gonorrhoeae and N. meningitidis. Publ. Hlth.
agar can be used for the isolation of most clinically significant Report, 81:559-562.
pathogens. The blood can be ‘chocolated’ if required. The medium
can be made selective for various groups by the addition of
appropriate antibiotic mixtures eg:
Streptococci – Colistin/Oxolinic acid (X013)
Gardnerella spp. – Colistin/Nalidixic acid (X011) Cooked Meat Granules
C. perfringens – Neomycin (XO15) (X016)
Staphylococci/streptococci – Colistin/Naladixic acid (X012) (for Cooked Meat Medium)
LAB 24
Formula g/litre
Columbia Peptone Mixture 23.0 Description
Dried minced ox heart which has been trimmed of excess fat and
Corn Starch 1.0 prepared according to the Martin and Lekpers modification of
Robertsons original formulation. The medium gives good growth of most
Sodium chloride 5.0 organisms and is especially useful in the recovery of fastidious
Agar No. 2 12.0 anaerobes. The medium is also suitable for prolonged storage of cultures.

Method for reconstitution

Method for reconstitution
Weigh 41 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving for 15 minutes at 121˚C. Cool to 48˚C and add 5-7%
sterile, defibrinated horse or sheep blood. Mix well before pouring.
Appearance: Cherry red if blood is fresh and well oxygenated.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080
S. pyogenes ATCC 19615
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation: Surface plating, streaking out for single colonies.
Incubation: 37˚C aerobically or microaerobically for 24 hours.
Anaerobically for 24 and 48 hours.
To make Cooked Meat Medium the granules should be added to
Nutrient Broth (LAB 14) or Fastidious Anaerobe Broth (LAB 71) in
the ratio of 15 grams of granules to 200ml. of broth. Cooked Meat
Medium should be dispensed into containers allowing at least 20-
25mm depth of meat particles and a broth supernatant of at least 10-
15mm. To sterilise autoclave at 121˚C for 15 minutes in capped tubes
which should be tightened after autoclaving to prevent re-oxygenation.
Medium prepared with Nutrient Broth LAB 14 should be
re-steamed when used after a period of storage. Medium made with
Fastidious Anaerobe Broth LAB 71 will not require
re-steaming after storage.
Storage of Prepared Medium: – up to 6 months at 15-20˚C in
the dark.
References
Cruickshank, R. 1972. Medical Microbiology. 11th Edn. Livingstone,
London.

Growth Characteristics
43
 Cooked Meat Medium Formula g/litre

LAB 127 Beef Extract 5.0

Balanced Peptone No. 1 5.0
Description
This is a complete version of Robertson’s Cooked Meat Broth which Lactose 10.0
was designed to grow all anaerobes found in battlefield wounds
during the First World War. The medium is based on LAB M Cooked Sodium citrate 5.0
Meat Granules – LAB 24 and Nutrient Broth – LAB 14. Sodium thiosulphate 5.0

Formulation g/litre Ferric citrate 1.0

Cooked meat granules 75.0 Sodium desoxycholate 2.5

Beef Extract 10.0 Neutral Red 0.025

Balanced Peptone No. 1 10.0 Agar No. 2 12.0

Sodium chloride 5.0 Method for reconstitution

Method for reconstitution
Use a calibrated scoop to distribute 0.9 gram amounts of granules into
tubes or bottles. Add 10ml deionised water. Sterilise at 121˚C for 10
minutes. Use proportionately more granules and water if greater
depths of medium are required.
Appearance: Granules covered in slightly opalescent pale
yellow liquid.
pH: 7.0 ± 0.2
Inoculation: Samples or swabs directly into the medium.
Incubation: 37˚C for mesophiles, appropriate temperature
for thermophiles.
Interpretation: Reddening of meat – saccharolytic organism –
Blackening and digestion – proteolytic organism.
References
Robertson, M., (1916) J. Path. & Bact. 20 327-349.
Weigh 45.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix, then bring to the boil with
frequent stirring. When the medium boils up into the neck of the
flask, quickly remove from the source of heat and allow the froth to
subside. Return to the heat and allow the foam to boil up into the neck
of the flask once more. Remove at once and cool to 47˚C approx.
before pouring plates. Dry the surface before inoculation. DO NOT
REMELT OR AUTOCLAVE THIS MEDIUM.
Appearance: Pale pink, translucent, a fine precipitate of
desoxycholate may be present which may clear if the pH is increased
by the growth of organisms.
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation: Surface, streaking for single colonies.
Incubation: 37˚C for 18-24 hours aerobically.

Cooked Meat Medium Tablets Growth Characteristics

colony size shape &
LAB 24Z organism (mm) surface colour other
Description S. typhi 0.5. 1.5 CV.E.G. transp (black centre)
Yellow
As Cooked Meat Granules (LAB 24) with Nutrient Broth (LAB 14)
incorporated and presented in tablet form. Other Salmonella
spp. 1.5-2.0 CV.E.G. transp (black centre)
Method for reconstitution (Opaque) (clearing
Add 2 tablets to 10mls of deionised water in a narrow container. Yellow around colony)
Allow to soak for 15 minutes then sterilise by autoclaving at 121˚C S. sonnei 1.5-2.0 CV.E.G. transp (more opaque
for 15 minutes. (pinkish) centre)
S. flexneri group 1.0-1.5 CV.E.G. transp-
(pinkish)
S. dysenteriae 0.5-1.0 CV.E.G. transp
D.C.A. E. coli P.P.-1.5 CV.E.D. Red/Pink ppt in medium
(Desoxycholate Citrate Agar) (uninhibited) (G)
Citrobacter spp. P.P.-2.0 CV.E.D. Red/Pink ppt in medium
LAB 29 (G) (black centre)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (black/grey
Description
centre)
This is Leifson’s original formulation of this selective medium for the fishy odour
isolation of Salmonella spp. and Shigella spp. from faeces and (clearing around
environmental samples. It has approximately half the quantity of colony)
inhibitors used in the Hynes modification. The medium uses sodium
citrate and sodium desoxycholate as inhibitors. Sodium thiosulphate Pseudomonas
is the substrate for the enzyme thiosulphate reductase being broken spp. 0.5-1.5 CV.E.D. Yellow/ (green pigment)
down to form sulphite and hydrogen sulphide. The hydrogen sulphide (G) Pink
reacts with the ferric ions to produce a black precipitate of ferrous
sulphide. This gives a typical black centre to the colonies of most References
species of Salmonella. Hynes. M. 1942. The isolation of intestinal pathogens by selective
media. J. Path. Bact. 54. 193-207.
Liefson. E. 1935. New culture media based on Sodium desoxycholate
for the isolation of intestinal pathogens and for the enumeration of
colon bacilli in milk and water. J. Path. Bact. 40: 581-589.

44
D.C.A. Hynes D.C.L.S. Agar
(Desoxycholate Citrate Agar -Hyne’s modification) (Desoxycholate Citrate Lactose Sucrose Agar)
LAB 65 LAB 3
Description Description
This modification of Leifson’s D.C.A. medium was introduced in A modification of Leifson’s D.C.A. medium which incorporates
1942. The medium was designed to be more inhibitory to commensal sucrose as an additional fermentable substrate to differentiate lactose
flora whilst allowing for adequate growth of Salmonella spp and negative sucrose positive coliforms from Salmonella spp. This
Shigella spp. The citrate and desoxycholate levels are significantly medium is unsuitable for the isolation of Yersinia spp. which are
increased. To keep the desoxycholate in solution the pH also had to sucrose positive.
be increased. The medium still uses lactose fermentation and
hydrogen sulphide production as differential indicators. Formula g/litre

Formula g/litre Balanced Peptone No. 1 7.0

Beef Extract 5.0 Beef Extract 3.0

Balanced Peptone No. 1 5.0 Lactose 5.0

Lactose 10.0 Sucrose 5.0

Sodium thiosulphate 5.4 Sodium citrate 10.5

Sodium citrate 8.5 Sodium thiosulphate 5.0

Ferric citrate 1.0 Sodium desoxycholate 2.5

Sodium desoxycholate 5.0 Agar No. 2 12.0

Neutral red 0.02 Neutral Red 0.03

Agar No. 2 12.0 Method for reconstitution

Weigh 50 grams of powder, disperse in 1 litre of deionised water.
Method for reconstitution Allow to soak for 10 minutes then heat gently with frequent mixing
Weigh 52 grams of powder, disperse in 1 litre of deionised water in a and bring to the boil. Simmer for 1 minute to complete dissolution of
two litre flask. Bring to the boil over a gauze, swirling frequently to the solids. Cool to 47˚C then distribute 20ml into 90mm Petri dishes.
prevent burning. Simmer for 30 seconds to dissolve. Cool to 47˚C Dry the surface by partial exposure, before use. DO NOT REMELT
before pouring plates. Dry the surface before inoculation. DO NOT OR AUTOCLAVE THIS MEDIUM.
REMELT OR AUTOCLAVE THIS MEDIUM.
Appearance: Pale Pink, clear.
Appearance: Pink, clear, bile aggregates may appear on the surface
pH: 7.2 ± 0.2
on refrigeration.
pH: 7.4 ± 0.2 Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli NCIMB 50034
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli NCIMB 50034 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Inoculation: Surface plating, streaking out to single colonies.
the dark.
Incubation: 37˚C aerobically for 24 hours.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C aerobically for 24 hours. Growth Characteristics
Growth Characteristics colony size shape &
organism (mm) surface colour other
colony size shape &
S. typhi 0.5-1.0 CV.E.G. Trans.
organism (mm) surface colour other colourless
S. sonnei 1.0-2.0 CV.E.G.(D) Colourless Other
- pale pink Salmonella spp. 1.5-2.0 CV.E.G. Slight
S. flexneri 1.0-2.0 CV.E.G. Colourless cloudy
colourless
Salmonella spp. 1.0-4.0 CV.E.G. Colourless (black centre)
S. sonnei 1.5-2.0 CV.E.G. Trans. (More opaque
S. typhi 0.5-1.5 CV.E.G. Colourless (Black/grey
Pinkish centre)
centre)
S. flexneri 0.5-1.0 CV.E.G. Trans.
E. coli P.P.-1.5 CV.CR.D. Red (No growth)
Pinkish
K. aerogenes 1.0-2.5 CV.E.G. Pink (mucoid)
S. dysenteriae 0.5 CV.E.G. Trans.
Proteus spp 0.5-2.0 CV.E.G. Colourless (Yellow) colourless
fishy odour
E. coli P.P.-1.5 CV.E.G.(D) Red (ppt around
P. aeruginosa 0.5-1.0 CV.CR.D. Colourless (green) (inhibited) colonies)
Citrobacter spp. P.P.-2.0 CV.E.D.(G) Red (ppt around
References (inhibited) colonies)
Hynes, M. 1942. The isolation of intestinal pathogens by selective Proteus spp. 1.0-2.0 CV.E.G. Yellow (Fishy odour)
media. J. Path. Bact, 54: 193-207
Pseudomonas
spp. 0.5-1.0 CV.E.D. Yellow (Green pigment)
Pink

45
 References
Hynes, M. 1942. The isolation of intestinal pathogens by selective
Dextrose Tryptone Agar
media. J. Path. Bact. 54: 193-207. LAB 20
Leifson, E. 1935. New culture media based on sodium desoxycholate
for the isolation of colon bacilli in milk and water. J. Path. Bact. Description
40: 581-589. A medium for the enumeration of thermophilic spore bearers in
foods. The medium was designed to detect the thermophilic bacteria
causing ‘flat sour’ spoilage of canned foods. The medium also detects
the ‘flat sour’ organism Bacillus stearothermophilus in sugar and
other sweetening agents used in the preparation of frozen dairy foods,
Dermatophyte Test Medium (D.T.M.) cereals and other food products.

LAB 117 Formula g/litre

Description Tryptone 10.0
A modification of the formulation of Taplin, Zaias, Rebell and Blank Glucose 5.0
for the detection of dermatophytic fungi. This medium helps in the
differentiation between saprophytic and environmental fungi. Bromocresol purple 0.04
Agar No. 2 12.0
Formula g/litre
Balanced Peptone No. 1 10.0 Method for reconstitution
Glucose 40.0 Weigh 27 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil to
Agar No. 2 12.0 dissolve agar before dispensing in 20ml amounts for poured plate
technique. Sterilise by autoclaving at 121˚C for 15 minutes.
Phenol Red 0.2
Appearance: Purple clear agar.
Method for reconstitution pH: 6.9 ± 0.2
Weigh 62 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes then bring to the boil with frequent Minimum Q.C. organisms: B. stearothermophilus
stirring. Dissolve 2 vials of Chloramphenicol X009 in ethanol and
add these to the agar, mix well and distribute into tubes or universal Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
containers. Sterilise at 121˚C for 15 minutes, allow to cool in the the dark. Capped container – up to 3 months at 15-20˚C in the dark.
sloped position. Inoculation: Pour plate technique, pre-heat sample by steaming for
Note: Do not exceed the times stated for sterilisation, overheated 20 minutes if a spore count is required.
acidified agar loses gel strength and the sugars are caramelised. Incubation: For Thermophiles – Aerobically for 48 hours at 55˚C.
Appearance: Yellow, clear gel. For Mesophiles – Aerobically for 48-72 hours at 30-32˚C.
pH: 5.5 ± 0.2 Interpretation: Count all colonies for total counts, count yellow
colonies for differential acid producer count. Non acid producing
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097 colonies are grey to colourless.
Trichophyton sp.
Growth Characteristics
Storage of Prepared Medium: Slopes – up to 1 month at 2-8˚C in
the dark. organism colony shape & colour
size (mm) surface
Inoculation: Surface plating or stab inoculation.
B. stearothermophilus 2.0 Rz.D Yellow zone
Incubation: 22-25˚C aerobically for 10-14 days. mauve centre
Interpretation: Dermatophytes appear as fluffy colonies, colour Bacillus spp. 1.5-3.0 Rz.D Mauve
varies with species, the medium is reddened. Fungi other than (Yellow halo)
dermatophytes cause the medium to become yellow due to acid
production. If incubation is prolonged the medium may become S. aureus 0.5-1.5 CV.E.G. Yellow
reddened. Yeasts appear as white creamy colonies. Blastomyces, E. coli 1.0-1.5 CV.E.G. Yellow
Histoplasma and Coccidiodes may also turn the medium red, though
these are rarely encountered in lesions associated with ring worm. Klebsiella spp. 1.5-2.5 CV.E.G. Yellow (mucoid)
Enterococci 0.5 CV.E.G. Yellow
References Proteus spp. 2.0-3.0 RzD Yellow (spreads)
Taplin, D., Zaias, N., Rebell, G., Blank, H. 1969. Isolation and
recognition of dermatophytes on a new medium. (DTM) Arch.
Dermatol. 99: 203-209. References
Williams, O. B. 1963. Tryptone Medium for the Detection of Flat
Sour Spores. Food Research 1, (3): 217-221.
American Public Health Association. 1972. Standard Methods for the
Examination of Dairy Products. 13th Edn. Ed. W. J. Hausler A.P.H.A.
Washington.
Tanner, F. W. 1946. The Microbiology of Food 2nd edn., Garrard
Press, Champners.
Baumgartner, J. G. and Hersom, A. C. 1956. Canned Foods. 4th Edn.
Churchill, London.

46
Diagnostic Semi-Solid Salmonella
Agar (Diassalm)
According to Van Netten and Van der Zee et al
LAB 537
Description
Diassalm, as developed by Van Netten et al (1991), is a semi-solid
differential medium for the isolation of Salmonella spp. from food
and water. It is an improved modification of MSRV (De Smedt and
Bolderdijk 1988) and SR (Perales and Audicana 1989) with regard to
the composition of the basal medium, selective system and the
introduction of a differential system.
The original basal medium was a commercially available sulphide
mobility-indole medium (SIM BBL) (Blazevic 1968). LAB M have
substituted their raw materials into Blazevic’s formula to create a
richer base for Diassalm. Selectivity is achieved by the use of
malachite green oxalate, magnesium chloride and novobiocin. The
diagnostic properties of Diassalm are based on the use of two
indicator systems; saccharose combined with bromocresol purple;
and ferro-iron in combination with thiosulphate.
The efficiency of Diassalm is due to the ability of salmonellae to
move through the highly selective mobility medium in a petri dish,
whilst the double diagnostic system allows visualisation of motile
and non-motile suspected salmonellae due to blacking zones against
the turquoise background. Diassalm can be seeded after pre-
enrichment or after 8hr enrichment in selective broth (De Smedt and
Bolderdijk 1987).
Formula g/litre
Tryptone 20.0
Meat Peptone 6.1
Ferrous ammonium sulphate 0.2
Sodium thiosulphate 5.0
Sucrose 7.5
Lactose 0.5
Bromocresol purple 0.08
Malachite green oxalate 0.037
Magnesium chloride anhyd. 11.0
Agar No.1 2.8
Preparation
Weigh 53.0 grams of powder, disperse in 1 litre of deionised water.
Mix well, bring quickly to the boil. Allow to cool to 47˚C and add 1
vial of Novobiocin supplement – X150 (10mg/vial). Mix well and
pour plates. Nitrofurantoin may be used instead of Novobiocin to
improve the isolation of S. enteritidis.
Physical properties
Appearance: - Green transparent, soft gel
pH - 5.5 ± 0.2
Shelf life: Ready-to-use medium – 7 days at 2-8˚C.
Inoculation method for samples
3 drops (0.1ml) of 8 to 20hr. incubated pre-enrichment broth are
inoculated in one spot in the centre of one plate of Diassalm.
Incubation method
At 42 ± 0.5 or 37˚C for 18-24 hours. Keep the lid uppermost at all
times.
Interpretation
After incubation the plates are examined for a mobility zone with a
purple/black colour change. When the mobility zone is absent, but the
centre is blackened, non-motile salmonellae may be present.
A loopfull of the motile zone which is the farthest from the sample
inoculum (or the blackened centre if non-motile) is sub-cultured onto
brilliant green agar and XLD agar. Futher biochemical and serological
identification are performed according to recognised procedure.
Direct latex agglutination may also be carried out from the edge of
the mobility zone.
Minimum QC organisms: Salmonella typhimurium
NCIMB 50076
Proteus mirabilis (Inhibition)
References
Blazevics, D.J. 1968 Appl. Microbiol. 16, 688
De Smedt J.M. et al 1987 J. Food Protection 50, 658
Perales, I and Audicana. Evaluation of semi-solid Rappaport medium
for detection of Salmonellae in meat products. J. Food Protection 52,
Van Netten, P., Van de Moosdijk, A., Perales, I. and Mossel, D.A.A.
Letters in Applied Microbiology
Van Netten, P., Van der Zee, H., and Van der Moosdijk, A., 1991. The
use of diagnostic selective semi-solid medium for the isolation of
Salmonella enteritidis from poultry. Proceedings of the 10th
Symposium on the quality of poultry meat, Spelderholt Beckbergen,
pp. 59-67.
Van der Zee, H., and Van Netten, P., 1992. Diagnostic semi-solid
media based on Rappaport-Vassiliadis Broth for the detection of
Salmonella spp. and S. enteritidis in foods. Proceedings of the
International Symposium of Salmonella and Salmonellosis.
Van der Zee, H., 1992. Detection of Salmonella spp. with the use of
a standard method, diagnostic semi-solid agars and immunocapture
kit. Proceedings Third World Congress Foodborne infections and
intoxications, Berlin.
D.N.’ase AGAR
LAB 95
Description
DN’ase agar provides a convenient means of identifying potentially
pathogenic staphylococci, based on the ability of coagulase-positive
species to split DNA. DN’ases produced by the organisms hydrolyse
the DNA molecule to a mixture of smaller mono and poly
nucleotides. DiSalvo observed perfect correlation between coagulase
activity and DN’ase production using S. aureus strains from clinical
specimens. Other publications have also reported a close correlation.
Formula g/litre
Tryptone 20.0
Deoxyribonucleic acid (DNA) 2.0
Sodium chloride 5.0
Agar No. 2 12.0
Method for reconstitution
Weigh 39 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving at 121˚C for 15 minutes. Allow to cool to 47˚C then pour
into petri dishes.
Appearance: Pale cream, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080
S. epidermidis NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days: at 2-8˚C in
the dark. Capped container – up to 1 month at 4˚C in the dark.
Inoculation: Use a heavy inoculum on a small area. Four or more
organisms can be tested on one 90mm petri dish.
Incubation: 37˚C aerobically for 18-24 hours.
Interpretation:
Having obtained good growth flood the plate with 1N hydrochloric
acid. This will precipitate the DNA in the medium. DN’ase producing
organisms will be surrounded by a clear area where the DNA has
been broken down into fractions which are not precipitated by the
Hydrochloric acid. Gram positive, catalase positive cocci that
47
 produce DN’ase can be provisionally classified as S. aureus, and
confirmed by tube coagluase or thermostable DN’ase tests. DN.’ase
is also produced by some Gram negative bacilli such as Serratia
marcescens, Pseudomonas aeruginosa. Some corynebacteria and
streptococci may also produce DN’ase.
References
Baird-Parker, A. C. 1965. The classification of staphylococci and
micrococci from world-wide sources. J. Gen. Microbiol. 38, 363-387.
Black, W. A., Hodgson, R. and McKechnie, A. 1971.
DiSalvo, J. W. 1958 Deoxyribunuclease and coagulase activity of
micrococci. Med. Tech. Bull. U.S. Armed Forces Med. J. 9, 191.
Martin, W. J and Ewing, W. H. 1967. The deoxryibonuclease test as
applied to certain gram-negative bacteria. Can. J. Microbiol. 13, 616-
618.
Messinova, O. V., Yusupova, D. V. and Shamsutdinov, N. S. 1963.
Deoxyribonuclease activity of Corynebacterium and its relation to
virulence. Fed. Proc. 22, T1033.
Streitfeld, M. M., Hoffmann, E. M. and Janklow, H. M. 1962.
Evaluation of extracellular deoxyribonuclease activity in
Pseudomonas. J. Bacteriol. 84, 77. Wannamaker, L. W. 1964.
Streptococcal deoxryribonuclease, pp. 140-165. J. W. Uhr (ed.). The
Streptococcus, Rheumatic Fever, Glomerulophritis. Baltimore:
Williams & Williams.
Weckman, B. G. and Catlin, B. W. 1957 Deoxryribonuclease activity
of micrococci from clinical sources. J. Bacteriol. 73, 747-753.
Zierdt, C. H. and Golde, D. W. 1970. Deoxryribonuclease-positive
Staphylococcus epidermidis strains. Appl. Microbiol. 20(1), 54-57.
Easter and Gibson
Pre-enrichment Broth
LAB 136
Description
A pre-enrichment broth for use in detection procedures utilising
conductance/impedance techniques. This broth is developed from
LAB 46 Buffered Peptone Water and will enhance the recovery of
sub-lethally damaged bacteria. Salmonella reduce T.M.A.O. to
produce a significant change in the medium that can be detected by
the conductance/impedance equipment.
Best results will be obtained by those customers using Pre-
Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)
bought from LAB M. These media are both manufactured with the
same peptones, so that organisms “switched on” to utilising the
substrates in LAB 136 can carry on metabolising the same substrates
in LAB 137, significantly shortening the lag time.
Formula g/litre
Tryptone 7.5
Meat Peptone 2.5
Sodium chloride 5.0
Disodium hydrogen phosphate 3.56
Potassium di-hydrogen phosphate 1.5
Mannitol 5.0
Method for reconstitution
Weigh 25 grams of powder, disperse in 1 litre of deionised water.
Heat gently to dissolve supplement, dispense into appropriate
containers, then sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Very pale yellow clear solution.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Capped container – up to 1 month at
15-20˚C in dark.
Inoculation: Homogenised food samples.
48
Incubation: 37˚C for 18 hours aerobically.
Sub-culture: Into LAB 137 Easter and Gibson Salmonella Medium.
References
Easter M. C. Gibson D. M. 1985. Rapid and automated detection of
Salmonella by electrical measurements. J. Hyg. 94, 245-262.
Gibson D. M. 1987. Some modifications to the media for rapid,
automated detection of salmonellas by conductance. J. Appl.
Bacteriol. 63, 299-304.
Ogden I. D., Cann P. C. 1987. A modified conductance medium for
the detection of Salmonella spp. J. Appl. Bacteriol. 63, 459-464.
Easter and Gibson Salmonella
Medium
LAB 137
Description
A medium for the rapid detection of Salmonella spp by
conductance/impedance techniques. This medium is based on LAB
55 Selenite Cystine Broth and is supplemented with dulcitol and
T.M.A.O. (trimethylamine-N-oxide). Salmonella reduce T.M.A.O. to
T.M.A. (trimethylamine) and, in so doing, increase the conductivity
of the medium which can be detected and measured by monitoring
equipment such as Malthus.
Best results will be obtained by those customers using Pre-
Enrichment Broth (LAB 136) and Salmonella Medium (LAB 137)
bought from LAB M. These media are both manufactured with the
same peptones, so that organisms ‘switched on’ to utilising the
substrates in LAB 136 can carry on metabolising the same substrates
in LAB 137 significantly shortening the lag time.
Formula g/litre
Meat Peptone 2.5
Tryptone 2.5
Sodium dihydrogen phosphate 10.0
Dulcitol 5.0
Sodium carbonate 5.0
Method for reconstitution
Weigh 2.5 grams of powder, disperse in 100mls of deionised water.
Add 1 vial of X137 T.M.A.O./Selenite supplement. Swirl to mix, heat
to boiling to effect sterilisation. When cool add 1ml of stock solution
of L-cystine. Distribute into sterile conductance tubes or bottles.
DO NOT AUTOCLAVE.
Stock solution of L-cystine
0.1gm L-cystine dissolved in 15mls of normal NaOH – add to 100mls
sterile distilled water. Keep refrigerated. Discard after 1 month.
Appearance: Clear reddish/orange solution. A slight precipitate
may form.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: capped container – up to 1 month at
15-20˚C in dark.
Inoculation: From LAB 136 Easter and Gibson Pre-Enrichment
Broth.
Incubation: 37˚C aerobically connected to monitoring equipment.
References
Easter M. C. Gibson D. M. 1985. Rapid and automated detection of
Salmonella by electrical measurements. J. Hyg. 94, 245-262.
Gibson D.M. 1987. Some modifications to the media for rapid
automated detection of salmonellas by conductance. J.Appl. Endo Agar
Bacteriol. 63, 299-304.
LAB 60
Ogden I.D., Cann P.C. 1987. A modified conductance medium for the
detection of Salmonella spp. J. Appl. Bacteriol. 63, 359-464. Description
This medium was developed in 1914 for the isolation of Salmonella
typhi; other media have since proved superior for this purpose, but
Endo agar has a role as a coliform medium. It is recommended by the
American Public Health Association as a standard medium for the
E.E. Broth enumeration of coliforms in water and dairy products. In this medium
acetaldehyde is produced by coliforms and then fixed by the sulphite
(Enterobacteriaceae Enrichment Broth) to produce a metallic sheen with the basic fuchsin dye. Most enteric
Gram negative organisms will grow well, whilst Gram positive
LAB 91 organisms are mostly inhibited.
Description
Formula g/litre
E.E. Broth is recommended as an enrichment medium when
examining food and feedstuffs for Enterobacteriaceae. It is a Balanced Peptone No. 1 10.0
modification of LAB 51 Brilliant Green Bile Broth, with an improved
buffering capacity to encourage early growth and prevent Lactose 10.0
autosterilization. E.E. Broth uses glucose instead of lactose to make Dipotassium phosphate 3.5
the medium a test for all enterobacteria including non lactose
fermenting organisms. Sodium sulphite 2.5
Agar No. 1 15.0
Formula g/litre
Balanced Peptone No. 1 10.0 Method for reconstitution
Dextrose 5.0 Weigh 41 grams of powder, disperse in 1 litre of deionised water. Add
4ml of a 10% w/v alcoholic solution of basic fuchsins (95% ethyl
Disodium hydrogen phosphate 6.45 alcohol). Bring to the boil with frequent swirling to dissolve the
solids. Sterilise by autoclaving at 121˚C for 15 minutes. Cool to 47˚C
Potassium dihydrogen phosphate 2.0 in a water bath before pouring. The precipitate typically associated
Bile Salts 20.0 with this medium should be dispersed by gentle swirling prior to
pouring the plates.
Brilliant green 0.0135 This medium is light sensitive and should therefore be stored in the
dark, preferably under refrigeration. The medium will become dark
Method for reconstitution red in colour if exposed to light.
Weigh 43.5 grams of powder and add to 1 litre of deionised water. Basic Fuchsin is a potential Carcinogen and care should be taken
Swirl to dissolve, warm gently if necessary, then distribute into when handling it to avoid inhalation of the powdered dye and
bottles or tubes and heat at 100˚C for 30 minutes only. Cool rapidly. contamination of the skin.
OVERHEATING THIS MEDIUM WILL ADVERSELY AFFECT
ITS PERFORMANCE. Appearance: Pale pink/orange
Appearance: Green, clear. pH: 7.5 ± 0.2
pH: 7.2 ± 0.2 Minimum Q.C. organisms: E. coli NCIMB 50034
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
B. subtilis (inhibition)
the dark.
Storage of Prepared Medium: capped containers – up to 3 months Inoculation: Surface, streaking out for single colonies.
at 15-20˚C in the dark. Incubation: 37˚C for 18-48 hours aerobically.
Inoculation: Add 1 part of sample suspension or dilution to 10 parts
of medium. Growth Characteristics
Incubation: 44˚C for 18 hours for thermotrophs. 32˚C for 24-48 colony size shape &
hours for mesotrophs. 4˚C for 10 days for psychrotrophs. organism (mm) surface colour other
Interpretation: Turbidity and a colour change to yellow-green is E. coli 1.0-2.0 CV.E.G. Deep Red (Metallic sheen)
presumptive evidence of Enterobacteriaceae. Subculture onto
confirmatory media e.g. LAB 88 V.R.B.G.A. must be carried out. K. aerogenes 1.0-2.5 CV.E.G. Red (mucoid)
Proteus spp 2.0-3.0 CV.E.G. Pale Pink
References colourless
Mossel, D. A. A., Visser, M. and Cornelissen, A. M. R. 1963. The Ps. aeruginosa 0.5-1.0 F.CR.D. Pale Pink
examination of foods for Enterobacteriaceae using a test of the type
generally adopted for the detection of salmonellae. J.Appl. Bacteriol. Shigella spp 0.5-1.0 CV.E.G. Pale Pink
26, 444-452. Salmonella spp 1.0-1.5
Mossel, D. A. E., Harrewijn, G. A. and Nesselrooy-van Zadelhoff, C. Gram positive no growth.
F. M. 1974. Standardisation of the selective inhibitory effect of organisms
surface active compounds used in media for the detection of
Enterobacteriaceae in food and water. Health Lab. Sci. 11, 260-267.
Richard, N. 1982. Monitoring the quality of selective liquid media by References
the official French dilution technique used for the bacteriological Endo, 1914, Centr. Bakt., Abt 1, Orig., 35: 109.
examination of foods. In: Quality assurance and quality control of
microbiological culture media, edited by J. E. L. Corry, G.I.T.-Verlag American Public Health Association, 1975. Standard Methods for the
Darmstadt, pp. 51-57. Examination of Water and Wastewater, 14th Edn. American Public
Health Association, Inc. Washington D.C.
American Public Health Association, 1972. Standard Methods for the
Examination of Dairy Products, 13th End., American Public Health
Association, Inc., Washington, D.C.

49
 Eosin Methylene Blue Agar (Levine) Fastidious Anaerobe Agar (F.A.A.)
LAB 61 LAB 90
Description Description
This medium was introduced in 1916 by Holt-Harris and Teague to A primary isolation medium capable of growing most clinically
differentiate Escherichia spp. and Aerobacter spp. It was modified by significant anaerobes. Developed by LAB M, comparisons have
Levine in 1918 who removed sucrose from the formula and increased shown this medium to be superior to other formulations as a primary
the lactose content. The distinctive metallic sheen produced by E. coli isolation medium for fastidious organisms. The peptones included
on this medium is due to acid production resulting in an amide have been chosen for maximum growth stimulation. Starch and
bonding between the eosin and methylene blue, other coliforms do sodium bicarbonate act as de-toxification agents whilst haemin
not produce enough acid to cause this reaction. Eosin inhibits most encourages pigment production in Porphyromonas melaninogenicus.
Gram positive organisms. The prepared medium is sensitive to light. Specific growth promoting agents are Cysteine for Fusobacterium
necrophorum, Propionibacterium acne and Bacteriodes fragilis,
Formula g/litre arginine for Eubacterium spp. soluble pyrophosphate for Porph.
gingivalis and Porph. assacchrolyticus. Pyruvate helps neutralise
Balanced Peptone No. 1 10.0 hydrogen peroxide and is also utilised by Veillionella spp. as an
energy source. Vitamin K and sodium succinate provide essential
Lactose 10.0 growth factors for some anaerobes as does the 0.1% glucose. The low
Dipotassium phosphate 0.7 level of glucose prevents the production of high levels of acids and
alcohols which would inhibit colonial development.
Monopotassium phosphate 1.3
Eosin Y 0.4 Formula g/litre

Methylene Blue 0.065 Peptone mix 23.0

Agar No. 2 15.0 Sodium chloride 5.0

Soluble starch 1.0
Method for reconstitution
Agar No. 2 12.0
Weigh 37.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by Sodium bicarbonate 0.4
autoclaving at 121˚C for 15 minutes. Cool to 50˚C and agitate gently
to ensure uniform distribution of the flocculant precipitate which is a Glucose 1.0
feature of this medium before pouring into petri dishes.
Sodium pyruvate 1.0
STORE IN THE DARK.
Appearance: Blue/purple with a light precipitate. Cysteine HCl monohydrate 0.5

pH: 6.8 ± 0.2 Haemin 0.01

Vitamin K 0.001
Minimum Q.C. organisms: E. coli NCIMB 50034
L-Arginine 1.0
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Soluble pyrophosphate 0.25

Inoculation: Surface, streaking for single colonies. Sodium succinate 0.5

Incubation: 37˚C aerobically for 24 hours.
Method for reconstitution
Weigh 46 grams of powder and add to 1 litre of deionised water.
Growth Characteristics Allow to soak for 10 minutes, swirl to mix then sterilise by
colony shape & autoclaving at 121˚C for 15 minutes. Cool to 47˚C then aseptically
organism size (mm) surface colour other add 5-10% of sterile defibrinated horse blood, mix well and pour into
petri dishes. This medium can be made selective for various species
E. coli 2.0-3.0 CV.E.G. Blue Black (Metallic of anaerobes by the addition of appropriate selective cocktails e.g.
sheen)
Gram negative anaerobes X090
Klebsiella spp. 3.0-4.0 CV.E.G. Brown
Blue (mucoid) Non-sporing anaerobes X091
Salmonella spp. 2.0-3.0 CV.E.G. Colourless Actinomyces spp. X092
Shigella spp. 1.0-2.0 CV.E.G. Colourless Clostridium difficile X093
Candida spp. 0.5-1.5 CV.Rz.G. Appearance: Red due to addition of blood. The blood will darken
(D) White (reduce) because of the presence of reducing agents.
S. aureus P.P. CV.E.G. Colourless pH: 7.2 ± 0.2
E. faecalis P.P. CV.E.G. Colourless Minimum Q.C. organisms: B. fragilis
P. anaerobious
References
American Public Health Association, American Water Works Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Association and Water Pollution Control Federation, 1975. Standard the dark.
Methods for the Examination of Water and Wastewater, 14th Edn., Inoculation: Surface plating, streaking out to single colonies.
Washington, D.C. American Public Health Association.
Incubation: 37˚C anaerobically with 10% CO2 for 48 hours to
Girolami, R. L. and Stamm, J. M. 1976. Inhibitory effect of light on 5 days.
growth supporting properties of Eosin Methylene Blue Agar. Appl.
Environ. Microbiol., 31:1 141-142.
Haesler, W. J. (ed) 1972. Standard Methods for the Examination of
Dairy Products, 13th edn., Washington, D.C., American Public
Health Association.
Levine, M. 1918. Differentiation of E. coli and B. aerogenes on a
simplified Eosin-Methylene Blue agar. J. Infect. Dis., 23: 43-47.

50
 Growth Characteristics (48 hours) Method for reconstitution
Weigh 29.7 grams of powder, disperse in 1 litre of deionised water.
colony shape & Allow to soak for 10 minutes, swirl to mix. Boil to dissolve the agar
organism size (mm) surface colour other then dispense into screw cap containers. Sterilise by autoclaving at
B. fragilis 1.0-2.0 CV.E.G. Grey 121˚C for 15 minutes. Tighten the caps as soon as possible after
autoclaving.
C. perfringens 1.0-2.0 CV.E.G. Grey ‘Target’
haemolysis Appearance: Pale straw, clear, viscous. May have a narrow band of
(non haemolytic) red/purple at the surface due to action of oxygen on the resazurin. If
the medium is reddish this indicates too much oxygen has been
F. necrophorum 1.0-2.0 CV.E.G.(D) trans- (grey) absorbed, the medium should be reheated to deoxygenate. Do not
parent (haemolytic) reheat more than once.
Porphyromonas assachrolyticus pH: 7.2 ± 0.2
1.0-2.0 CV.E.G. Grey/
Brown (clearing) Minimum Q.C. organisms: B. fragilis
B. ureolyticus 0.5 F.E.D. translucent pitting
Storage of Prepared Medium: Capped containers – up to 3 months
Prop acne 0.5 CV.E.G. White
at 15-20˚C in the dark.
Pept. anaerobius 0.5-2.0 CV.E.G. White/
Inoculation: If used as a blood culture medium a minimum dilution
Grey
of 1:10 should be used.
A. israeli 0.5-1.0 CV.E.G. White (‘molar tooth’)
(smooth) Incubation: 37˚C for 24-72 hours. Keep the container airtight.
Growth indicators: The broth may become turbid or individual
colonies may form suspended in the medium.
References
Brazier, J. S. (1986). Yellow fluorescence of Fusobacteria Letters in
Applied Microbiol. 2: 124-126. References
Gould, J. H., Duerden, B. I. 1983. Blood culture – current state and
Brazier, J. S. 1986. A note on ultra violet red fluorescence of future prospects. J. Clin. Pathol. 36: 963-977.
anaerobic bacteria in vitro. J. Appl. Bact. 60: 121-126.
Ganguli, G. A., O’Hare, W., Hyde, W. A. 1984. Rapid Detection of
Eley, A., Clarry, T., Bennett, K. W. 1989. Selective and differential Bacteraemia by early subculture. J. Med. Microbiol. 17: 311-315.
medium for isolation of Bacteriodes ureolyticus from clinical
specimens. European Journal of Clinical Microbiology, Infectious Ganguli, L. A., Keaney, M. G. L., Hyde, W. A., Fraser, B. J. 1985.
Diseases. 8: 83-85. More Rapid identification of bacteraemia by manual rather than
radiometric methods. J. Clin. Pathol. 38: 1146-1149.
Wade W. Griffiths, M. 1987. Comparison of Media for cultivation of
subgingival bacteria. J. Dent. Res. 66: no. 4 abstract 334. Junt, G. H., Price, E. H. 1982. Comparison of a home made blood
culture broth containing a papain digest of liver, with four
Heginbotham M., Fitzgerald T. C., and Wade W. G. (1990). commercially available media, for the isolation of anaerobes from
Comparison of solid media for the culture of anaerobes. J. Clin. Path. simulated paediatoic blood cultures. J. Clin. Pathol. 35: 1142-1149.
43: 253-256.
Ganguli, L. A., Turton, L. J., Tillotson, G. S. 1982. Evaluation of
Fastidious Anaerobe Broth as a blood culture medium. J. Clin. Pathol.
35: 458-461.
Tillotson, G. S. 1981. Evaluation of ten commercial blood culture
Fastidious Anaerobe Broth (F.A.B.) systems to isolate a pryridoxal dependent streptococcus. J. Clin.
Pathol. 34: 930-934.
LAB 71
Description
F.A.B. was developed by LAB M working in conjunction with the
microbiology department of a University of Manchester teaching Fluid Thioglycollate Medium (U.S.P.)
hospital. The medium was designed to give optimum growth of
fastidious anaerobes and has found applications as a blood culture LAB 25
medium and an enrichment broth for the isolation of anaerobes. The
medium is very rich in nutrients from the specially selected peptone Description
mixture. Vitamin K. haemin and L-cysteine are all growth factors A medium for sterility tests, prepared according to the specification
required by some anaerobes. L-cysteine together with sodium of the United States Pharmacopeia. Aerobic and anaerobic organisms
thioglycollate reduce the Eh of the medium and the agar content grow well in this medium even from small inocula. In appropriate
inhibits absorption of oxygen and convection currents. Resazurin is a tubes or bottles the thioglycollate ensures adequate anaerobic
redox indicator. Several published evaluations show F.A.B. to be the conditions. The low level of agar reduces oxygen diffusion into the
liquid medium of choice for fastidious anaerobes. medium. The thioglycollate will also serve to inactivate any
mercurial compounds used as preservatives.
Formula g/litre
Peptone mixture 15.0 Formula g/litre

Yeast Extract 10.0 Tryptone 15.0

Sodium thioglycollate 0.5 L-Cystine 0.5

Sodium chloride 2.5 Glucose 5.5

Agar No. 1 0.75 Yeast Extract 5.0

L-Cysteine HCl 0.5 Sodium chloride 2.5

Resazurin 0.001 Sodium thioglycollate 0.5

Sodium bicarbonate 0.4 Resazurin 0.001

Haemin 0.005 Agar 0.75

Vitamin K 0.0005

51
 Method for reconstitution
Weigh 29.75 grams, disperse in 1 litre of deionised water. Soak for 10
minutes, swirl to mix, then bring to the boil to dissolve and dispense
15ml into 6mm x 150mm tubes. Sterilise by autoclaving for 15
minutes at 121˚C. Store at ambient temperature in the dark, but not in
the refrigerator. If more than 30% of the medium turns pink
(oxidised) the Eh may be restored (once only) by heating in a boiling
water bath or by free-steaming.
Appearance: Pale straw colour, clear. Surface may be pink/blue due
to oxidation of Resazurin.
pH: 7.1 ± 0.2
Minimum Q.C. organisms: C. sporogenes
S. aureus NCIMB 50080
Storage of Prepared Medium: Capped container – up to 3 months
at 15-20˚C in the dark.
Incubation: 30-35˚C aerobically for 14 days.
Growth indicators: Turbidity, colonies in medium.
References
The Pharmacopeia of the United States of America. 21st End. 1985.
References
King, E. O., Ward, M. K. and Raney, D. E. 1954. 2 simple media for
demonstration of pyocyanin and fluorescein, J. Lab. Clin Med., 44:
301-307.
Brown, V. I. and Lowbury, E. J. L. 1965. Use of an improved
cetrimide agar medium and other culture methods for Pseudomonas
aeruginosa. J. Clin. Path. 18: 752-756.
Fraser Broth
LAB 164
Description
Developed as a modification of UVM II medium, Fraser broth is a
secondary enrichment broth for the isolation of Listeria spp., and is
similar to Palcam broth in that it contains aesculin to indicate the
presence of a potential Listeria isolate. It also contains lithium
chloride in an attempt to suppress the growth of enterococci in the
medium (as does Palcam). Fraser broth may also be used as a primary
enrichment medium by incorporating 1/2 strength supplement into
the broth base (X164 or X564).

Formula g/litre
Peptone mixture 15.0
Fluorescence Agar
Yeast extract 5.0
LAB 16 Aesculin 1.0
Description Disodium hydrogen phosphate 9.6
This medium is a modification of King, Ward and Raney’s medium
formulated for the demonstration of the fluorescein pigment Potassium dihydrogen phosphate 1.35
produced by many strains of Pseudomonas. The pyocyanin pigment Sodium chloride 20.0
produced by most strains of Pseudomonas aeruginosa is also
produced. Can be made selective by the addition of selective agents Lithium chloride 3.0
such as X108 Cetrimide Fucidin Cephaloridine.
Method for reconstitution
Formula g/litre Weigh 55 grams of power and add to 1 litre of deionised water (add
Balanced Peptone No. 1 20.0 to 900ml if preparing 1/2 Fraser). Allow to soak for 10 minutes, swirl
to mix and sterilise at 121˚C for 15 minutes. Cool to 47˚C and add 2
Dipotassium phosphate 1.5 vials of Fraser supplement X165 (or 2 vials of 1/2 Fraser supplement
X164), mix well and aseptically dispense into sterile tubes or bottles.
Magnesium sulphate 1.5
Appearance: Straw opalescent broth with precipitate (clears on
Agar No. 2 12.0 storage)
pH 7.2 + 0.2
Method for reconstitution
Weigh 35 grams of powder, disperse in 1 litre of deionised water Minimum Q.C. organisms: Listeria sp NCIMB 50007
containing 10ml Glycerin B.P. Allow to soak for 10 minutes, swirl to E.coli (inhibition) NCIMB 50034
mix then sterilise for 15 minutes at 121˚C. Mix well before pouring.
Slant over a generous butt if required. Storage of Prepared Medium: Bottles – up to 14 days at 2-8˚C.
Appearance: Straw coloured, clear gel. Inoculation: 1/2 Fraser – Add 25g sample to 225ml of 1/2 Fraser
pH: 7.2 ± 0.2 broth and homogenise Fraser – Subculture 0.1ml of primary
enrichment broth (UVM I or 1/2 Fraser) into 10ml of Fraser broth.
Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067 Incubation: 1/2 Fraser – 30˚C aerobically for 24hrs
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in Fraser – 35˚C aerobically for 24hrs and 48hrs. Subculture onto
the dark. selective agars at 24 and 48hrs.
Inoculation: Surface spreading. Interpretation
Incubation: 30-37˚C for 24 and 48 hours aerobically. Blackening of the broth indicates the presence of a potential Listeria
and should be subcultured onto Listeria isolation medium (Oxford)
Growth Characteristics LAB122 or Palcam agar LAB148. All broths should be subcultured
before discarding, irrespective of colour change.
colony size shape &
organism (mm) surface colour other References
Ps. aeruginosa 0.5-2.5 F.CR.D. Grey (colony size varies Fraser J.A., and Sperber W.H., (1988) Rapid detection of Listeria spp
Green- with strain) in food and environmental samples by esculin hydrolysis. J.Food
fluorescent-(non pigmented) Protection 51 (10) 762-765
pigment (mucoid) McClain D., and Lee W.H. (1989) FSIS method for isolation of
Ps. fluorescens 1.0-2.5 F.CR.D. Grey L.monocytogenes from processed meat and poultry products.
fluorescent Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,
pigment Microbiol. Div.

52
G.C. Agar Base Hektoen Enteric Agar
LAB 67 LAB 110
Description Description
A nutritious agar base described by Thayer and Martin for the A medium developed at the Hektoen Institute in Chicago for the
isolation of Neisseria gonorrhoeae. The right peptone mixture is enhanced recovery of shigellae from clinical specimens. This
enhanced by the use of corn starch to absorb toxic metabolites and a medium has high levels of peptones and sugar which counteract some
buffering system is used to maintain neutral pH. The medium is made of the toxic effects of bile salts used to make the medium selective.
selective by the use of various antibiotic cocktails. Thayer and Martin This allows the shigellae to grow as well as the salmonellae. Salicin
originally recommended the use of vancomycin, colistin and nystatin is fermented by many coliforms including those that do not ferment
(X067 V.C.N.) but the addition of trimethoprim (X068 V.C.N.T.) is lactose and sucrose. The medium employs a double indicator system
useful in preventing the swarming of proteus. More recently the similar to that used in LAB 6 C.L.E.D., (Bevis) and an H2S indicator
emergence of vancomycin sensitive gonococci has made the New system similar to that used in LAB 32 XLD. Although intended
York City selective agents (lincomycin, colistin, amphoteracin, primarily for clinical use this medium is quoted in B.S. 4285 as
trimethoprim X070 LCAT) the combination of choice. Enrichment of suitable for the examination of dairy products for salmonellae.
the base is usually by the addition of lysed blood. Alternatively
chocolated blood or haemoglobin powder and Thayer and Martin’s Formula g/litre
mixture of vitamins, amino acids and coenzymes can be used.
Meat Peptone 12.0
Formula g/litre Yeast Extract 3.0
Special Peptone 15.0 Lactose 12.0
Corn Starch 1.0 Sucrose 12.0
Sodium chloride 5.0 Salicin 2.0
Dipotassium hydrogen phosphate 4.0 Bile Salts No. 3 7.0
Potassium dihydrogen phosphate 1.0 Sodium desoxycholate 2.4
Agar No. 2 10.0 Sodium chloride 5.0

Method for reconstitution Sodium thiosulphate 5.0

Weigh 36 grams of powder, disperse in 1 litre of deionised water. Ammonium ferric citrate 1.5
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 48˚C and add 50-70ml Acid fuchsin 0.1
of lysed blood and 2 vials of X070 selective agent. Mix well and pour Bromothymol blue 0.065
into petri dishes.
Agar No. 1 14.0
Appearance: Dependent on blood supplement used.
pH: 7.2 ± 0.2 Method for reconstitution
Minimum Q.C. organisms: N. gonorrhoeae ATCC CDC98 Weigh 76 grams of powder, disperse in 1 litre of deionised water.
E. coli (inhibition) NCIMB 50034 Allow to soak for 10 minutes, swirl to mix then heat gently and bring
to the boil. Cool to 47˚C and pour plates. DO NOT AUTOCLAVE
OR OVERHEAT THIS MEDIUM.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Appearance: Green, clear.
Inoculation: Surface, streaking out for single colonies. pH: 7.5 ± 0.2
Incubation: 37˚C microaerobically for 24-48 hours. Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
Shigella sp.
Growth Characteristics E. coli (some inhibition)
NCIMB 50034
colony size shape &
organism (mm) surface colour other
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
N. gonorrhoeae 1.0-2.0 CV.E.G. Transp variations in the dark.
arent colony size
Inoculation: Surface plating, streak out to single colonies.
N. lactamica 1.0-2.0 CV.E.G. Grey
Incubation: 37˚C aerobically for 18-24 hours.
N. meningitidis 2.0-3.0 CV.E.G. Grey
B. catarrhalis 2.0-3.0 CV.E.G. Cream
Other organisms should not grow with the exception of antibiotic
resistant variants.

References
Young, H. 1978. Cultural diagnoses of gonorrhoea with modified
New York City (MNYC) medium. Brit. Journ. Ven. Dis. 54: 36-40:
Thayer, J. D. and Martin, J. E. 1966. Improved medium selective for
the cultivation of N. gonorrhoeae and N. Meningitidis: Public Health
rep. 81: 559-562.

53
 Growth Characteristics Method for reconstitution
Weigh 45.0 grams of powder and disperse in 1 litre of deionised
colony size shape & water. Soak for 10 minutes, swirl to mix and sterilise at 121˚C for 15
organism (mm) surface colour other minutes. Cool to 47˚C and add 100ml of Horse Serum BP 50, and 2
H2S +ve 2-3 CV.E.G. Green+ vials of VCA supplement X040. Mix well and pour, continuing to
Salmonella Black mix whilst pouring to keep the charcoal in suspension.
H2S -ve 2-3 CV.E.G. Green Appearance: Black Agar
Salmonella pH: 7.4 ± 0.2
S. sonnei 2-2.5 CV.E.G. Green (Rough)
Minimum Q.C. organisms: Helicobacter Pylori
S. flexneri 1.0-2.5 CV.E.G. Green S.aureus (inhibition) NCIMB 50080
S. dysenteriae 1-2 CV.E.G. Green
Storage of prepared medium: Plates – up to 7 days at 2-8˚C
E. coli 0.5-2 CV.E.G. Salmon ppt. (Rough)
the dark.
around (No growth)
colonies Inoculation: Surface streaking for single colonies.
Citrobacter spp. 1.0-2.0 CV.E.G. Salmon (Rough) Incubation: 37 ˚C for 72 hours.
Klebsiella spp. 0.5-2.0 CV.E.G. Salmon (Mucoid)
Proteus spp. 1.0-2.0 CV.E.G. Green/ (No growth
Growth Characteristics
Black brownish centre) Organisms Colony Size Shape and Colour
centre (mm) Surface
Pseudomonas H. Pylori 1.0-1.5 CVEG Grey
spp. 0.5-1.5 F.Rz.D. Green (No growth)
B. Catarrhalis 1.0-2.0 CVEG White/Cream
References
King, S. and Metzger, W. I. 1967. A new medium for the isolation of
References
Salmonella and Shigella species. Bact. Proc. Am. Soc. Microbiol. 77. Bolton et al. Public Health Laboratory, Preston. Personal
King, S. and Metzger, W. I. 1968. A new plating medium for the communication.
isolation of enteric pathogens. Hektoen Enteric Agar, Appl.
Microbiol., 16(4), 577.
King, S. and Metzger, W. I. 1968. A new plating medium for the
isolation of enteric pathogens. II. Comparison of Hektoen Agar with
SS and EMB agar. Appl. Microbiol., 16(4), 579.
Hoyle’s Medium
Speck, M. L. (ed.). 1976. Compendium of Methods for the LAB027
Microbiological Examination of Food. Washington, D.C.: American
Public Health Association. Description
A highly selective culture medium for the isolation and
differentiation of Corynebacterium diphtheriae types gravis, mitis
and intermedius. Hoyle’s medium gives rapid growth of all types of
C. diphtheriae, which results in most specimens giving adequate
Helicobacter Pylori Medium growth with overnight incubation.

LAB 140 Formula g/litre

Description Beef Extract 10.0

A selective medium for the isolation of Helicobacter Pylori, the Peptone 10.0
causative agent of chronic gastritis.
Sodium chloride 5.0
This campylobacter-like organism was described in 1983 colonising
the gastric mucosa, a site previously thought to be sterile due to the Agar 12.0
low PH. The high level of Urease production by this organism
appears to be the major pathogenicity factor enabling it to withstand Method of Reconstitution
the strongly acidic environment. Weigh 37 grams of powder and disperse in 1 litre of deionised water.
Helicobacter Pylori medium is a modification of CCDA Medium for Allow to soak for 10 minutes, and sterilise by autoclaving at 121˚C
the isolation of Campylobacter spp. Formulated by Bolton et al at for 15 minutes. Cool to 47˚C, add 50ml of lysed horse or sheep blood
Preston Public Health Laboratory, it incorporates a rich agar base and 10ml of X027 potassium tellurite solution. Mix well before
supplemented with horse serum to promote optimum growth, and pouring.
Vancomycin, Cefsulodin, and Amphoteracin as selective agents.
Appearance: Dark Red, clear gel.
Formula g/litre
pH: 7.8 ± 0.2
Beef Extract 10.0
Meat Peptone 5.0 Inoculation
Spread the entire surface with the swab or sample under
Tryptone 5.0 investigation. Hoyle’s medium is very selective and spreading for
single colonies using a wire loop is not necessary. Use of a non-
Sodium chloride 5.0 selective blood agar alongside Hoyle’s is recommended.
Charcoal 4.0 Incubation: 37˚C for 18-48 hrs, aerobically
Acid Hydrolysed Casein 3.0
Ferrous sulphate 0.25
Sodium pyruvate 0.25
Sodium carbonate 0.4
Agar no.2 12.0

54
 Interpretation Minimum Q.C. organisms: E. faecalis NCIMB 50030
colony size shape & E. coli (inhibition) NCIMB 50034
organism (mm) surface colour other
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
C.diphtheriae 1.5-2.5 CV.CR.D Grey Colonies difficult the dark.
var gravis (daisy head) to emulsify
Inoculation: Surface, spread 0.1ml to 0.5ml over entire surface of
C.diphtheriae 0.5-2.0 CV.E.G. Grey Easily plate.
var mitis (dark centre) emulsified
Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
C.diphtheriae 0.5-1.0 CV.E.G. Grey
var intermedius (dark centre) Interpretation: Count all white/grey colonies, approx 2mm
diameter, surrounded by a black halo to give presumptive
C. Ulcerans 1.0-1.5 CV.E.G. Grey Streptococcal enterococcus/faecal streptococcus count.
(dark centre) appearance
in Gram stain
References
C.hoffmanii 0.5-1.0 CV.E.G. Black White/grey
Mossel, D. A. A., P. H. G. Bijken, I. Eelderink, and K. A. van
(confluent growth)colonies
Spreekens, 1978. Streptococci, edited by F. A. Skinner and L. B.
C.xerosis 0.5-2.5 CV.E.G. Black Quesnel, SAB Symposium Series No. 7 Academic Press, London.
Streptococcus pp - 1.5 CV.E.G. Black Enterococci may
spp. be larger
H.influenzae pp - 1.5 CV.E.G. Grey/black Some strains
no growth Kanamycin Aesculin Azide Broth
Storage: Plates – up to 7 days at 2-8˚C (K.A.A. Broth)
Minimum Q.C. Organisms C.diphtheriae var mitis LAB 107
(non-toxigenic)
E. coli NCTC 10418 (inhibition) Description
An enrichment and isolation medium for enterococci. The medium
can be used with the M.P.N. technique to enumerate enterococci in
Reference: food. This broth is identical to LAB 106 K.A.A. agar with the
Hoyle L. (1941) A Tellurite Blood Agar Medium for the Rapid omission of the agar.
Diagnosis of Diphtheria. Lancet 1 175-
176. Elek S.D. (1948) The Recognition of Toxigenic Bacterial Strains Formula g/litre
in vitro. Brit. Med. J. 1 493-496. Tryptone 20.0
Yeast Extract 5.0
Sodium chloride 5.0
Kanamycin Aesculin Azide Agar Sodium citrate 1.0
(K.A.A. Agar) Aesculin 1.0

LAB 106 Ferric ammonium citrate 0.5

Sodium azide 0.15
Description
A selective isolation and enumeration medium for enterococci Kanamycin sulphate 0.02
(Lancefield group D streptococci) in food. Sodium azide and
kanamycin provide the selective inhibition required whilst aesculin Method for reconstitution
and iron salts form an indicator system for the presumptive Weigh 33 grams of powder, disperse in 1 litre of deionised water.
identification of enterococci. Incubation at 42˚C will increase the Allow to soak for 10 minutes, warm gently to dissolve completely
medium’s selectivity. then disperse into tubes or bottles. Sterilise by autoclaving at 121˚C
for 15 minutes.
Formula g/litre
Appearance: Light straw, clear.
Tryptone 20.0 pH: 7.0 ± 0.2
Yeast Extract 5.0
Minimum Q.C. organisms: E. faecalis NCIMB 50030
Sodium chloride 5.0 E. coli (inhibition) NCIMB 50034
Sodium citrate 1.0
Storage of Prepared Medium: Capped containers – up to 3 months
Aesculin 1.0 at 15-20˚C in the dark.
Ferric ammonium citrate 0.5 Inoculation: Inoculate tubes with decimal dilutions of food
suspension.
Sodium azide 0.15
Incubation: 37˚C or 42˚C aerobically for 18-24 hours.
Kanamycin sulphate 0.02
Interpretation: Blackening of the medium suggests the presence of
Agar No. 1 10.0 enterococci/faecal streptococci.

Method for reconstitution References

Weigh 43 grams of powder, disperse in 1 litre of deionised water. Mossel, D. A. A., P. H. G. Bijken, I. Eelderink, and K. A. van
Allow to soak for 10 minutes, swirl to mix then sterilise by Spreekens. 1978. Streptococci, edited by F. A. Skinner and L. B.
autoclaving at 121˚C for 15 minutes. Cool to 47˚C, then dispense into Quesnel, SAB Symposium Series No. 7 Academic Press, London.
petri dishes.
Appearance: Pale straw, clear.
pH: 7.0 ± 0.2

55
 Kirchner’s T.B. Medium Incubation: 37˚C aerobically for up to six weeks.

LAB 123 Growth Characteristics

organism
Description
The cultivation of non-sputum specimens for mycobacteria requires M. tuberculosis white floccular ‘snowflake’ colonies
special attention. Specimens such as C.S.F., tissue or body fluids M. scrofulaceum- ‘ropey’ deposit
obtained by surgical procedures are not easily repeated and may
contain small numbers of mycobacteria. It is important, therefore, M. kansasii- fine granular deposit.
that as large a portion of specimen as possible be inoculated into
suitable growth media. References
Historically, such specimens were inoculated into guinea pigs; Kirchner, O., 1932. Erfahrungen bei diagnosticher Verwendung der
however improvements in culture methods have led to a situation Tiefenkulter. Sbl fur Bact. Abt 1. Originale 124, 409-412.
where comparable results, can be obtained from the use of liquid
culture media. Marks, J. 1972, Ending the routine guinea pig test. Tubercle 1972. 53:
31-34.
Selective Kirchner’s Medium is a liquid culture media which is made
selective by the addition of an antibiotic cocktail, and has been shown Mitchison, D. A. Allen, B. W., Manickavasagar, D. 1983. Selective
to be sufficient in the recovery of mycobacteria from non-sputum Kirchner’s medium in the culture of specimens other than sputum for
specimens. mycobacteria. J.Clin. Path. 36: 1357-1361.

Formula g/litre
Sodium dihydrogen phosphate 7.54
Potassium dihydrogen phosphate 2.0 Kligler Iron Agar
Magnesium sulphate 0.34 LAB059
Trisodium citrate 2.5 Description
L-Asparagine 5.0 A differential medium for the recognition of enteric pathogens by
their ability to ferment glucose and/or lactose, and liberate sulphides.
Phenol red 0.012 Fermentation liberates acid, with or without gas, turning phenol red
indicator yellow. Fermentation of glucose only, is followed by
Method for reconstitution reversion in pH on the slope, from initial acidity to final alkalinity
Weigh 17.4 grams disperse in 1 litre deionised water, add 20ml. (red colour), but not in the anaerobic conditions of the butt, which
Glycerol A.R. Mix well and dissolve by gentle heat. Dispense 9ml. remains acid (yellow). Fermentation of lactose as well as glucose,
volumes into Macartney bottles. Sterilise at 121˚C for 10 minutes. produces acidity in both slope and butt (yellow). Liberation of
Before use add 1ml. sterile heat in activated horse serum and sulphide results in the formation of iron sulphide (blackening of
selective agents, if required. either slope or butt).

Selective Agents Formula g/litre

Polymixin B 200 units/ml Peptone 20.0
Carbenicillin 100 µg/ml Lactose 10.0
Trimethoprin 10 µg/ml
Glucose 1.0
Amphoterocin 10 µg/ml
Sodium chloride 5.0
Appearance: Pale red, clear fluid.
Ferric ammonium citrate 0.5
pH: 7.0 ± 0.2
Sodium thiosulphate 0.3
Minimum Q.C. organisms: M.tuberculosis (H37RV strain)
E. coli (inhibition) Phenol red 0.025
NCIMB 50034
Candida sp. (inhibition) Method of reconstitution
NCIMB 50010 Weigh 49 grams of powder and mix with 1 litre of distilled water.
Bring to the boil with frequent stirring to dissolve completely.
Storage of Prepared Medium: up to 3 months at 2-8˚C in the dark. Dispense into tubes and sterilise for 15 minutes at 121˚C. Cool in a
slanted position such that slopes are formed over deep butts approx.
Inoculation: Kirchner’s Medium is suitable for the cultivation of 3cm in depth.
mycobacteria from the following specimens:
Pre-treatment Required Appearance: Reddish brown agar.
Sputum, Urine & Faeces Concentration and acid or pH: 7.4±0.2
alkali decontamination.
C.S.F., Pus with Minimum Q.C. Organisms Salmonella typhimurium
no other bacteria None. NCIMB 50076
Tissue Disintegration followed by Pseudomonas aeruginosa
acid or alkali decontamination, NCIMB 50067
if necessary.
The acidified or alkaline de-contaminated material may be added
directly to the Kirchner. The pH is then adjusted using the appropriate Inoculation
normal solution observing colour or indicator. Subcultures for further identification are picked from the centre of
isolated colonies on selective media and streaked across the slant and
Although Kirchner’s Medium has been shown to give higher stabbed deep into the butt of tubes of Kligler Iron Agar.
isolation rates than solid media, it is recommended that specimens
should also be inoculated onto Lowenstein Jensens slopes for the Incubation: 37˚C aerobically for 18-24 hours.
following reasons: – Growth suitable for further characterisation may
be obtained more rapidly – Some Mycobacterium species including
M.intracellulare, M.kansasii and M.scrofulaceum may be inhibited
by the antibiotic cocktail.
56
 Interpretation Liquid Baird Parker Medium
Organism Butt Slope Sulphide
Salmonella typhi Acid Alkaline + LAB 158
S. paratyhi A + B Acid Alkaline - Description
Other Salmonella Acid/gas Alkaline + Developed by Van Doorne et al in 1981, this medium is essentially
Baird Parker Agar LAB 085 without the agar and egg yolk
E. coli Acid/gas Acid - components. This medium was chosen for development to overcome
Proteus spp Acid/gas Alkaline + the problems of other selective enrichments and non-selective
enrichments, in the isolation of Staphlococcus aureus.
Shigella sonnei Acid Alkaline -
Other selective broth media suffer from the potential of inhibiting sub
S. flexneri Acid Alkaline - lethally damaged S. aureus cells where the salt content is greater than
40g/l. Even Giolitti Cantoni Medium may be inhibitory to some
Storage: Tightly capped containers - up to 3 months at 15-20˚C in the strains of S. aureus.
dark. Non selective enrichments have been suggested, but are not ideal due
to potential inhibition of S. aureus by microbial antagonism in a
References: mixed bacterial population.
Kligler, I.J. 1917. A Simple Medium for the Differentiation of
Liquid Baird Parker Medium is the ideal solution for detecting low
Members of the Typhoid - Paratyphoid Group. Am. J. Publ. Hlth,
numbers (<20/g) of S. aureus as it has been shown to give acceptable
7:1042-1044.
selectivity whilst being non-inhibitory to injured cells.
Bailey, S.F. and Lacey, G.R. 1927. A modification of the Kligler Lead
Acetate Medium. J. Bact. 13:182-189. Formulation g/litre
Meat Peptone 8.0
Tryptone 2.0
Lactose Broth Beef Extract 5.0

LAB 126 Yeast Extract 1.0

Sodium pyruvate 10.0
Description
A medium for the performance and confirmation of the Presumptive Glycine 12.0
Test for members of the coliform group in water and dairy products,
recommended by the U.S.P. Lithium chloride 5.0

Formula g/litre Method for Reconstitution

Double Strength Medium: Weigh 86 grams of powder and disperse
Beef Extract 3.0 in 1 litre of deionised water. Soak for 10 minutes, mix to dissolve and
dispense 10ml amounts in tubes. Sterilise at 121˚C for 15 minutes.
Gelatin Peptone 5.0
Before use, heat to 100˚C in a water bath or steamer for 15 minutes
Lactose 5.0 to remove oxygen, cool and add 0.2ml of 1% potassium tellurite
solution to each tube.
Method for reconstitution Single Strength Medium: Weigh 43 grams of powder and proceed
Weigh 13 grams of powder, disperse in 1 litre of deionised water, heat as above. When cool, add 0.1ml of 1% potassium tellurite solution to
to dissolve then distribute into bottles with Durham tubes. Sterilise by each tube.
autoclaving at 121˚C for 15 minutes. Appearance: Clear straw broth.
Appearance: Straw coloured, clear. pH: 6.8 ± 0.2.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: S. aureus NCIMB 50080
Minimum Q.C. organisms: E. coli NCIMB 50034 E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: See methods for standard techniques.
Incubation: 35˚C aerobically for 48 hours.
Interpretation: Coliforms are presumptively identified by their
ability to ferment lactose and produce gas within 48 hours at 35˚C.
References
American Public Health Association. 1975. Standard Methods for the
examination of water and waste water, 892. Washington. United
States Pharmocopeia, XXI, 1985.
Storage of prepared medium: Up to 1 month at 2-8 ˚C (without
tellurite added).
Inoculation: Double strength; 10ml of sample homogenate to each
tube. Single strength; 1ml of sample homogenate to each tube.
Incubation: 37˚C anaerobically for 48 hours. Anaerobic conditions
can be achieved using an anerobic jar (container caps must be loose
fitting) or by overlaying the surface of the broth with 1cm of molten
agar or liquid parafin.
Interpretation: Culture tubes showing growth (blackening of the
medium and turbidity) onto Baird Parker Agar LAB 085.
References
Van Doorne, H., Baird, R.M., Hendriksz, D.T., Van der Kreek, D.M.,
Pauwels, H.P. (1981) Liquid Modification of Baird Parker’s Medium
for the Selective Enrichment of Staphylococcus aureus. Antonie Van
Leeuwenhoek 47 267-278.

57
 Listeria Enrichment Broth Listeria Isolation Medium
LAB 138 (Oxford Formulation)
Description LAB 122
A medium for the selective enrichment of food and environmental
samples for Listeria spp. first described in 1987 by J. Lovett. The Description
medium offers more rapid enrichment than the low temperature A selective identification medium for the isolation of Listeria
enrichment techniques. This medium is now recommended by the monocytogenes from food and clinical material. Columbia agar is the
Commission of European Communities and the International Dairy nutrient base to which selective inhibitors have been added. Lithium
Federation for the examination of soft cheeses for Listeria chloride is used to inhibit enterococci and acriflavine to inhibit some
monocytogenes. The medium is incubated at 30˚C and utilises Gram negative and Gram positive species. Further selective agents
acriflavine, nalidixic acid and cyclohexamide as selective agents. may be added after autoclaving to increase the selectivity; these are
colistin, fosfomycin, cefotetan and cyclohexamide. Aesculin is
included in the formula as a differential indicator. L. monocytogenes
Formula g/litre
will hydrolyse aesculin to aesculutin which reacts with the iron salt to
Tryptone 17.0 give a black precipitate around the colonies.
Soy Peptone 3.0 LAB M’s formulation has been used to successfully isolate Listeria
from such diverse products as chicken giblets and dairy cheeses. The
Sodium chloride 5.0 advisability of using this medium at two levels of selectivity has been
recognised.
Dipotassium hydrogen phosphate 2.5
Glucose 2.5 Formula g/litre
Yeast Extract 6.0 Columbia Agar Base 41.0
Aesculin 1.0
Method for reconstitution
Weigh 36 grams of powder and add to 1 litre of deionised water. Ferric ammonium citrate 0.5
Allow to soak for 10 minutes then swirl to mix and sterilise by Lithium chloride 15.0
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and add 2 vials of
X138 reconstituted in 50% alcohol. Aseptically dispense into sterile
tubes or bottles. Method for reconstitution
Weigh 57.5 grams of powder. Add to 1 litre of deionised water. Allow
Appearance: Yellow, clear.
to soak for 10 minutes, swirl to mix then sterilise by autoclaving at
pH: 7.3 ± 0.2 121˚C for 15 minutes. Allow to cool to 47˚C, add 2 vials of selective
supplement X122, mix well and pour plates.
Minimum Q.C. organisms: L. monocytogenes NCIMB 50007
Appearance: Pale yellow, clear.
E. coli (inhibition) NCIMB 50034
pH: 7.2 ± 0.2
Storage of Prepared Medium: Capped containers – up to 14 days at
2-8˚C in the dark. Minimum Q.C. organisms: L. monocytogenes NCIMB 50007
E. coli (inhibition) NCIMB 50034
Inoculation: Homogenised samples of food, 25 grams of
homogenate to 225mls of broth.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Incubation: 30˚C aerobically for up to 48 hours. the dark.
Subculture: After 24 and 48 hours onto Listeria Isolation Medium – Inoculation: Surface, streak out to single colonies. This medium is
LAB 122. highly selective, a heavy inoculum can be used.
Incubation: 30˚C aerobically for 24-48 hours.
References
Lovett, J. Frances, D. W. Hunt, J. M. J. Food Protect. 50: 188-192.
Bolton, F. J. Personal Communication Public Health Laboratory, Growth Characteristics
Preston U.K. colony size shape &
organism (mm) surface colour other
L. monocytogenes 0.5-1.0 CV.E.G. Black Black/brown
around colonies
diffusion
Gram-ve
Bacilli No growth
Enterococci p.p. 0.5 CV.E.G. Black Usually no growth

References
Garayzabal, J.F.F., Rodriguez, L.D., Boland, J.A.V., Cancelo, J.L.B.,
Fernandez, G.S. 1986. Listeria monocytogenes dans le lait pasteurise.
Can. J. Microbiol. 32: 149-150.
Donnelly. C.W., Gregory J. Baigent 1986. Method for flow
cytometric detection of Listeria monocytogenes in milk. Appl. &
Environ. Microbiol.Oct. 689-695.
Bolton, C.F.J. Preston P.M.L. Personal communication. Lovett, J.
Francis, D.W. Hunt. J.M. (1987). Listeria monocytogenes in raw
milk: Detection, Incidence and Pathogenicity. Journ. Food Protect.
Vol. 50. No. 3: 188-192.
Van Netten, P., Van de Van, A., Perales, I., Mossel, P.A.A. 1988. A
selective and diagnostic medium for use in enumeration of Listeria
spp. in foods. International Journal of Food Microbiology 6:187-198.

58
M17 Agar MacConkey Agar
LAB 92 (With Salt)
Description LAB 30
A medium for the enumeration of lactic streptococci (Streptococcus
lactis, Streptococcus cremoris, Streptococcus diacetylactis) in dairy Description
products. The medium can also be used to investigate the A selective medium for the isolation of bile tolerant organisms from
bacteriophage susceptibilities of these organisms. Another faeces, urine, sewage and foodstuffs. Bile-tolerant Gram positive
application is for the enumeration of Streptococcus thermophilus in organisms as well as Gram negative organisms will grow on this
yoghurts. medium. This formula is recommended by W.H.O. and other bodies
for the examination of water and milk. Some strains of Proteus spp.
will spread on this medium making interpretation difficult, for this
Formula g/litre
reason LAB 2 MacConkey Agar (without salt) may be preferred as it
Balanced Peptone 5.0 is less prone to this phenomenon.
Soy Peptone 5.0
Formula g/litre
Yeast Extract 2.5
Peptone 20.0
Beef Extract 5.0
Lactose 10.0
Lactose 5.0
Bile Salts 5.0
Sodium glycerophosphate 19.0
Sodium chloride 5.0
Magnesium sulphate 0.25
Neutral red 0.05
Ascorbic acid 0.5
Agar No. 2 12.0
Agar No. 2 15.0
Method for reconstitution
Method for reconstitution Weigh 52 grams of powder, disperse in 1 litre of deionised water.
Weigh 57.2 grams of powder, disperse in 1 litre of deionised water. Allow to soak for 10 minutes, swirl to mix then sterilise by
Allow to soak for 10 minutes, swirl to mix then bring to boil to autoclaving at 121˚C for 15 minutes. Cool to 47˚C and mix well
dissolve agar before dispensing in 15ml aliquots. Sterilise by before pouring into petri dishes.
autoclaving at 115˚C for 20 minutes. Appearance: Pink/red, clear
Appearance: Pale straw, translucent agar. pH: 7.4 ± 0.2
pH: 7.1 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
Minimum Q.C. organisms: S. lactis
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
Storage of Prepared Medium: Capped containers – up to 1 month dark.
at 15-20˚C in the dark. Inoculation: Surface plating, streaking out to single colonies.
Inoculation: Pour plate technique. Incubation: 37˚C aerobically for 24 hours.
Incubation: 30˚C for 48-72 hours for mesophilic streptococci, 37˚C
for 48 hours for Streptococcus thermophilus. Growth Characteristics
Interpretation: Count all colonies. Streptococci form colonies of approx. size shape &
1-2mm in diameter. organism (mm) surface colour other
References E. coli 2.0-3.0 CV.E.G. Red (ppt around colony)
Terzaghi, B. E. Sandine, W. E.. 1975. Improved medium for lactic K. aerogenes 2.0-4.0 CV.E.G. Pink/Red (mucoid)
Streptococci and their Bacteriophages. Appl. Microbiol. 29 No. 6 pp
Citrobacter spp. 2.0-4.0 CV.E.G. Pink/Red (ppt around
807-813. colony)
Proteus spp. 1.5-2.5 CV.E.G. Yellow
(spreading)
Salmonella spp. 1.5-2.5 CV.E.G. Colourless
Shigella spp. 1.0-2.0 CV.E.G. Colourless
(pink)
Transp.
S. aureus 0.5-2.0 CV.E.G. White/ (dependent on
Pink lactose
fermentation
Orange and pigment
Opaque production)
Enterococcus
spp. P.P.-0.5 CV.E.G. Pink/Deep
Red
Opaque
P. aeruginosa 1.0-3.0 F.CR.D. Transp. (colonial-
Pinkish variation)

References
Ministry of Health. Public Health Laboratory Service Water
Committee 1969. The Bacteriological Examination of Water
Supplies, 4th Edn. report No. 71, H.M.S.O., London.

59
 World Health Organisation 1971. International Standards for
Drinking Water. 3rd Edn. W.H.O., Geneva.
Taylor, E. W. 1958. The Examination of Water and Water Supplies.
7th Edn. Churchill, London.
Cruikshank, R. 1973. A Guide to the Laboratory Diagnosis and
Control of Infection. Medical Microbiology. 12th Edn. Churchill.
MacConkey, A. T. 1908 Bile salt media and their advantages in some
bacteriological examinations, J.Hyg. (Camb.), 8: 322-341.
Ministry of Health, Public Health Laboratory Service Water
Committee 1969. The Bacteriological Examination of Water
Supplies, 4th Edn. report No. 71. H.M.S.O., London.
World Health Organisation 1971, International Standards for
Drinking Water, 3rd Edn. W.H.O., Geneva. Taylor, E. W. 1958. The
Examination of Water Supplies, 7th Edn. Churchill, London.

MacConkey Agar
(without salt)
LAB 2
Description
A medium first introduced by MacConkey in 1905 for the isolation
and differentiation of lactose and non lactose fermenting enteric
bacteria. The medium has since been modified to improve the
recovery of staphylococci and enterococci, it is used for culturing a
wide range of clinical material and has applications in food, water
and dairy bacteriology.
MacConkey Agar No. 3
LAB 45
Description
A modification recommended by the W.H.O. and the American
Public Health Association for the isolation of Enterobacteriaceae
from waters and sewage. The medium has been made more selective
than MacConkey’s original formula by the use of crystal violet as
well as bile salts. Gram positive organisms will not grow on this
medium.

Formula g/litre Formula g/litre

Mixed Peptones 20.0 Peptone 20.0
Lactose 10.0 Lactose 10.0
Bile 5.0 Bile Salts No. 3 1.5
Neutral red 0.05 Sodium chloride 5.0
Agar No. 2 13.5 Neutral red 0.03
Crystal violet 0.001
Method for reconstitution
Weigh 48.5 grams of powder, disperse in 1 litre of deionised water. Agar No. 2 15.0
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving for 15 minutes at 121˚C. Cool to 47˚C and mix well Method for reconstitution
before pouring plates. Prior to inoculation, dry the surface of the agar
Weigh 51.5 grams of powder and add to 1 litre of deionised water.
by partial exposure at 37˚C.
Allow to soak for 10 minutes, swirl to mix then sterilise for 15
Appearance: Pink/red, clear. minutes at 121˚C. Cool to 47˚C and pour into petri dishes. Dry the
surface before inoculation.
pH: 7.4 ± 0.2
Appearance: Pale red slight violet tinge.
Minimum Q.C. organisms: E. coli NCIMB 50034
pH: 7.1 ± 0.2
S. aureus NCIMB 50080
Minimum Q.C. organisms: E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in Ent. faecalis (inhibition)
the dark. NCIMB 50030
Inoculation method: Surface inoculation, streaking for single
colonies. Storage of prepared medium: Plates – up to 7 days at 2-8˚C in
Incubation: 37˚C aerobically for 24 hours. the dark.
Inoculation: Surface, streaking for single colonies.
Growth Characteristics Incubation: 37˚C aerobically for 18-24 hours.
colony size shape &
organism (mm) surface colour other Growth Characteristics
E. coli 2.0-3.0 CV.E.G. Red (non lactose colony size shape &
fermenting organism (mm) surface colour other
yellow)
E. coli 3.0-4.0 CV.E.G. Red (red ppt around
K. aerogenes 3.0-4.0 CV.E.G. Pink-Red (mucoid) (D) colony)
Proteus spp. 2.0-3.0 CV.E.G. Yellow fishy odour Kleb. aerogenes 4.0-5.0 CV.E.G. Pink-red (mucoid)
Ps. aeruginosa 2.0-4.0 F.CR.D. Pink- characteristic Proteus spp. 3.0-4.0 CV.E.G. Pale- (fishy odour)
Yellow- odour if green yellow
(Green)
Ps. aeruginosa 1.0-1.5 F.CR.D. Yellow-
Shigella spp. 1.0-3.0 CV.E.G. Yellow green
Salmonella spp. 2.0-3.0 CV.E.G. Yellow Shigella spp. 2.0-3.0 CV.E.G. Pale- (pink)
S. aureus 0.5-1.0 CV.E.G. Pink- (lactose-negative) (D) yellow
Orange Staph. aureus No growth
Enterococcus spp. 0.5 CV.E.G. Pink- Other
Deep Red Staphylococcus
spp. No growth
References Enterococcus
MacConkey, A. T. 1905 Lactose-fermenting bacteria in faeces. J.Hyg. spp. No growth
(Camb), 5: 333-379.
60
References
American Public Health Association 1950. Diagnostic Procedures
Malt Extract Agar
and Reagents. 3rd edn. A.P.H.A., New York. American Public Health LAB 37
Association 1946. Standard Methods for the examination of Water
and Sewage. 9th edn. A.P.H.A., New York. Description
An acidic medium which will support the growth of most yeasts and
moulds whilst inhibiting most bacteria. It was first described by
Thom and Church in 1926 in a study of Aspergillus spp. claiming the
MacConkey Broth high carbohydrate content ensured rapid growth. Selectivity can be
increased by further lowering the pH with the addition, after
(Purple) sterilisation, of XO37 Lactic Acid. It should be noted that excess
heating of this medium together with its low pH can easily result in
LAB 5 hydrolysis of the agar gel producing soft plates.

Description Formula g/litre

This medium is used in the detection and enumeration of faecal
Malt Extract 30.0
coliforms (37˚C) and E. coli (44˚C). The replacement of neutral red
used in the original formulation by bromocresol purple makes the Mycological Peptone 5.0
colour change caused by acid producing organisms easier to read.
Agar No. 2 15.0
Formula g/litre
Method for reconstitution
Peptone 20.0 Weigh 50 grams of powder, disperse in 1 litre of deionised water,
Lactose 10.0 allow to soak for 10 minutes, swirl to mix then sterilise at 115˚C for
10 minutes. If the addition of XO37 Lactic Acid is required this
Bile Salts 5.0 should be done after sterilisation. One 5ml vial of XO37 will lower
the pH of 250ml of medium to 3.5-4.0. Cool to 47˚C before making
Bromocresol purple 0.01 additions and pouring plates.

Method for reconstitution Appearance: Pale brown/straw, clear.

Weigh 35 grams of powder, disperse in 1 litre of deionised water. Mix pH: 5.4 ± 0.2 (if XO37 is added pH 3.5-4.0)
well and dispense into tubes or bottles with inverted Durham tubes.
Sterilise by autoclaving for 15 minutes at 121˚C. Prepare double Minimum Q.C. organisms: Candida spp. NCIMB 50010
strength broth (70g/l) if 50ml or 10ml amounts of inoculum are to be
added to equal volumes of broth. Prepare single strength broth (35g/l) Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
if 1ml or 0.1ml amounts of inoculum are to be added to 10ml of broth. the dark. Capped container – up to 1 month at 15-20˚C in the dark.
Appearance: Purple, clear. Inoculation: Pour plate technique or surface streaking for single
colonies.
pH: 7.3 ± 0.2
Incubation: 25˚C aerobically for 5 days.
Minimum Q.C. organisms: E. coli NCIMB 50034
B. subtilis (inhibition) Growth Characteristics
Storage of Prepared Medium: Capped containers – up to 1 month colony size shape &
at 15-20˚C in the dark. organism (mm) surface colour other
Incubation: 37˚C aerobically for coliforms, 44˚C aerobically for Candida albicans 4 CV.E.D. White
E. coli. Use Durham tubes to detect gas production for E. coli. Candida krusei 10 F.CR.D. White
Growth Indicators: Turbidity, gas production. Lactose-fermenting Penicillium
organisms cause a colour change from purple to yellow. notatum 25 Green (white/yellow -
velvet strain dependent)
References
Aspergillus niger 25 White (yellow/black
Ministry of Health 1937. Bacteriological Tests for Graded Milk, border, centre)
Memo 139/Foods. H.M.S.O., London. black
Minister of Health, Public Health Laboratory Service Water centre
Committee 1969. The Bacteriological Examination of Water
Supplies, 4th Edn. report No. 71. H.M.S.O., London. References
World Health Organisation 1971. International Standards for Galloway, L.D. and Burgess, R. 1952. Applied Mycology and
Drinking Water, 3rd Edn, W.H.O., Geneva. Bacteriology, Leonard Hill, London. Thom and Church, 1926.
The Aspergilli.

61
 Malt Extract Broth Minimum Q.C. organisms: S. aureus
E. coli (inhibition)
LAB 159
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Description the dark.
A liquid medium of low pH for the growth of yeasts and moulds,
Inoculation Method: Surface plating, streak out for single colonies.
typically employed as part of sterility testing protocols for various
products. The high carbohydrate content of the medium ensures rapid Incubation: 37˚C for 48 hours aerobically.
growth of yeasts and moulds.
Growth Characteristics
Formula g/litre
colony size shape &
Malt Extract 17.0 organism (mm) surface colour other
Mycological Peptone 3.0 S. aureus 1.5-2.0 CV.E.G. Bright
Yellow
Method for reconstitution Other
Weigh 20g of powder and disperse in 1 litre of deionised water. Allow Staphylococci 1.0-1.5 CV.E.G. White or (some ferment
to soak for 10 minutes, swirl to dissolve and dispense into final Yellow mannitol)
containers. Sterilise by autoclaving at 115˚C for 10 minutes. Enterobacteriaceae no growth
Appearance: Pale brown/straw, clear broth. Vibrio spp.
pH 5.4 + 0.2 & other 1.5-4.0 various usually (yellow if
halophiles Pink mannitol +ve)
Inoculation:
Inoculate samples direct into tubes of broth according to the References
particular method being employed. American Public Health Association 1966. Recommended Methods
for Microbiological Examination of Foods, 2nd Edn. (ed. J. M. Sharf)
Incubation: A.P.H. Washington.
25˚C (or 37˚C) for up to 7 days aerobically, depending upon protocol Davis, J. G., 1959. Milk Testing 2nd edn, Dairy Industries, London.
used.Subculture turbid tubes onto solid media for identification of
growth.

Minimum QC organism: Candida albicans NCIMB 50010

Aspergillus niger NCIMB 50097 Maximum Recovery Diluent
(Peptone/Saline diluent)
References:
Galloway L D, and Burgess R (1952) Applied Mycology and LAB 103
Bacteriology, 3rd ed, Leonard Hill, London pp 54 & 57.
Description
An osmotically controlled solution which is an alternative to, and a
replacement for, 1/4 strength Ringers LAB 100. The presence of a
low level of peptone lessens the physiological shock normally
Mannitol Salt Agar experienced by bacterial cells when they are introduced to a diluent
such as Ringers Solution. The level of peptone is such that
LAB 7 multiplication of the organisms is not possible in the time in which
the sample will be present in the diluent (1-2 hours). This formula is
Description recommended by ISO 6887: BS5763.
A medium for staphylococci which is selective because the high
sodium chloride level inhibits most other species with the exception Formula g/litre
of halophilic vibrios. The majority of Staphylococcus aureus ferment
mannitol producing yellow colonies, occasional strains of coagulase Peptone 1.0
negative staphylococci may also ferment mannitol. It is necessary to
confirm the identity of presumptive S. aureus colonies by other Sodium chloride 8.5
means e.g. coagulase, protein A, DN’ase, thermonuclease or latex
agglutination. Method for reconstitution
Dissolve 9.5 grams of powder in 1 litre of deionised water, heat
Formula g/litre gently to dissolve then distribute into final containers. Sterilise by
autoclaving at 121˚C for 15 minutes.
Beef Extract 1.0
Appearance: Clear fluid.
Balanced Peptone No. 1 10.0
Storage of Prepared Medium: Capped containers – up to 3 months
Sodium chloride 75.0 at 15-20˚C in the dark.
D-Mannitol 10.0 References
Agar No. 2 12.0 Straka, R. P. and Stokes, J. L. 1957. Rapid destruction of bacteria in
commonly used diluents and its elimination. Appl. Microbiol. 5: 21-
Phenol Red 0.025 25. ISO 6887. 1983. Microbiology-General guidance for the
preparation of dilutions for microbiological examination. BS5763
Method for reconstitution Part 6. Preparation of dilutions.
Weigh 108 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, mix by swirling. Sterilise by
autoclaving for 15 minutes at 121˚C. Cool to 47˚C before pouring
into petri dishes.
Appearance: Red, clear gel.
pH: 7.4 ± 0.2

62
Membrane Lauryl Sulphate Broth Milk Agar
LAB 82 LAB 19
Description Description
This medium superseded Membrane Enriched Teepol broth when An approved formulation for the enumeration of micro-organisms in
Shell Chemicals withdrew Teepol 610 from sale. Sodium lauryl milk, rinse waters and dairy products. With the addition of a further
sulphate was found to be an adequate reproducible substitute and this 5 g/l Agar No. 1 the medium is suitable for the preparation of Roll-
medium is recommended for the enumeration of coliform and Tubes using established mechanical equipment. Also see Milk Plate
organisms in water and sewage. Count Agar LAB 115. Milk Agar can also be used with the P-INC
supplement (X019, X219) for accelerated shelf-life determination of
Formula g/litre dairy products, (see page 103).

Peptone 39.0 Formula g/litre

Yeast Extract 6.0 Yeast Extract 3.0
Lactose 30.0 Peptone 5.0
Phenol red 0.2 Antibiotic Free Skim Milk Powder 1.0
Sodium lauryl sulphate 1.0 Agar No. 1 15.0

Method for reconstitution Method for reconstitution

Weigh 76.2 grams of powder, disperse in 1 litre of deionised water. Weigh 24 grams of powder, disperse in 1 litre of deionised water.
Distribute into screw cap containers and sterilise by autoclaving at Allow to soak for 15 minutes, swirl to mix then sterilise for 15
115˚C for 10 minutes. minutes at 121˚C. Cool to 45˚C before mixing with sample dilutions.
Appearance: Red, clear solution. Appearance: White, opalescent gel.
pH: 7.4 ± 0.2 pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034 Minimum Q.C. organisms: E. coli NCIMB 50034
S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark. Storage of Prepared Medium: Capped containers – up to 3 months
Inoculation: E. coli and coliform counts should be made on separate at 15-20˚C in the dark.
samples of water. The volumes should be chosen so as the number of Inoculation: Pour plate technique.
colonies on the membrane lies between 10 and 100. With waters
expected to contain less than 1 coliform perml, a sample of 100ml Incubation: Aerobically at 30˚C for 72 hours.
should be filtered. The membrane filter should be placed face
upwards on a pad soaked in Membrane Lauryl Sulphate Broth, after References
filtration. These membranes should be incubated in a container which Ministry of Health 1937. Bacteriological Tests for Graded Milk.
does not allow evaporation to occur. Water tight metal containers Memo 139/Foods H.M.S.O., London. British Standard 4285:
placed in an accurate water bath are required for incubation of Methods of Microbiological Examination for Diary Purposes.
membranes at 44˚C.
Incubation: E. coli 4 hours at 30˚C 14 hours at 44˚C; Coliforms 4
hours at 30˚C 14 hours at 35˚C.
Interpretation: No colonies:- assume a nil count. Small colonies of
an intermediate colour:- return to incubation for a full period.
E. coli: Yellow-coloured colonies from membranes incubated at 44˚C
should be subcultured to Lactose Broth LAB 126 and Tryptone
Water, LAB 129 to confirm gas and indole production respectively,
after 24 hours incubation at 44˚C.
Coliform organisms: Yellow colonies from membranes incubated at
35˚C or 37˚C should be subcultured into Lactose Broth LAB 126.
After 48 hours incubation at 37˚C a result should be obtained
regarding the production of gas.
Full details of the methodology can be found in The Bacteriological
Examination of Water Supplies 71, 1969.

References
Burnham, N.P. 1967. Proc. Soc. Wat. Treat, Exam. 16:40. Report 71.
1994. The Bacteriological Examination of Water Supplies. 4th Ed.
London, HMSO. Windle Taylor E. 1961 Glutamic acid medium, 40th
Ann Rep. Div. Water Exam. Met. Water Board London pp 18-22.

63
 Milk Plate Count Agar Calcium chloride (CaCl2.2H2O) 0.02

LAB 115 Dipotassium hydrogen phosphate 1.8

Bromocresol purple 0.02
Description
A medium recommended by the British Standards Institute and the LAB 80b
International Organisation for Standardisation for the enumeration of
viable bacteria in milk and other dairy products. Glutamic acid (sodium salt) 12.7

Formula g/litre Method for reconstitution

Double strength: Dissolve 22.7 grams of base medium (LAB 80a)
Tryptone 5.0 together with 12.7 grams of sodium glutamate (LAB 80b) in 1 litre of
deionised water containing 5 grams of ammonium chloride eg BDH
Yeast Extract 2.5
cat no. 27149. Dispense 10ml and 50ml volumes into tubes with
Dextrose 1.0 inverted Durham tube.
Antibiotic Free Skim Milk Powder 1.0 Single strength: Dissolve in 11.35 grams of base medium (LAB 80a)
together with 6.35 grams of sodium glutamate (LAB 80b) in 1 litre of
Agar No. 1 10.0 distilled water containing 2.5 grams ammonium chloride. Dispense
5ml volumes into tubes with inverted Durham tubes.
Method for reconstitution Sterilise by autoclaving for 10 minutes at 115˚C, alternatively heat to
Weigh 19.5 grams of powder, disperse in 1 litre of deionised water. 100˚C for 30 minutes on three successive days.
Allow to soak for 10 minutes, swirl to mix then bring to the boil to
Appearance: Purple, clear solution.
dissolve the agar. Allow to cool to 47˚C and dispense into suitable
containers. Sterilise by autoclaving at 121˚C for 15 minutes. pH: 6.7 ± 0.2
Appearance: Pale cream, opalescent gel. Minimum Q.C. organisms: E. coli NCIMB 50034
pH: 6.9 ± 0.2
Storage of Prepared Medium: Capped containers – up to 3 months
Minimum Q.C. organisms: E. coli NCIMB 50034 at 15-20˚C in the dark.
S. aureus NCIMB 50080
Inoculation: Use the Most Probable Number technique. With 10ml
and 50mls of sample add to equal volumes of double strength
Storage of Prepared Medium: Capped container – up to 3 months medium. With 1ml volumes of sample add to 5mls of single strength
at 15-20˚C in the dark. medium. Ensure the Durham tube is free of bubbles.
Inoculation: Pour plate technique. Incubation: 37˚C for 18-24 hours aerobically.
Incubation: 30˚C aerobically for 72 hours. Interpretation Tubes showing the production of acid (medium turns
Interpretation: Count all colonies. Calculate back to determine yellow) and gas in the Durham’s tube are considered presumptive
viable organisms per ml. positive. Each presumptive positive tube should be subcultured to
Brilliant Green Bile Broth LAB 51 with Durham tube and incubated
References at 44˚C for 24 hours and examined for gas production. A tube
British Standards Institute. 1984. BS 4285 Section 1.2. International of Tryptone Water LAB 129 should also be inoculated and incubated
Organisation for Standardisation Draft International Standard. 1982 at 44˚C for 24 hours for the production of indole. The production
ISO/DIS 6610. D.I.N. 10192. at 44˚C of gas from lactose and the formation of indole are evidence
of E. coli.

References
Gray, R. D. 1964. An improved formate lactose glutamate medium
Minerals Modified Glutamate Medium for the detection of Escherichia coli and other coliform organisms in
water. J. Hyg. Camb. 62: 495-508.
LAB 80A & LAB 80B PHLS Water Sub-Committee. 1958. A comparison between
MacConkey broth and Glutamic acid media for the detection of
Description coliform organisms in water. J. Hyg. Camb. 56: 377-388.
This medium was developed for use with the Most Probable Numbers
Technique (M.P.N.) for the enumeration of coliforms in water PHLS Standing Committee on Bacteriological Examination of Water
supplies. The medium is an improved version of the chemically Supplies. 1968. Comparison of MacConkey Broth, Teepol Broth and
defined glutamic acid medium described by Gray in 1964. The Glutamic Acid Media for the enumeration of Coliform organisms in
product is supplied in two parts because it has been shown that water. J. Hyg. Camb. 66: 67-87.
separating the sodium glutamate from the base improves its stability. Report 71. 1969. The Bacteriological Examination of Water Supplies.
4th Edn., London, HMSO.
Formula g/litre
LAB 80a (double strength)
Lactose 20.0
Sodium formate 0.5
L-Cystine 0.04
L(-) Aspartic acid 0.048
L(+) Arginine 0.04
Thiamine 0.002
Nicotinic acid 0.002
Pantothenic acid 0.002
Magnesium sulphate (MgSO4.7H20) 0.2
Ferric ammonium citrate 0.02

64
M.L.C.B. Agar Growth Characteristics
(Mannitol Lysine Crystal Violet Brilliant Green Agar) colony size shape &
organism (mm) surface colour
LAB 116 Salmonella spp. 2.0-3.0 CV.E.G. Black

Description Salmonella spp. 2.0-3.0 CV.E.G. Pale

(H2S negative)
A medium for the selective isolation of Salmonella spp. (with the
exception of S. typhi and S. paratyphi A) from food and faeces. Proteus spp. 1.0-2.0 CV.E.G. Grey brown
Salmonella colonies are recognised by distinctive colonial Shigella spp. mainly
appearance and H2S production and like the Bismuth Sulphite Agar inhibited
of Wilson & Blair, this medium will detect lactose and sucrose
fermenting strains. Some problems may occur with H2S negative Citrobacter spp. 0.5-2.5 CV.E.G. Mainly
strains, eg S. pullorum, S. senftenberg, S. sendai and S. berta. This inhibited
medium should not be used to detect S. typhi and S. paratyphi A, as pale may
these strains are more susceptible to the brilliant green dye. have black
centre
Formula g/litre E. coli mainly inhibited
Yeast Extract 5.0 Klebsiella spp mainly inhibited

Tryptone 5.0 Gram positive

organisms mainly inhibited
Meat Peptones 7.0
Sodium chloride 4.0 References
Inove et al. 66th meeting of the Japanese Vet. Medicine Society.
Mannitol 3.0
L-Lysine HCL 5.0
Sodium thiosulphate 4.0
Ferric ammonium citrate 1.0
M.R.S. Agar
Brilliant green 0.012 (de Man, Rogosa and Sharpe Agar)
Crystal violet 0.01 LAB 93
Agar No. 2 15.0 Description
This medium was developed for the cultivation and enumeration of
Method for reconstitution Lactobacillus spp. from various sources and is intended as a
Weigh 49 grams of powder, disperse in 1 litre of deionised water. substitute for Tomato Juice Agar. The medium is suitable for most
Allow to soak for 10 minutes, swirl to mix then bring to the boil with lactic acid bacteria. When acidified to pH 5.4 M.R.S. Agar can be
frequent agitation to completely dissolve the powder. Cool to 47˚C used to enumerate Lactobacillus bulgaricus in yoghurts. Tween 80,
and pour plates. DO NOT AUTOCLAVE OR OVERHEAT. sodium acetate, magnesium and manganese sulphates act as growth
stimulants.
Appearance: Pale purple, translucent gel.
pH: 6.8 ± 0.2 Formula g/litre
Minimum Q.C. organisms: Salmonella spp. NCIMB 50076 Mixed Peptones 10.0
E. coli (inhibition) NCIMB 50034
Yeast Extract 5.0
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in Beef Extract 10.0
the dark.
Glucose 20.0
Inoculation: Surface plating, streaking for single colonies.
Inoculation can be carried out directly, or from enrichment broths. Potassium phosphate 2.0
Because of the low selectivity of this medium the inoculum should
Sodium acetate 5.0
not be heavy, and it is recommended that this medium should be used
in conjunction with other more selective media. Ammonium citrate 2.0
Incubation: 37˚C aerobically for 24 hours. Magnesium sulphate 0.2
Manganese sulphate 0.05
Tween 80 1.08
Agar No. 1 15.0

Method for reconstitution

Weigh 70 grams of powder and add 1 litre of deionised water. Allow
to soak for 10 minutes, swirl to mix. Heat to dissolve and sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and adjust pH if
the acidified medium is required.
Appearance: Light amber, clear.
pH: 6.4 ± 0.2

Minimum Q.C. organisms: Lactobacillus acidophilus

65
 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculation: Surface, spread to cover surface, or use pour plate
technique.
Incubation: 25˚C microaerobically for 2-5 days.
Interpretation: Count all colonies exhibiting typical morphology.
Interpretation: For enumeration purposes count tubes showing signs
of growth as positive.
References
de Man, J. C., Rogosa, M. and Sharpe, M. E. 1960. A medium for the
cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-138.

Growth Characteristics MSRV

colony size shape & (Semi-solid Rappaport Medium)
organism (mm) surface colour
Lactobacillus 0.5-2.5 F.E.G. White Rough LAB 150
acidophilus
Description
Lactobacillus sake 0.5-1.0 F.E.G. White MSRV was developed in 1986 by De Smedt, Bolderdijk and Rappold
Streptococcus lactis 0.5-1.5 CV.E.G. White as a rapid means of Salmonella detection. The medium, based upon
Rappaport Vassiliadis broth, is inoculated directly from the pre-
Lactobacillus enrichment medium, in the centre of the plate. Motile organisms
bulgaricus 1.0-1.3 CV.E. G. White spread from the centre in the semi-solid agar, but non-salmonellas are
inhibited by the selective agents.
References After overnight incubation the use of polyvalent salmonella antisera
de Man, J. C., M. Rogosa and M. E. Sharpe, 1960. A medium for the or a latex kit can confirm the presence of a Salmonella. Alternatively,
cultivation of lactobacilli. J. Appl. Bacteriol. 23: 130-135. a paper disc wetted with polyvalent H antiserum can be placed 1/3 of
the way from the edge of the dish, and will signal the presence of a
Salmonella by inhibiting the mobility of the organism around the
disc.
M.R.S. Broth Using this medium De Smedt and Bolderdijk have reported the
possibility of detecting Salmonella in 24hrs (1987)
(de Man, Rogosa and Sharpe Broth)
Formula g/litre
LAB 94
Tryptone 2.3
Description
Meat Peptone 2.3
A medium for the cultivation and enumeration of Lactobacillus spp.
this product has the same formulation as LAB 93 M.R.S. agar with Acid Hydrolysed Casein 4.7
the omission of agar. The medium can be used for confirmatory tests
on organisms isolated on M.R.S. agar. The medium can also be used Sodium chloride 7.3
for enumeration by the Miles and Misra technique. Potassium dihydrogen phosphate 1.5

Formula g/litre Magnesium chloride 10.9

Mixed Peptones 10.0 Malachite green 0.037

Yeast Extract 5.0 Agar No. 1 2.5

Beef Extract 10.0 Method for reconstitution

Glucose 20.0 Weigh 31.5 grams of powder and disperse in 1 litre of deionised
water. Soak for 10 minutes, swirl to mix and bring to the boil. Cool
Potassium phosphate 2.0 to 47˚C. and add 2 vials of X150 novobiocin supplement (10mg/vial).
Sodium acetate 5.0 Mix well before dispensing.
Appearance: Turquoise/blue, clear, soft gel.
Magnesium sulphate 0.2
pH: 5.2 0.2
Manganese sulphate 0.05
Tween 80 1.08 Minimum Q.C organisms: Salmonella typhimurium
NCIMB 50076
Ammonium citrate 2.0 E.coli (inhibition) NCIMB 50034

Method for reconstitution Storage of prepared medium: Plates – up to 7 days at 4˚C.

Weigh 55 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then warm to completely
dissolve solids. Dispense into suitable tubes or bottles then sterilise
by autoclaving at 121˚C for 15 minutes.
Appearance: Light amber, clear.
pH: 6.4 ± 0.2
Minimum Q.C. organisms: Lactobacillus acidophilus
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: From pre-enrichment broth (6-24hrs) adding 0.1ml to
the centre of the plate.
Incubation: 37˚C. or 42 ±0.5˚C. for 18-24 hours. Keep lid uppermost
at all times.
Interpretation: A spreading growth indicates a Salmonella may be
present, substantiated if a disc with polyvalent H antiserum has been
added and is inhibiting the zone. This should be confirmed by
subculturing from the edge of the mobility zone onto XLD and
brilliant green agar and performing biochemical and serological tests.
Direct latex agglutination may be carried out from the edge of the
mobility zone.

Inoculation: Either with suspect colonies from M.R.S. agar or with
serial dilutions of test material.
Incubation: 25˚C microaerobically for 2-5 days.
66
References
De Smedt J.M. and Bolderdijk R.F. (1987): ‘One Day Detection of
Salmonella from Foods and Environmental Samples by Mobility
Enrichment’. Fifth International Symposium on Rapid Methods and
Automation in Microbiology and Immunology, Florence 1987. Brixia
Academic Press.
De Smedt J.M. and Bolderdijk R.F., Rappold H. and Lautenschlaeger
D. Rapid Salmonella Detection in Foods in Mobility Enrichment on Mueller Hinton Broth II
a Modified Semi-Solid Rappaport-Vassiliadis Medium. Journal of
Food Protection 49 510-514. 1986. LAB 114
De Smedt J.M. and Bolderdijk R.F. Dynamics of Salmonella Description
Isolation with Modified Semi-Solid Rappaport-Vassiliadis Medium. This medium is the broth version of Mueller Hinton Agar. It is an
Journal of Food Protection 50 658-661. 1987. antagonist free medium for use in the tube dilution technique for the
De Smedt J.M. and Bolderdijk R.F. Collaborative Study of the determination of antibiotic M.I.C. values. The medium is carefully
International Office of Cocoa. Chocolate and Sugar Confectionery on standardised to meet N.C.C.L.S. standards for antimicrobial
the Use of Mobility Enrichment for Salmonella Detection in Cocoa susceptibility tests on bacteria which grow aerobically.
and Chocolate. Journal of Food Protection 53 659-664. 1990.
Goossens H., Wauters G., De Boeck M., Janssens M., and Butzler J.P. Formula g/litre
Semi-solid selective mobility enrichment medium for isolation of Beef Extract 2.0
Salmonella from faecal specimens J. Clin. Microbiol 19 940-941.
1984. Acid Hydrolysed Casein 17.5
Starch 1.5
Calcium ions 50 mg/litre

Mueller Hinton Agar II Magnesium ions 20 mg/litre

LAB 39 Method for reconstitution

Description
A medium for antimicrobial sensitivity testing by the disc diffusion
method. This medium, used in the technique of Bauer and Kirby, has
been adopted by the National Committee for Clinical Laboratory
Standards (NCCLS) in the USA as the definitive method for
susceptibility testing. The medium has a very low thymine and
thymidine content, making it suitable for trimethoprim and
sulphonamide testing, controlled to ensure correct zone sizes with
aminoglycoside and tetracyline antibiotics. The medium was
originally formulated as a heat labile protein free medium for the
isolation of pathogenic Neisseriaceae.
Formula g/litre
Beef Extract 2.0
Acid Hydrolysed Casein 17.5
Weigh 21 grams powder, disperse in 1 litre distilled water. Allow to
soak for 10 minutes, swirl to mix then heat gently to dissolve.
Distribute into tubes or bottles, and sterilise at 121˚C for 10 minutes.
Appearance: Pale straw , clear.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus ATCC 25923
E. coli ATCC 25922
(M.I.C. values)
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: Standard inocula are required. As described by NCCLS.
Incubation: As recommended by methodology for particular
organisms and antibiotics by NCCLS.

Starch 1.5 References

Agar No. 1 17.0 MacFaddin, J. 1985. Media for isolation cultivation, identification
maintenance of medical bacteria. Williams & Williams, Baltimore.
Calcium ions 50-100mg/litre
N.C.C.L.S.-M7-A. 1985. Methods for dilution antimicrobiol
Magnesium ions 20-35mg/litre susceptibility tests for bacteria that grow aerobically. Approved
Standard.
Method for reconstitution
Weigh 38 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise at 121˚C for
15 minutes. Cool to 47˚C, mix well and pour plates.
Appearance: Straw coloured, clear gel.
pH: 7.3 ± 0.1

Minimum Q.C. organisms: E. coli ATCC 25922

S. aureus (antibiotic sensitivity
zones) ATCC 25923
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation: Surface, inoculum as described by N.C.C.L.S.
Incubation: As recommended by methodology for particular
organisms and antibiotics by NCCLS.

References
Mueller, J.H. and Hinton, J. 1941. Protein-free medium for primary
isolation of gonococcus and meningococcus. Proc. Soc. Exp. Biol.
and Med., 48: 330-333.
Goodale, W.I., Gould G. and Schwab, L. 1943. Laboratory
Identification of sulphonamide resistant gonococcic infection. J.Am.
Med. Ass., 123: 547-549.
American Public Health Association. 1950. Diagnostic Procedures
and Reagents. 3rd edn., A.P.H.A., New York.
NCCLS. 1986. Performance standards for antimicrobial
susceptibility testing – second informational supplement.

67
 Mueller Kauffman Tetrathionate Broth Nu Sens Agar
LAB 42 LAB 74
Description Description
A selective enrichment broth for salmonellae first described by This is a sensitivity test medium developed by LAB M in response to
Mueller in 1923 then modified by Kauffman in 1935 with the requests for a nutritionally rich sensitivity medium. This medium will
addition of Brilliant Green and Ox Bile increasing its selectivity. grow all organisms not having a specific need for blood, giving
Organisms which can utilise tetrathionate, such as most Salmonella, reproducible zone sizes. It is composed of specially selected peptones
flourish. However some salmonellas will be missed in this medium with a small amount of glucose, solidified with a very pure agar and
either because they are sensitive to Brilliant Green or cannot utilise is free from antagonists.
tetrathionate (included in this group is S. typhi.) This medium is used
in the standard European Community Salmonella Isolation Procedure Formula g/litre
and in International Standards Organisation (ISO) Methods.
Acid Hydrolysed Casein 2.0
Formula g/litre Peptone 7.5
Tryptone 7.0 Beef Extract 5.0
Soy Peptone 2.3 Sodium chloride 5.0
Sodium chloride 2.3 Glucose 2.0
Calcium carbonate 25.0 Agar No. 1 15.0
Sodium thiosulphate anhydrous 40.7
Ox Bile 4.75
Method for reconstitution
Weigh 36.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
Method for reconstitution autoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour plates.
Weigh 82 grams of powder, disperse in 1 litre of deionised water.
Appearance: Transparent, almost colourless.
Bring to the boil and cool to below 45˚C. Prior to use, add 30ml 1N
iodine solution (or 19ml of a solution containing iodine 20g, pH: 7.4 ± 0.2
potassium iodine 25g, distilled water 100ml) and 9.5ml of brilliant
green 0.1% w/v solution. Mix well and dispense into sterile Minimum Q.C. organisms: S. aureus NCTC 6571
containers, keeping the chalk in suspension. E. coli NCTC 10418
Appearance: Green, turbid solution which precipitates on standing. (antibiotic sensitivity zones)

pH: 7.8 ± 0.2 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark.
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli (inhibition) NCIMB 50034 Inoculation method: Joan Stokes method, surface inoculum for semi
confluent growth, or breakpoint technique.
Storage of Prepared Medium: Capped container – up to 7 days at Incubation: 37˚C aerobically for 18-24 hours.
2-8˚C in the dark.
Inoculation: 1 part sample to 9 parts medium or 1 part pre- References
enrichment medium to 10 parts medium. Ericsson, H. M., Sherris, J. C. 1971. Antibiotic sensitivity testing.
Report of an international collaborative study. Acta. Pathol.
Incubation: 43˚C aerobically for 24-48 hours. Microbiol. Scand. Suppl. 217: 1-90.
Subculture: Subculture onto selective agars. e.g. B.G.A. LAB 34, Garrod, L. P. and Waterworth, P. M. 1969. Effect of medium
XLD LAB 32, M.L.C.B. LAB 116. composition on the apparent sensitivity of Pseudomonas aeruginosa
to gentamicin. J. Clin. Pathol. 22: 534-538.
References
Mueller. 1923. Un nouveau milieu d’enrichissment pour la recherche
du bacille typhique et des paratyphiques C.R. Soc. Biol (Paris) 89:
434. 1975.
International Organisation for Standardisation. Meat and meat
products. Detection of salmonellae (Reference method).
ISO 3565 (E). International Organisation for Standardisation
Microbiology-1981 General guidance on methods for the detection of
Salmonella. ISO 6579(E).
International Organisation for Standardisation. Milk and milk
products- 1985. Detection of Salmonella. ISO 6785(E).
Edel, W. and E.H. Kampelmacher, 1968. Comparative studies on
Salmonella isolations in eight European laboratories, Bull. Wld. Hlth.
Org. 41: 297-306.
Kauffman, F., 1935. Weitere Erfahrungen mit dem kombinierten
Anreicherungsverfahren fur Salmonellabacillen. Z. Hyg. 117: 26-32.
van Leusden, F.M., M. van Schothorst and H.J. Beckers, 1982. The
standard Salmonellae isolation method. In: Isolation and
identification methods for food poisoning organisms, edited by J.E.L.
Corry, D. Roberts and F.A. Skinner. SAB Technical Series, No. 17
35-49. Academic Press, London.

68
Nutrient Agar Nutrient Broth No. 2 B.P.
LAB 8 LAB 14
Description Description
A general purpose medium for the cultivation of organisms that are A general purpose broth which can be used for sterility testing for
not demanding in their nutritional requirements e.g. organisms that aerobic organisms as recommended by the British Pharmacopoeia.
can be isolated from air, water, dust etc. Nutrient Agar is suitable for This broth can also be used as the suspending medium for cooked
teaching and demonstration purposes, it is isotonic and can be meat granules for the cultivation of anaerobic organisms.
enriched with biological fluids such as sterile blood and egg yolk.
Formula g/litre
Formula g/litre
Beef Extract 10.0
Peptone 5.0
Balanced Peptone No. 1 10.0
Beef Extract 3.0
Sodium chloride 5.0
Sodium chloride 8.0
Agar No. 2 12.0 Method for reconstitution
Weigh 25 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then dispense into tubes
Method for reconstitution
or bottles, and sterilise for 15 minutes at 121˚C.
Weigh 28 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by Appearance: Pale straw, clear.
autoclaving for 15 minutes at 121˚C. Cool to 47˚C, mix well then pH: 7.3 ± 0.2
pour plates.
Appearance: Buff, opalescent gel. Minimum Q.C. organisms: S. aureus NCIMB 50080
E. coli NCIMB 50034
pH: 7.3 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080
E. coli NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation Method: Surface streaking for single colonies.
Incubation: Temperature and time to suit organisms. Usually
aerobic.
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Incubation: 37˚C for 18-24 hours aerobically.
References
British Pharmacopoeia. 1973. H.M.S.O., London. Cruikshank, R.
1972. Medical Microbiology. 11th edn. Livingstone, London.

Growth Characteristics
colony size shape &
Nutrient Broth “E”
organism (mm) surface colour other LAB 68
S. aureus 1.0-2.0 CV.E.G. White-
Yellow Description
An inexpensive broth for the growth of nutritionally non-demanding
other organisms. Ideal for teaching purposes.
Staphylococcus 0.5-2.0 CV.E.G. White-
spp. Yellow
Formula g/litre
Strep. pyogenes P.P.-0.5 CV.E.G. Transp.
Beef Extract 1.0
E. coli 1.5-2.5 CV.E.G. Grey
Proteus spp. spreading - Grey fishy odour Yeast Extract 2.0
Klebsiella spp. 2.0-4.0 CV.E.G. Grey mucoid Peptone 5.0
Bacillus spp. 2.0-6.0 various Grey may spread Sodium chloride 5.0
Ps. aeruginosa 2.0-4.0 F.CR.D. Grey-Green odour if
pigmented Method for reconstitution
Weigh 13 grams of powder, add to1 litre of deionised water. Heat to
dissolve then dispense into bottles or tubes. Sterilise by autoclaving
at 121˚C for 15 minutes.
Appearance: Straw coloured, clear.
pH: 7.4 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080

E. coli NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 3 months

at 15-20˚C in the dark.
Inoculation and incubation: To suit chosen organism.
Growth indicator: Turbidity.

69
 O157 Broth MTSB Orange Serum Agar
(Modified Tryptone Soy Broth) LAB 147
LAB 165 Description
A medium developed for the investigation of organisms involved in
Description the spoilage of citrus products including fruit juices and citrus
Modified tryptone soy broth has emerged as the medium of choice for concentrates. The low pH of these products restricts the growth of
the enrichment of E.coli O157:H7 in red meats1,2. As concern organisms to those capable of tolerating an acid environment such as
regarding this organism has grown due to the severity of the disease yeasts and moulds and bacteria belonging to the genera Bacillus,
syndromes caused, and the increase in foodborne infection3, so too has Lactobacillus, Leuconostoc, Streptococcus and Clostridium. By
the need to optimise methods for its efficient isolation. Symptoms having a low pH and incorporating orange extract, Orange Serum
start with severe stomach cramps and watery, bloody diarrhoea, and a Agar is the ideal isolation medium.
percentage of individuals infected will develop Haemolytic Uraemic
Syndrome (HUS) leading to acute renal failure4. In a comparison of 4
Formula g/litre
different selective broth media, MTSB was the most productive and
selective for the isolation of E.coli O157:H71. MTSB is made Tryptone 10.0
selective for O157:H7 by including bile salts in the dehydrated
medium, and the addition of novobiocin supplement (X150). Yeast Extract 3.0
Orange Extract 5.0
Formulation g/litre
Glucose 4.0
Tryptone 17.0
Di-potassium phosphate 3.0
Sodium chloride 5.0
Agar No.2 7.0
K2HPO4 4.0
Dextrose 2.5 Method for reconstitution
Weigh 42 grams of powder, disperse in 1 litre of deionised water.
Soy Peptone 3.0 Allow to soak for 10 minutes. Bring to the boil swirling frequently,
Bile Salts No.3 1.5 cool to 47˚C. and dispense into containers. Sterilise by autoclaving at
115˚C. for 15 minutes.
pH: 7.2 ± 0.2 Appearance: Amber, slightly opalescent gel.
Appearance: Clear straw broth pH: 5.5 ± 0.2

Method for reconstitution Minimum Q.C. organisms L. acidophilus

Weigh 33 grams of powder and add to 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix and autoclave at 121˚C for
15 minutes. Cool to 47˚C and add 2 vials of Novobiocin supplement
X150. Mix well and distribute aseptically into sterile containers.
Inoculation
Add 25g sample to 225ml of supplemented MTSB and homogenise
for 2 minutes.
Incubation
42˚C aerobically for 24hrs. Subculture onto CT-SMAC (LAB161
plus X161) and examine for non-sorbitol fermenting colonies. Some
workers1 recommend the use of an immunomagnetic separation step
after 6hrs incubation.
Interpretation
Turbidity in the broth indicates growth. All broths should be
subcultured to selective media whether turbid or not.
Minimum QC organisms: E.coli O157:H7
(non-toxigenic) NCTC 12900
E.coli NCIMB 50034
(inhibition)
Pen. roquefortii
Storage of Prepared Medium: Plates – up to 7 days at 4˚C. in the
dark. Capped containers – up to 3 months at 15-20˚C. in the dark.
Inoculation: Pour plate technique.
Incubation: 3 days at 30˚C. for bacteria, 5 days at 30˚C for yeasts
and moulds.
Interpretation: Count bacterial colonies and yeast/moulds
separately. Calculate the colony forming units (C.F.U.’s) per ml of the
sample allowing for dilution factors.
References
Hays G.L. (1951) The isolation, cultivation and identification of
organisms which have caused spoilage in frozen concentrated orange
juice. Proc. Florida State Hort. Soc.
Hays G.L. and Reister D.W. (1952) The control of ‘off-odour’
spoilage in frozen concentrated orange juice. Food Tech 6 p386.
Murdock D.I., Folinazzo J.F., and Troy V.S. (1952) Evaluation of
plating media for citrus concentrates. Food Tech. 6 p181.

References
1) Bolton E.J., Crozier L., Williamson J.K. (1995) Optimisation of
methods for the isolation of Escherichia coli O157 from beefburgers.
PHLS Microbiology Digest 12 (2) 67-70.
2) Willshaw G.A., Smith H.R., Roberts D., Thirlwell J., Cheasty T.,
Rowe B. (1993) Examination of raw beef products for the presence
of verocytotoxin producing Escherichia coli, particularly those of
serogroup O157. J.Appl.Bacteriol. 75 420-426.
3) Sharp J.C.M., Coia J.E., Curnow J., Reilly W.J. (1994) Escherichia
coli O157 infections in Scotland. J.Med.Microbiol 40 3-9.
4) Doyle M.P. (1991) Escherichia coli O157:H7 and its
significancein foods. Int.J.Food Microbiol. 12 289-302.

70
Oxytetracycline-Glucose-Yeast Extract Palcam Agar Base
Agar Base (Polymixin, Acriflavine, Lithium chloride, Ceftazidine,
Aesculin, Mannitol)
(O.G.Y.E.)
LAB 89 LAB 148
Description
Description Palcam Agar was developed by Van Netten et al in 1989 as an
A selective medium for the enumeration of yeasts and moulds in improved selective differential medium for the isolation of Listeria
food, introduced by Mossel in 1970. Unlike many selective media for monocytogenes from food, clinical and environmental specimens.
yeasts O.G.Y.E. has a neutral pH and it has been shown to give better
recovery rates than those media with a low pH. Oxytetracycline is Improved selectivity is achieved by the combination of antibiotic
used to inhibit bacteria, certain high protein foods may reduce the supplements and microaerobic incubation, whilst the double indicator
system of aesculin hydrolysis and mannitol fermentation aids
effectiveness of this antibiotic as a selective agent. Rose Bengal differentiation of Listeria spp from enterococci and staphylococci
Chloramphenicol LAB 36 is recommended in these instances. which can be confused with Listeria spp on other types of culture
media.
Formula g/litre
Yeast Extract 5.0 Formula g/litre

Dextrose 20.0 Columbia Peptone Mix 23.0

Biotin 0.001 Sodium chloride 5.0

Agar No. 2 12.0 Corn Starch 1.0

Yeast Extract 3.0
Method for reconstitution
Glucose 0.5
Weigh 37 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by Mannitol 10.0
autoclaving at 115˚C for 10 minutes. Cool to 47˚C and aseptically
add two vials of X089 Oxytetracycline selective supplement. Mix Aesculin 0.8
thoroughly and pour into petri dishes. Lithium chloride 15.0
Appearance: Pale yellow, clear.
Ferric ammonium citrate 0.5
pH: 7.0 ± 0.2
Phenol red 0.08
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097 Agar No. 2 12.0
Saccharomyces cerevisiae

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the

Method for reconstitution
dark. Capped containers – up to 1 month at 4˚C in the dark. Weigh 71 grams of powder and disperse in 1 litre of deionised water.
Soak for 10 minutes, swirl to mix then sterilise by autoclaving at
Inoculation: Surface spreading or pour plate. 121˚C. for 15 minutes. Cool to 47˚C and add 2 vials of P.A.C.
Supplement – X144. Mix thoroughly before dispersing.
Incubation: 25˚C aerobically for 5 days.
Appearance: Red, translucent
Growth Characteristics pH: 7.2 ± 0.2
colony size shape & Minimum Q.C. organisms L. monocytogenes
organism (mm) surface colour NCIMB 50007
Candida spp. 3.0-4.0 CV.E.D. Cream E. coli (inhibition) NCIMB 50034
Candida krusei 7.0-8.0 F.CR.D. White
Storage of Prepared Medium: Plates – up to 7 days at 4˚C. in
S. cerevisiae 3.0-4.0 CV.E.D. White the dark.
Pen. notatum 1.5 Green/blue centre Inoculation: 0.1ml of sample selectively enriched in Palcam Broth
Pen. flavescens 1.5-2.0 Yellow centre (or other enrichment medium) spread over surface of plate.
Aspergillus niger 3.0-5.0 Yellow with Incubation: 30˚C. aerobically or microaerobically for 24-48 hours.
black centre
Rhizopus spp. confluent Aerial hyphae with Growth Characteristics
black conidia colony size shape &
Tetracycline resistant bacteria may grow, colonial morphology organism (mm) Surface colour other
dependent upon species. L. monocytogenes 1.5-2.0mm F.E.D. Grey/green Black halo,
(draughtsman
References colonies).
Mossel, D. A. A. et al. 1970. O.G.Y.E. for Selective Enumeration of
Moulds and Yeast in Foods and Clinical Material. J. Appl. Bact. 35: Other
454-457. Listeria spp. 0.5-2.0mm F.E.D. Grey/green Black halo,
(draughtsman
Banks, J. G., Board, R. G. 1987. Some factors influencing the colonies).
recovery of yeasts and moulds from chilled foods. Int. J. Food
Microbiol. 4: 197-206. Enterococci Inhibited - - (Small yellow
colonies with
yellow/green
halo).
Staphylococci Inhibited - - (Small white/
yellow colonies
with yellow/
green halo)
Bacillus spp. Inhibited - - -

71
 References
Van Netten P., Perales I., Curtis G.D.W., Mossel D.A.A. (1989)
Peptone Water
Liquid and solid selective differential media for the enumeration of LAB 104
L. Monocytogenes Int. J. Food Micro. 8 (4) 299-316.
Description
A general purpose growth medium that can be used as a base for
carbohydrate fermentation studies. The medium has a high level of
Palcam Broth tryptone making it suitable for use in the indole test. Adjustment of
the pH to 8.4 makes the medium an enrichment broth for Vibrio spp.
(Liquid, Polymixin, Acriflavine, Lithium chloride,
Ceftazidime, Aesculin, Mannitol) Formula g/litre
Peptone 5.0
LAB 144
Tryptone 5.0
Description
Sodium chloride 5.0
Developed by Van Netten et al (1989) L-Palcam is a selective
differential medium for the enrichment of Listeria spp. in food,
environmental and clinical samples. It is unique amongst Listeria Method for reconstitution
enrichment media in that it contains an indicator system (aesculin) Weigh 15 grams of powder, and disperse in 1 litre of deionised water.
which will signal the presence of a possible Listeria by a Allow to dissolve then distribute into final containers. Sterilise by
browning/blackening of the broth; the result being the indication of a autoclaving at 121˚C for 15 minutes. If sterile additions are to be
potential problem up to 48 hours before growth on plating media can made to this medium e.g.: carbohydrates, the volume of water for
be observed. reconstitution must be reduced accordingly. A pH indicator may be
added to detect acid production from carbohydrate utilisation.
Formula g/litre Appearance: Clear, colourless.
Columbia Peptone Mix 23.0 pH: 7.2 ± 0.2
Yeast Extract 5.0 Minimum Q.C. organisms: E. coli NCIMB 50034
Peptonised Milk 5.0
Storage of Prepared Medium: Capped containers – up to 3 months
Sodium chloride 5.0 at 15-20˚C in the dark.
Mannitol 5.0 Inoculation: A light inoculum from a pure culture.
Aesculin 0.8 Incubation: According to organism.
Ferric ammonium citrate 0.5
References
Phenol red 0.08 Bergey’s Manual of Systematic Bacteriology, Vol. 1, 1984. Williams
and Wilkins, Baltimore/London.
Lithium chloride 10.0
MacFadden, J. F. 1983. Biochemical Tests for the Identification of
Medical Bacteria, 2nd edn. Williams and Wilkins, Baltimore/London.
Method for Reconstitution
Weigh 54.4 grams of powder and disperse in 1 litre of deionised
water. Soak for 10 minutes, swirl to mix and autoclave at 121˚C for
15 minutes. Cool to 47˚C and add two vials of X144. Mix well and
dispense into sterile tubes or bottles. Perfringens Agar
Appearance: Clear red broth
(O.P.S.P.)
pH: 7.2 ± 0.2
LAB 109
Minimum Q.C. organisms: L. Monocytogenes
NCIMB 50007 Description
E. coli (inhibition) NCIMB 50034 This medium was developed by Handford in 1974 to overcome some
of the problems associated with enumerating Clostridium perfringens
Storage of prepared medium: Capped containers – up to 7 days in foods. The medium is buffered and utilises sodium metabisulphite
at 4˚C. and liver extract as sources of H2S with ferric ammonium citrate as
Inoculation: Sample or pre-enriched sample added to the broth in the the indicator. The medium is made selective with the addition of
ratio 1 : 10. X109 Sulphadiazine and X110 Oleandomycin/Polymixin
supplements.
Incubation: 30˚C for 24 hours and 48 hours.
Subculture: Onto Palcam Agar – LAB 148. If low numbers of Formula g/litre
Listeria are present the medium may not produce the brown black
colour. All tubes should be subcultured onto selective agar before a Tryptone 15.0
sample is scored as negative. Yeast Extract 5.0

References Soy Peptone 5.0

Van Netten P., Perales I., Curtis G.D.W., Mossel D.A.A. (1989) Liver Extract 7.0
Liquid and solid selective differential media for the enumeration of
L. Monocytogenes Int. J. Food Micro. 8 (4) 299-316. Ferric ammonium citrate 1.0
Sodium metabisulphite 1.0
Tris buffer 1.5
Agar No. 2 10.0

72
Method for reconstitution References
Weigh 45.5 grams of powder, disperse in 1 litre of deionised water. Reasoner D.J., Geldreich E.E. (1985) A New Medium for the
Allow to soak for 10 minutes then sterilise by autoclaving at 121˚C Enumeration and Subculture of Bacteria from potable water. App &
for 15 minutes. Allow to cool to 47˚C before adding 2 vials each of Env. Microbiol. Jan. 1985 p.1-7.
selective supplements X109 and X110.
American Public Health Association (1985) Standard Methods for the
Appearance: Pale straw, clear. Enumeration of Water and Wastewater. 16th Edition. American
Public Health Association Inc. Washington D.C.
pH: 7.3 ± 0.2
Report on Public Health and Medical Subjects No. 71. (1994)
Minimum Q.C. organisms: C. perfringens Methods for the Examination of Waters and Associated Materials.
E. coli (inhibition) HMSO.

Storage of Prepared Medium: Use on day of preparation.

Inoculation: Pour plates, 1ml sample plus 9ml medium. When set
overlay with sterile medium. Plate Count Agar (A.P.H.A.)
Incubation: 37˚C anaerobically for 24 hours.
(Standard Methods Agar, Tryptone Glucose Yeast Agar)
Interpretation: Count large black colonies, presumptively identified
as C. perfringens. LAB 10
References Description
Handford, P. M. 1974. A new medium for the detection and Formulated to A.P.H.A. specifications, this medium is used for
enumeration of C. perfringens in foods. J. Appl. Bact. 37: 559-570. establishing total viable counts for aerobes in food, dairy and water
bacteriology. The product uses agar of very high gel strength in order
Shahidi, S. A. and Ferguson, A. R. 1971. A new quantitative and
that it can be used in pour-plate as well as surface inoculation
confirmatory medium for C. perfringens in food. Appl. Microbiol.
techniques. The product can be remelted prior to use although it
21: 500-506.
should not be held for a prolonged period in the molten stage.
Marshall, R. S., Steenberger, J. F. and McClung, L. S. 1965. Appl.
Microbiol. 13: 559. A rapid technique for the enumeration of C. Formula g/litre
perfringens.
Tryptone 5.0
Pharmacopoeia of culture media for food microbiology. 1987. Int. J.
Food Microbiol. 5: 3: 240-241. Yeast Extract 2.5
Glucose 1.0
Agar No. 1 15.0
Plate Count Agar Method for reconstitution
LAB 149 Weigh 23.5 grams of powder, disperse in 1 litre of deionised water.
Bring to the boil with frequent stirring to dissolve. Dispense into
Description tubes and sterilise by autoclaving at 121˚C for 15 minutes. Cool to
A medium designed for use with the spiral plating system and other 44-46˚C for not more than 3 hours prior to use.
surface inoculation techniques. The formula is equivalent to A.P.H.A. ROLL-TUBES. Add an additional 10g/litre Agar No. 1 prior to
Plate Count Agar and is suitable for the determination of total viable reconstitution of the medium.
counts in food products by surface count and pour plate methods.
Appearance: Pale straw coloured, clear gel.
Formula g/litre pH: 7.0 ± 0.2
Tryptone 5.0 Minimum Q.C. organisms: S. epidermidis NCIMB 50082
Yeast Extract 2.5 E. coli NCIMB 50034

Glucose 1.0 Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Agar No. 2 12.0
Inoculation method: Pour plate technique or surface inoculation.
Method for reconstitution Incubation: 30˚C aerobically for 48 hours for aerobic mesotroph
Weigh 20.5 grams of powder, disperse in 1 litre of deionised water. count. 6˚C aerobically for 10 days for aerobic psychrotroph count.
Allow to soak for 10 minutes, swirl to mix then sterilise by 55˚C aerobically for 48 hours for aerobic thermotroph count.
autoclaving at 121˚C for 15 minutes. Cool to 47˚C then pour into Interpretation: Count all colonies and calculate the number of
petri dishes. organisms (or ‘colony forming units’ c.f.u.) per ml of sample
Appearance: Pale straw colour, clear. allowing for dilution factors.
pH: 7.0 ± 0.2
References
Minimum Q.C. organisms: S. epidermidis NCIMB 50082 American Public Health Association 1972. Standard Methods for the
E. coli NCIMB 50034 Examination of Dairy Products. 13th edn. (ed. W. J. Hausler)
A.P.H.A., Washington.
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the American Public Health Association 1966. Recommended Methods
dark. for the Microbiological Examination of Foods, 2nd edn. (ed. J. M.
Inoculation: Surface, or pour plate. Sharf) A.P.H.A., Washington.

Incubation: 30˚C aerobically for 48 hours for aerobic mesotroph American Public Health Association 1976. Standard Methods for the
count. 6˚C aerobically for 10 days for aerobic psychrotroph count. Examination of Water and Waste Water, 14th edn. (ed. M. A. Franson)
55˚C aerobically for 48 hours for aerobic thermotroph count. A.P.H.A., Washington.

Interpretation: Count all colonies or use spiral plating colony count

equipment.

73
 Potato Dextrose Agar Pseudomonas Agar Base
LAB 98 (C.F.C./C.N. Agar)
Description LAB 108
Potato Dextrose Agar is recommended by the American Public
Health Association for the enumeration of yeasts and moulds in Description
examination of dairy products, soft drinks, dried and frozen foods and The base medium is a modification of King’s medium A which uses
other types of product. Depending on whether the medium is to be magnesium and potassium salts to enhance production of the
used as a selective or non-selective agar it can be used with or pigments pyocyanin (green) and fluorescein (detected by U.V./blue
without acidification. light). The medium is made selective for Pseudomonas aeruginosa
by the addition of X107 C.N. supplement. Alternatively the medium
can be made selective for Pseudomonas species generally by the
Formula g/litre
addition of X108 C.F.C. supplement.
Potato Extract 4.0
Formula g/litre
Dextrose 20.0
Acid Hydrolysed Casein 10.0
Agar No. 1 15.0
Gelatin Peptone 16.0
Method for reconstitution Potassium sulphate 10.0
Weigh 39 grams of powder, disperse in 1 litre of deionised water, then
sterilise at 121˚C for 15 minutes. Mix well before pouring into sterile Magnesium chloride 1.4
Petri dishes. In certain cases it may be desirable to lower the pH of
the medium to 3.5 in order to suppress bacterial growth. This can be Agar No. 2 11.0
done by adding 10ml of sterile 10% Lactic Acid X037, to one litre of
Potato Dextrose Agar LAB 98. This addition must be after Method for reconstitution
autoclaving and cooling to 47˚C. Once the pH has been lowered the Weigh 48.4 grams of powder and disperse in 1 litre of deionised
medium may not be heated again without resultant loss of gel water. Add 10mls of glycerol. Sterilise by autoclaving at 121˚C for 15
strength caused by agar hydrolysis. minutes. Allow the medium to cool to 47˚C then add the contents of
Appearance: Translucent white agar. 2 vials of either X107 C.N. supplement or X108 C.F.C. supplement.
Mix well and pour into petri dishes.
pH: 5.6 ± 0.2 (3.5-4.0 if X037 is added)
Appearance: Pale straw, opaque.
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097 pH: 7.1 ± 0.2
Saccharomyces cerevisiae
Minimum Q.C. organisms: Ps. aeruginosa NCIMB 50067
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in E. coli (inhibition) NCIMB 50034
the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Inoculum: Pour plate technique. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Incubation: 21˚C aerobically for 5 days.
Inoculation: Surface, spread 0.1 to 0.5mls of sample over entire
surface.
Growth Characteristics
Incubation: 25-30˚C aerobically for 48 hours.
colony size shape &
Interpretation: Count all colonies as Pseudomonas species.
organism (mm) surface colour
Colonies that exhibit the pyocyanin and fluorescein pigments count
Candida spp. 2.0 C.V.E.D. White as Ps. aeruginosa.
Candida kruseii 2.0 F.Rz.D. Grey/White
Tryc. mentagrophytes 4.0 Fluffy Yellow
Growth Characteristics
White obverse colony size shape &
Tryc. verrucusum 5.0 Fluffy Yellow organism (mm) surface colour fluorescence
White obverse Ps. aeruginosa 2.0-3.0 CV.Cr.D. Green/Blue yes
Toro. glabrata 3.0 C.V.E.G. White Ps. fluorescens 2.0-3.0 CV.Cr.D. Yellow yes
Asp. niger 4.0 Black spores Yellow Ps. fragi 1.0-3.0 CV.Cr.D. Grey no
centre obverse
White surround Ps. maltophila 2.0-3.0 CV.Cr.G. Grey no
Pen. notatum 4.0 Green spores Green Ps. putida 2.0-3.0 F.Cr.D. Grey no
centre Ps. cepacia 2.0-3.0 F.Cr.D. Grey no
White surround obverse
References
References Burton, M. O., Campbell, J. J. R. and Eagles, B. A. 1948. The mineral
Association of Official Analytical Chemists (AOAC). Bacteriological requirement for pyocyanin production. Can. J. res. Sect. C. Bot. Sci.
Analytical Manual, 5th ed. 1978. Washington D.C. Hausler, 26:15.
W. J. (ed.).
King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple media
Standard Methods for the Examination of Dairy Prod. 14th edn., for the demonstration of pyocyanin and fluorescein. J. Lab. Clin.
Washington D.C.: American Public Health Association, 1976. Med. 44: 301.
Goto, S. and Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72.
Mead, G. C. and Adams, B. W. 1977. Br. Poult. Sci. 18: 661-667.

74
R2A Medium Rappaport Vassiliadis (R.V.) Single
LAB 163 Component Enrichment Broth
Description LAB 86
R2A medium was developed to determine the bacterial count in
potable waters during treatment and distribution, and has been shown Introduction
to give significantly higher counts than plate count agar (PCA) or Rappaport Vassiliades Broth (R10 modification) was born out of a
similar high-nutrient media. The standard plate count (SPC) method long series of experiments carried out to determine the correct levels
using PCA provides an enumeration of bacteria which grow best at, of malachite green and magnesium chloride that would allow
or near, body temperature and this estimation at best may correlate to Salmonella to multiply freely yet still inhibit the other enteric
the coliforms present in the sample. However, there will be a organisms.
population of heterotrophic bacteria which cannot grow at all under This formulation has been shown to be superior to Mueller
the conditions of the SPC method or may grow so slowly that the Kauffmann and Selenite Broth for the isolation of Salmonella from
colonies fail to reach a size detectable to the eye in the 48-h meat products.
incubation period. In order to enumerate this section of the bacterial
population in water, a medium of low nutritional content (R2A) and The development work carried out on the formulation shows that it is
extended incubation times are required. R2A medium is extremely efficient in detecting small numbers of Salmonella in
recommended in Report 71, Methods for the Examination of Waters heavily contaminated products. This formulation is very hygroscopic
and Associated Materials, and Standard Methods for the Enumeration and will produce a slight exothermic reaction when mixed with water.
of Water and Wastewater.
Formula g/litre
Formulation g/litre
Soy Peptone 4.5
Yeast Extract 0.5
Sodium chloride 7.2
Meat Peptone 0.5
Potassium dihydrogen phosphate 1.26
Casamino acids 0.5
Dipotassium hydrogen phosphate 0.18
Glucose 0.5
Magnesium chloride anhydrous 13.58
Starch 0.5
Malachite green 0.033
Dipotassium hydrogen Phosphate 0.3
Magnesium sulphate 0.05 Method for reconstitution
Weigh 26.8 grams powder, disperse in 1 litre of deionised water, swirl
Sodium pyruvate 0.3 to mix, when dissolved dispense in 10ml. volumes in screw capped
bottles and sterilise by autoclaving at 115˚C for 15 minutes.
Agar No.2 15.0
Appearance: Clear, green fluid.
pH: 7.2 + 0.2 pH: 5.2 ± 0.2
Appearance: Clear opalescent gel
Minimum Q.C. organisms: E. coli (inhibited) NCIMB 50034
Method for reconstitution S. enterididis NCIMB 50073
Weigh 18 grams of powder and disperse in 1 litre of deionised water.
Swirl to mix and sterilise at 121˚C for 15 minutes. (If required, bring Storage of Prepared Medium: Capped container – 6 months
to the boil to dissolve the agar, and pour into smaller volumes before at 2-8˚C
sterilising). Cool to 44-46˚C for not more than 3 hours before use. Inoculation: From pre-enrichment broth in the proportions of 1-part
Inoculation: Pour 15ml into a petri dish containing 1ml of sample, inoculum to 99 parts R.V. Broth. Sub-culture onto either XLD Agar,
mix well and allow to set. Pour a further 10ml as an overlay and again M.L.C.B. Agar or other salmonella selective agars.
allow to set. Alternatively it may be used as a spread plate, Incubation: 41.5 ± 0.5˚C for 24 hours (incubator) or 42 ± 0.1˚C for
inoculating 0.1ml onto the plate and spreading over the entire surface 24hrs (water bath).
of the medium. It can also be used with membrane filters if required.
Incubation: When plates have set, incubate at 22˚C for 5-7 days or References
30˚C for 3 days. Other incubation temperatures between 20˚C and Vassiliades P., 1983 The Rappaport Vassiliades (R.V.) Enrichment
28˚C may be used. Medium for the Isolation of salmonellas: An overview J. Appl.
Bacteriol. 56 69-76.
Interpretation: Count all colonies and report the number of bacteria
in the original sample as the heterotrophic plate count Vassiliades P., Mavromatti CH. Efstratiou, M. and Chronas G. 1985.
A note on the stability of Rappaport-Vassiliades Enrichment Medium
Minimum QC organisms: Pseudomonas fluorescens, J. Appl. Bacteriol. 59 143-145.
Aeromonas hydrophila Bolton F.G. Preston P.H.L. Personal communication.
Int. J. Food Micro. Pharmacopoeia of culture media for Food
References Microbiology.
Reasoner D.J., Geldreich E.E. (1985) A New Medium for the Peterz M., Wiberg C., and Norberg P. 1989. The effect of incubation
Enumeration and Subculture of Bacteria from potable water. App & temperature and magnesium chloride concentration on growth of
Env. Microbiol. Jan. 1985 p. 1-7. Salmonella in home-made and in commercially available dehydrated
American Public Health Association (1985) Standard Methods for the Rappaport-Vassiliadis broths.
Enumeration of Water and Wastewater. 16th Edition. American
Public Health Association Inc. Washington DC.
Report on Public Health and Medical Subjects No. 71. (1994)
Methods for the Examination of Waters and Associated Materials.
HMSO.

75
 Reinforced Clostridial Agar Reinforced Clostridial Medium
LAB 23 (R.C.M.)
Description LAB 22
This is a solidified version of R.C.M. (LAB 22) and can be used for
the enumeration of anaerobes by pour plate, shake tube or membrane Description
filtration methods. When solidified in tubes or bottles with minimal This medium was formulated by Hirsch and Grinstead to recover
head space it can be used for anaerobic culture without the need for small numbers of Clostridium spp. from a variety of sources. Various
anaerobic atmosphere. workers have reported its efficiency with many products and
specimens, R.C.M. is rich, non-selective and uses cysteine
hydrochloride and glucose as reducing agents. The small amount of
Formula g/litre
agar reduces diffusion of oxygen through the fluid.
Yeast Extract 3.0
Formula g/litre
Beef Extract 10.0
Yeast Extract 3.0
Peptone 10.0
Beef Extract 10.0
Glucose 5.0
Peptone 10.0
Soluble Starch 1.0
Soluble Starch 1.0
Sodium chloride 5.0
Glucose 5.0
Sodium acetate 3.0
L-Cysteine hydrochloride 0.5
L-Cysteine hydrochloride 0.5
Sodium chloride 5.0
Agar No. 2 12.0
Sodium acetate 3.0
Method for reconstitution Agar No. 1 0.75
Weigh 49.5 grams of powder, disperse in 1 litre of deionised water,
allow to soak for 10 minutes, swirl to mix then sterilise for 15
minutes at 121˚C. Cool to 47˚C. and distribute into sterile dishes or Method for reconstitution
tubes containing decimal dilutions of the sample under test. Weigh 38 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil to
Appearance: Pale straw, translucent gel. dissolve. Distribute 25ml into 1oz Universal containers. Sterilise for
pH: 6.8 ± 0.2 15 minutes at 121˚C.
Appearance: Straw coloured, clear.
Minimum Q.C. organisms: C. perfringens NCIMB 50027
pH: 6.8 ± 0.2
Storage of Prepared Medium: Capped container – up to 3 months
at 15-20˚C in the dark. Minimum Q.C. organisms: C. perfringens NCIMB 50027
Inoculation: Pour plate technique or tube culture. Storage of Prepared Medium: Capped containers – up to 3 months
Incubation: 30˚C for up to 72 hours. Anaerobic conditions for pour at 15-20˚C in the dark.
plate. Count as early as possible as prolonged incubation may result Inoculation: Homogenised food sample to a ratio of 1:10
in the medium being disrupted due to gas production. with R.C.M.
Interpretation: Count all colonies as presumptive clostridia. Incubation: 30˚C for up to 72 hours.

References Growth Indicators

Miller, N. J., Garrett, O. W. and Prickett, P. S. 1939. Anaerobic
technique – a modified deep agar shake. Food Res. 4: 447-451.
Ingram, M. and Barnes, E. M. 1956. A simple modification of the
deep shake tube for counting anaerobic bacteria. Lab. Pract. 5: 145.
Turbidity, colonies in medium, gas production.
References
Hirsch, A. and Grinstead, E. 1954. Methods for the growth and
enumeration of anaerobic spore-formers from cheese. J. Dairy Res.
21: 101-110.
Barnes, E. M. and Goldberg, H.S. 1962. The isolation of anaerobic
Gram-negative bacteria from poultry. J. Appl. Bact. 25: 94-106.

76
Ringers Solution (1/4 strength)
LAB 100 LAB 100Z (TABLETS)
Description
An osmotically controlled solution for the preparation of suspensions
of food samples and for use as a diluent in dilution techniques for
bacterial enumeration. The solution can also be used in the sampling
of food production apparatus by the rinse and swab method.
References
Min. of Health, 1937. Bacteriological Tests for Graded Milk. Memo
139/Foods. H.M.S.O. London. Davis, J. G. 1956. Laboratory Control
of Dairy Plant. Dairy Industries, London. Higgins, M. 1950.
Comparison of recovery rate of organisms from cotton wool and
calcium alginate wool swabs. Mon. Bull. Min. Health, P.H.L.S., 9:
50-51.

Formula g/litre Rose Bengal Chloramphenicol

Sodium chloride 2.25 Agar Base
Potassium chloride 0.105
LAB 36
Calcium chloride 0.12
Description
Sodium bicarbonate 0.05
A selective medium for the enumeration of moulds and yeasts in
foods. The original formulation of Jarvis (1973) used
Method for reconstitution chlortetracycline, this has been substituted by chloramphenicol
LAB 100 – Dissolve 2.5 grams of powder in 1 litre of deionised because of superior selectivity. The Rose Bengal dye is taken up by
water, when completely dissolved dispense into containers as the growing colonies making them easier to see and inhibiting their
required and sterilise by autoclaving at 121˚C for 15 minutes. spreading. Rose Bengal becomes increasingly toxic on exposure to
LAB 100Z – Dissolve 1 tablet in 500mls of deionised water. light so it is important to store plates in the dark.
Proceed as for LAB 100.
Formula g/litre
Mycological Peptone 5.0
Dextrose 10.0
Ringers Solution (Calgon) Dipotassium phosphate 1.0
LAB 101 Magnesium sulphate 0.5
Description Rose bengal 0.05
For use with calcium alginate swabs. The sodium hexametaphosphate Agar No. 2 12.0
in the formula will enable the alginate swabs to completely dissolve.
This ensures total release of all the organisms taken up by the swab
to increase the accuracy of a quantitative bacterial count. Method for reconstitution
Weigh 28.5 grams of powder, disperse in 1 litre of deionised water,
Formula g/litre allow to soak for 10 minutes, swirl to mix then sterilise for 15
minutes at 121˚C. Allow to cool to 47˚C then add 2 vials of X009.
Sodium chloride 2.25 Chloramphenicol (or 2 vials X089 oxytetracycline). Mix well, then
pour into petri dishes. This medium should be protected from light.
Potassium chloride 0.105 Chloramphenicol may be added before autoclaving.
Calcium chloride 0.12 Appearance: Deep purple/red, clear.
Sodium bicarbonate 0.05 pH: 7.2 ± 0.2
‘Calgon’ (Sodium hexametaphosphate) 10.0 Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097
Sacchromyces cerevisiae
Method for reconstitution E. coli (inhibition) NCIMB 50034
Dissolve 12.5 grams of powder in 1 litre of deionised water. Dispense
10ml amounts into screw cap containers and sterilise by autoclaving Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
at 121˚C for 15 minutes. the dark. Capped Container – up to 1 month at 2-8˚C in the dark.
Inoculation: Surface spreading.
Incubation: 25˚C aerobically for 24 hours to 5 days.

Ringers Solution (Thiosulphate) Growth Characteristics

LAB 102 colony size shape &
organism (mm) surface colour other
Description Rhizopus spp. 13.5 Fluffy White
An isotonic rinse for use in hygiene studies on plant and equipment
that have been treated with chlorine disinfectants. 100ml of LAB M Mucor spp. 12.5 Fluffy White
Thiosulphate Ringer Solution will neutralise 7mg of chlorine. Fusarium spp. 10 Fluffy White
Aspergillus flavus 8 Flat & Yellow/ hyphae
Formula g/litre hyphae Green
Sodium chloride 2.15 Candida spp. 1.5 CV.E.G. White
Potassium chloride 0.075 Sacchromyces
spp. 1.0 CV.E.G. White
Calcium chloride 0.12
E. coli no growth
Sodium thiosulphate 0.5
Ent. faecalis no growth
Method for reconstitution B. subtilis no growth
Dissolve 2.8 grams of powder in 1 litre of deionised water. Dispense Staphylococcus
into containers as required then sterilise by autoclaving at 121˚C for spp. no growth
15 minutes.
77
 References References
Jarvis, B, 1973. Comparison of an improvised Rose-Bengal Sabouraud, R. 1910. Les Teignes Paris. Pagano. J., Levin, J. D. and
Chlortetracycline Agar with other media for the selective isolation Trejo, W. 1957-8. Diagnostic medium for the differentiation of
and enumeration of moulds and yeasts in food. J. Appl. Bact. 36: 723- species of Candida. Antibiotics Annual, 137-143.
727.
Overcast, W.W. and Weakley, D.L. 1969. An aureomycin rose-bengal
agar for the enumeration of yeast and mould in cottage cheese. J.Milk
and Fd.Tech. 32: 442-445.
Banks, J. G. Board, R. G. 1987. Some factors influencing the
Sabouraud Liquid Medium U.S.P.
recovery of yeasts and moulds from chilled foods. Int. J. Food (Fluid Sabouraud Medium)
Microbiol. 4: 197-206.
LAB 33
Description
A liquid sterility test medium for the detection of yeasts, moulds and
Sabouraud Dextrose Agar acidophilic bacteria in pharmaceutical products. This medium
conforms with the United States Pharmacopeia. It can also be used as
LAB 9 a growth medium for the determination of fungistatic activity in
pharmaceutical products.
Description
Introduced by Sabouraud in 1910 as a selective medium for fungi and Formula g/litre
yeasts. The acidic pH (5.6) of this medium inhibits many species of
bacteria. The medium can be made more selective by the addition of Pancreatic digest of casein 5.0
chloramphenicol supplement (X009). Diagnostic features, such as Peptic digest of fresh meat 5.0
sporing structures and pigmentation are well developed on this
medium. Because of its low pH this medium is very sensitive to Glucose 20.0
overheating which will soften the agar and caramelise the
carbohydrate.
Method for reconstitution
Weigh 30 grams of powder, disperse in 1 litre of deionised water,
Formula g/litre allow to soak for 10 minutes, swirl to mix. Heat to dissolve, then
Balanced Peptone No. 1 10.0 dispense into final containers before sterilising at 121˚C for 15
minutes.
Dextrose 40.0
Appearance: Pale straw clear
Agar No. 2 12.0 pH: 5.7 ± 0.2

Method for reconstitution Minimum Q.C. organisms: Candida sp. NCIMB 50010
Weigh 62 grams of powder, disperse in 1 litre of deionised water. E. coli (inhibition) NCIMB 50034
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving for 15 minutes at 121˚C. NOTE: The gel strength of the Storage of Prepared Medium: Capped containers – up to 3 months
medium may diminish if recommended sterilising time or at 15-20˚C in the dark.
temperature is exceeded.
Inoculation: As recommended in the U.S.P.
Cool to 47˚C, mix well then pour plates.
Incubation: 22-25˚C aerobically for 10 days.
Appearance: Buff opalescent gel.
pH: 5.6 ± 0.2 References
The Pharmacopeia of the United States of America. XVII 1965.
Minimum Q.C. organisms: Candida sp. NCIMB 50010
E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark. Capped container – up to 3 months at 15-20˚C in the dark. Sabouraud Maltose Agar
Inoculation method: Surface streaking for single colonies or stab
method. LAB 111
Incubation: Aerobically, yeasts 37˚C for 48 hours; fungi 25-30˚C for Description
up to 3 weeks. This is a modification of Sabouraud Dextrose Agar, substituting
maltose for dextrose, recommended by the American Public Health
Growth Characteristics Association.
colony size shape &
Formula g/litre
organism (mm) surface colour other
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell Balanced Peptone 10.0
C. krusei 1.0-3.0 F.CR.D. Grey- Yeasty smell Maltose 40.0
White
Agar No. 2 12.0
T. rubrum 25 White- Reverse-
fluffy shades of Method for reconstitution
red
Weigh 62 grams of powder, disperse in 1 litre of deionised water,
T. mentagraphytes 25 White- Reverse- allow to soak for 10 minutes, swirl to mix then autoclave at 121˚C for
fluffy yellow- 15 minutes. Do not overheat or the agar gel will be softened and the
orange carbohydrate will be caramelised. This medium may be made
M. canis 25 White- reverse- selective by the addition of 2 ampoules X009 Chloramphenicol
centre- yellow- selective supplement which may be added either before or after
yellow autoclaving. Cool to 47˚C and mix well before pouring plates.
radial Appearance: Cream/yellow, translucent.
E. floccosum 25 White- Reverse- pH: 5.6 ± 0.2
fluffy tan
78 Minimum Q.C. organisms: Candida sp. NCIMB 50010
 E. coli (inhibition) NCIMB 50034
Selenite Broth
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in LAB 44A & LAB 44B
the dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Surface streaking or stab inoculum. Description
A medium for the selective enrichment of salmonellae from faeces,
food and sewage. First described by Leifson in 1936 the medium is a
Incubation: Aerobic; yeasts 37˚C for 48 hours; other fungi 25˚C for peptone lactose broth, moderately buffered, which utilises sodium
up to 3 weeks. biselenite as a selective agent. This medium can be incubated at
various temperatures from 35-43˚C to vary the selectivity.
Growth Characteristics Subcultures should be performed after no more than 24 hours
incubation as there is an increasing loss of selectivity if incubation is
colony size shape & prolonged.
organism (mm) surface colour other
C. albicans 0.5-2.0 CV.E.D. White Yeasty smell Formula g/litre
C. krusii 1.0-3.0 F.CR.D. Grey- Yeasty smell Selenite Broth Base LAB 44A:
White
Peptone 5.0
T. rubrum 25mm White- Reverse-
fluffy shades of Lactose 4.0
Red
Sodium phosphate 10.0
T.mentagraphytes 25mm White- Reverse-
fluffy yellow- Sodium biselenite LAB 44B:
orange Sodium hydrogen selenite 4.0
M. canis 25mm White- Reverse-
centre yellow- Method for reconstitution
yellow orange Dissolve 4 grams of sodium biselenite in 1 litre of cold deionised
radial
waer. Add 19 grams of Selenite Broth Base and warm to dissolve.
E. floccosum 25mm White- Reverse- Distribute into tubes or bottles and sterilise for 5-10 minutes in a
fluffy tan boiling water bath, or by free steaming. DO NOT AUTOCLAVE.
Appearance: Pale orange/red with slight precipitate (overheating
References will cause excessive precipitate and loss of selectivity).
Sabouraud R. 1910. Les Teignes. Paris. Pagano. J., Levin. J. D. and pH: 7.1 ± 0.2 (complete medium)
Trejo, W. 1957-8 Diagnostic medium for the differentiation of species
of Candida. Antibiotics Annual. 137-143. Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 3 months

at 15-20˚C in the dark.
Salt Meat Broth Tablets Inoculation: Approximately 0.5-1 gram of sample per 10ml tube.
LAB 113Z Incubation: Up to 24 hours aerobically at 35-43˚C.
Description Subculture: Onto two or more selective agars.
An enrichment medium for the isolation of staphylococci from
heavily contaminated samples. It can also be used for the isolation of References
halophilic micrococci which contaminate hides and raw salt. Leifson, E. 1936. New selenite enrichment media for isolation of
typhoid and paratyphoid (Salmonella) bacilli. Amer. J.Hyg. 24: 423-
Formula g/litre 432.

Beef Extract 10.0 MacFaddin, J.F. 1985. Media for the isolation, cultivation,
identification of Medical Bacteria Vol 1. Williams and Wilkins,
Meat Peptone 5.0 Baltimore.
Tryptone 5.0
Cooked Meat Granules 30.0 WARNING: SODIUM BISELENITE
Sodium Chloride 100.0 Toxic by inhalation and ingestion.
Danger of cumulative effects. Causes
Method for reconstitution severe burns. After contact with skin
Add 2 tablets to 10ml of deionised water in a narrow container. Allow wash with water immediately. If you
to soak for 15 minutes then sterilise by autoclaving at 121˚C for 15 feel unwell seek medical advice.
minutes.
Appearance: Straw broth over meat particles
pH: 7.6 ± 0.2

Minimum Q.C. organisms: S. aureus NCIMB 50080

E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Capped containers – up to 3 months

at 15-20˚C in the dark.
Inoculation: Up to 1g of sample to 10ml of medium.
Incubation: 37˚C aerobically. Subculture at 24 and 48 hours.
Subculture: On to Baird Parker Medium LAB 85, Mannitol Salt
Agar LAB 7 or 4S agar LAB 84.

79
 Selenite Cystine Broth
LAB 55A & 44B
Description
This formulation is a result of the investigation of North and Bartram
in 1953. They examined the effect of varying concentrations of
cystine and phosphate on the recovery of salmonellae in egg products
using selenite broth. It was found that the addition of 10
micrograms/ml of cystine to Leifson’s selenite broth enhanced
recovery of salmonellae.
Sensitivity Test Agar
(S.T.A.)
LAB 12
Description
A medium formulated for antibiotic susceptibility testing by the Joan
Stokes technique. S.T.A. is inhibitor-free, very rich and includes
various nucleotides to enable fastidious organisms to be tested. It is
necessary to add lysed or ‘chocolated’ blood for some organisms.

Formula g/litre Formula g/litre

Selenite Cystine Broth Base LAB 55A Peptone-Infusion Solids 21.5

Balanced Peptone No. 1 5.0 Starch 0.6

Lactose 4.0 Sodium chloride 5.0

Sodium phosphate 10.0 Disodium citrate 1.0

L-Cystine 0.01 Adenine sulphate 0.01

Sodium biselenite LAB 44B Guanine hydrochloride 0.01

Sodium hydrogen selenite 4.0 Uracil 0.01

Method for reconstitution
Dissolve 4 grams of Sodium biselenite (LAB 44B) in 1 litre of
deionised water. Add 19 grams of Selenite Cystine Broth Base and
heat to dissolve. Distribute into tubes or bottles, and sterilise for 10
minutes in a boiling water bath, or steamer. DO NOT AUTOCLAVE
THIS MEDIUM.
Appearance: Pale straw colour, clear with slight precipitate. (A brick
red precipitate indicates overheating).
pH: 7.0 ± 0.2
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: Add sample to broth in the ratio of 1:10. Use a pre-
enrichment broth if damaged organisms are to be recovered.
Incubation: 37˚C for 24-48 hours aerobically. Subculture onto
Salmonella selective media.
References
International Organisation for Standardization. Microbiology 1981.
General guidance on methods for the detection of Salmonella.
ISO, 6579-1981.
International Organization for Standardization. Milk and milk
products 1985. Detection of Salmonella. ISO 6785-2985 (E). ICMSF,
1978. Micro-organisms in foods. 1. Their significance and methods
of enumeration, 2nd edn., University of Toronto Press, Toronto, Ont.
Leifson, E., 1936. New selenite enrichment media for the isolation of
typhoid and paratyphoid (Salmonella) bacilli. Am, J. Hyg. 24, 423-
432.
North, W. R. and M. T. Bartram, 1953. The efficiency of selenite
broth of different compositions in the isolation of Salmonella. Appl.
Microbiol. 1, 130, 134.
Speck, M. L. 1984. Compendium of methods for the microbiological
examination of foods, 2nd edn., American Public Health Association.
Xanthine 0.01
Aneurine hydrochloride 0.01
Uridine 0.1
Agar No. 2 12.0
Method for reconstitution
Weigh 40 grams of powder, disperse in 1 litre of deionised water,
allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving for 15 minutes at 121˚C. To prepare blood agar cool to
45˚C and add 7% lysed blood or 6% defibrinated blood according to
preference. Mix well then pour plates.
Appearance: Dependent upon the blood additive.
pH: 7.4 ± 0.2
Minimum Q.C. organisms: S. aureus NCTC 6571
E. coli NCTC 10418
(antibiotic sensitivity zones)
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Inoculation method: Surface, according to technique.
Incubation: 37˚C, atmosphere to suit organisms metabollic
requirements.
Interpretation: There are no defined zone sizes as in Mueller
Hinton, but all antibiotics should give adequate zone sizes when
compared to controls using standard organisms, e.g. S. aureus NCTC
6571, E. coli NCTC 10418, Ps. aeruginosa NCTC 10662.
References
Stokes, E. J. (1968). Clinical Bacteriology 3rd edn. Arnold, London.
Committee of the A.C.P. 1965. Report on the Antibiotic Sensitivity
test trial organised by the bacteriology committee of the Association
of Clinical Pathologists. J.Clin. Pathol., 18: 1-5.
Hanus, F. J. Sands, J. G. and Bennett, E. O. 1967. Antibiotic activity
in the presence of agar. Appl. Microbiol., 15: 31-34.
Bechtle, R. M. and Scherr. G. H. 1958. A new agar for in vitro
antimicrobial sensitivity testing. Antibiot. Chemother., 8: 599-606.

WARNING: SODIUM BISELENITE

Toxic by inhalation and ingestion.
Danger of cumulative effects. Causes
severe burns. After contact with skin
wash with water immediately. If you
feel unwell seek medical advice.

80
Simmons Citrate Agar Single Step Staphyloccocus Selective
LAB 69 Agar (4-S medium)
Description LAB 84
A medium devised by Simmons in 1926 to help in the differentiation
of enteric bacteria and in the isolation of fungi. Certain Description
Enterobacteriacae have the ability to utilize citrate as the sole source This staphylococcal isolation and identification medium is based on
of carbon and utilize inorganic ammonium salts as the sole source of the ability of S. aureus to grow and produce lecithinase in the
nitrogen resulting in an increase in alkalinity. Bromothymol Blue is presence of a high sodium chloride concentration and potassium
used as a pH indicator. tellurite at 42˚C. The high temperature inhibits the production of
lipase (which causes clearing of egg protein) allowing the effect of
Formula g/litre lecithinase (which causes opacity of egg protein) to be more easily
detected. For the recovery of heat stressed staphylococci a 3 hour
Magnesium sulphate 0.2 preincubation in Brain Heart Infusion Broth LAB 49 at 37˚C is
Ammonium dihydrogen phosphate 1.0 recommended.

Dipotassium phosphate 1.0 Formula g/litre

Sodium citrate 2.0 Tryptone 4.0
Sodium chloride 5.0 Yeast Extract 3.0
Bromothymol blue 0.08 Dextrose 10.0
Agar No. 2 15.0 Sodium chloride 50.0

Method for reconstitution Agar No. 2 13.0

Weigh 24 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then heat to dissolve the
agar and solids. Dispense into tubes or bottles then sterilise by
autoclaving at 121˚C for 15 minutes. Allow to set as slopes.
Appearance: Green, opalescent.
pH: 6.9 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
C. freundii
Method for reconstitution
Weigh 80 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
autoclaving at 121˚C for 15 minutes. Cool to 48˚C and add
aseptically 30mls X073 egg yolk emulsion and 1ml X027 3.5%
potassium tellurite solution. Mix well then pour plates.
Appearance: Pale cream, opaque.
pH: 6.7 ± 0.2

Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: Streak on surface and stab into the butt.
Incubation: 37˚C aerobically for 24-48 hours, with loose caps to
allow gaseous exchange.
Interpretation: Utilisation of citrate and ammonium salts results in
growth and a change in colour of the medium from green to blue.
Minimum Q.C. organisms: S. aureus NCIMB 50080
S. epidermidis NCIMB 50082
Storage of Prepared Medium: Capped container – up to 3 months
at 15-20˚C in the dark.
Inoculation: Surface spreading or streak out to single colonies.
Incubation: 42˚C aerobically for 48 hours.

Growth Characteristics Growth Characteristics

organism growth colour of medium colony size shape &
organism (mm) surface colour other
(most) Salmonella spp yes blue
S. aureus 0.5-1.0 CV.E.G. Grey (Opaque zone of
S. typhi no green ppt around colony)
S. cholerasuis no green S. epidermidis 0.5 CV.E.G. White/ (No ppt zone)
S. gallinarum no green grey
S. paratyphi A no green S. faecalis P.P. CV.E.G. Grey (Usually no
growth)
Serratia spp. yes blue
Most other organisms are inhibited by this medium at 42˚C.
Shigella spp. no green
Yersinia spp. no green References
Klebsiella spp. yes blue Mintzer-Morgenstern, L. and Katzenelson, E. 1982. A Simple
Proteus mirabilis yes blue Medium for Isolation of Coagulase-Positive Staphylococci in a
Single Step. J. Food Protect. 45:3:218-22.
P. vulgaris yes blue
Providencia rettgeri no green
P. providenciae no green
E. coli no green
C. freundii yes blue

References
Simmons, J. S. 1926. A Culture medium for differentiating organisms
of typhoid – colon aerogenes groups and for isolation of certain fungi.
J.Inf. Dis. 39: 209-215.
Koser, S. A. 1923. Utilisation of the salts of organic Acids by the
Colon-aerogenes group. J. Bact. 8: 493-520. MacFaddin, J. F. 1983.
Biochemical Tests for Identification of Medical Bacteria. Williams
and Wilkins.
81
 Slanetz and Bartley Medium Sorbitol MacConkey Agar
(Membrane Enterococcus Agar) (SMAC, CT-SMAC)
LAB166 LAB 161
Description Description
This medium was originally described by Slanetz and Bartley for the This is a selective differential medium for the isolation of
enumeration of enterococci from water samples using a membrane Escherichia coli 0157:H7, the primary serovar associated with
filtration technique, but it may also be used as a spread plate for the haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS).
examination of other sample types. Enterococci reduce tetrazolium Pathogenicity of the organism is linked to the production of
chloride to the insoluble red dye formazan, producing colonies which verocytotoxins (VT1 and VT2), but it should be noted that not all
are dark red or maroon on the surface of the membrane or agar. This strains of 0157:H7 produce verocytotoxins, and that strains from
reaction is not exclusive to enterococci, and the count at this stage other serovars can be toxin producers (e.g. 026, 0111, 0113, 0145).
should be considered presumptive. Colonies may be confirmed as 0157:H7 has been associated epidemiologically with food poisoning
enterococci by demonstrating aesculin hydrolysis using Kanamycin outbreaks involving beefburgers and cold cooked meats 3, 4, 5, 6.
Aesculin Azide Agar LAB106 The medium is a modification of MacConkey Agar No. 3 with the
substitution of the fermentable carbohydrate from lactose to sorbitol.
Formula g/litre 0157:H7 is sorbitol negative and produces translucent colonies
whereas most other E.coli strains are sorbitol positive and so produce
Tryptone 20.0 pink/red colonies. Selectivity of the medium can be increased by
adding Cefixime-Tellurite (C.T.) supplement X161.
Yeast Extract 5.0
Glucose 2.0 Formula g/litre

Dipotassium hydrogen phosphate 4.0 Peptone 20.0

Sodium azide 0.4 Sorbitol 10.0

2,3,4 Tetrazolium chloride 0.1 Bile salts no.3 1.5

Agar 12.0 Sodium chloride 5.0

Neutral red 0.03
Method for reconstitution Crystal violet 0.001
Weigh 43.5 grams of powder and mix with 1 litre of deionised water.
Bring to the boil with frequent stirring to dissolve completely. Cool Agar 12.0
to 47˚C and pour into sterile petri dishes. DO NOT AUTOCLAVE,
OVERHEAT, OR LEAVE FOR GREATER THAN 4hr AT 47˚C. Method for reconstitution
Appearance: Rose coloured gel. Weigh 48.5 grams of powder and add to 1 litre of de-ionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise by
pH: 7.2 ± 0.2 autoclaving at 121˚C for 15 minutes. Cool to 47˚C, add 2 vials of CT
supplement X161, and pour plates. Dry the surface prior to
Mininmum QC organisms: Enterococcus faecalis inoculation.
NCIMB 50030
Appearance: Pale red, slight violet tinge.
Escherichia coli NCIMB
50034. pH: 7.1 + 0.2

Storage: Plates – upto 7 days at 2-8˚C. Storage in bottles is not Minimum QC organisms: E.coli 0157:H7 (non-toxigenic)
recommended as re-melting the medium will cause damage. NCTC 12900 (translucent)
E.coli NCIMB 50034 (Pink/red)
Ent. faecalis NCIMB 50030
Inoculation (inhibition)
Water: Filter 100ml of the water through a suitable membrane, and
place this on the surface of a properly dried Slanetz and Bartley plate. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
Other samples: Dilute as necessary and spread 0.5ml over the surface the dark
of the plate using a spreader, and allow to soak into the agar. Inoculation: Surface streak for single colonies

Incubation Incubation: 37˚C aerobically for 18-24 hr.

Water: at 37˚C for 48hr if testing potable waters or processed foods.
At 37˚C for 4hr then 44˚C for 44hr if testing untreated waters or raw Growth Characteristics:
materials. Organism Size (mm) Shape Colour
E.coli 0157:H7 2.5 – 4.0 CV.E.G Translucent
Interpretation
Other E.coli 2.5 – 4.0 CV.E.G Pink/red
Count all red and maroon colonies as presumptive enterococci.
Confirmation of isolates can be achieved by demonstration of a Sorbitol +ve
positive aesculin reaction on KAAA LAB106. organisms 2.5 – 5.0 Any Pink/red

Reference:
Slanetz L.W., and Bartley C.H. (1957) J.Bact. 74 591-595
Report on Public health and Medical Subjects No.71 Methods for the
Examination of Waters and Associated Materials. The Microbiology
of Water 1994, Part 1 – Drinking Water. HMSO 1SBN 011 753010 7.
82
References
Law D., Ganguli L.A., Donohue-Rolfe A., Acheson D.W.K. (1992)
J.Med.Micro. 36 198-202.
Hitchins A.D., Hartman P.A., and Todd E.C.D. (1992) in
“Compendium of methods for the microbiological examination of
foods” Ch.24. Published by American Public Health Association.
Varnam A.H., Evans M.G., (1991) Foodborne Pathogens an
Illustrated Text. Published by Wolfe Publishing Ltd.
Riley L.W. (1991) Ann.Rev.Micro. 41 383-408
Riley L.W. et al (1983) New Eng.J.Med 308 681-685
Salmon R.L., Farrel I.D., Hutchinson J.G.P. (1989) Epid.Inf. 103
249-254
S.S. Agar References
Isenberg, H. D., Kominos, S., and Siegel, M. 1969. Isolation of
(Salmonella Shigella Agar) salmonellae and shigellae from an artificial mixture of fecal bacteria.
Appl. Microbiol., 18: 4, 656-659.
LAB 52 Leifson, E. 1935. New culture media based on sodium desoxycholate
for the isolation of intestinal pathogens and for the enumeration of
Description colon bacilli in milk and water. J. Pathol. Bacteriol., 40: 581-589.
This medium is a modification of Leifson’s DCA Medium first
described in 1941 by Mayfield and Goeber shortly before Hynes Taylor, W. I., Harris, B. 1965 Isolation of shigellae. II. Comparison of
described his modification of DCA. The selectivity of the medium plating media and enrichment broths. Am. J. Clin. Pathol. 44: 4, 476-
was increased by the addition of extra bile salts, sodium citrate and 479.
the addition of brilliant green dye. There is also the extra thiosulphate
giving good H2S production which reduces the ferrous ammonium
sulphide giving black centred colonies with H2S positive organisms.
The selectivity of this medium can be such that it was suggested by
Taylor et al in 1965 to be unsuitable for the isolation of Shigella
Sugar Free Agar
species. Greater understanding of the selection mechanisms involved LAB 87
enable us to adjust the reaction and allow the more delicate Shigella
to grow without unduly impairing the medium’s selective properties. Description
A formula described by the International Dairy Federation for the
Formula g/litre enumeration of psychrotrophic and mesophilic Gram negative rods in
butter and other dairy products. The Gram negative rods are able to
Beef Extract 5.0 deaminate proteins as a carbon source, whilst some enterococci are
Balanced Peptone No. 1 5.0 inhibited by this formula. The medium conforms to the formulation
of the International Dairy Federation (I.D.F.).
Lactose 10.0
Bile Salts No. 3 8.5 Formula g/litre

Sodium citrate 8.5 Gelatin Peptone 7.5

Sodium thiosulphate 8.5 Tryptone 7.5

Ferric citrate 1.0 Sodium chloride 5.0

Brilliant Green 0.00033 Agar No. 1 14.0

Neutral Red 0.025 Method for reconstitution

Agar No. 2 13.5 Weigh 34 grams of powder and disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, boil to dissolve and disperse into tubes
Method for reconstitution or flasks. Sterilise by autoclaving at 121˚C for 15 minutes.
Weigh 60 grams of powder, disperse in 1 litre of deionised water, and Appearance: Light straw, clear.
allow to soak for 10 minutes. Swirl to mix, then bring to the boil, and pH: 7.6 ± 0.2
allow to cool to 47˚C. Mix well then pour plates. Dry the surface
before incubation. DO NOT AUTOCLAVE THIS MEDIUM. Minimum Q.C. organisms: E. coli NCIMB 50034
Appearance: Pale Pink, clear.
Storage of Prepared Medium: Capped container – up to 3 months
pH: 7.0 ± 0.2
at 15-20˚C in the dark.
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076 Inoculation: 0.2ml of butter fat in a pour plate technique.
E. coli (inhibition) NCIMB 50034
Incubation: 30˚C for 2 days then 20˚C for a further two days –
aerobically.
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in
the dark. Interpretation: Count colonies.
Inoculation method: Surface plating, streaking for single colonies.
References
Incubation: 37˚C aerobically for 18-24 hours. International Dairy Federation (1964). International standard count
of contaminating organisms in butter. International Standard
Growth Characteristics FIL-IDF30.
colony size shape & Ritter, P. and Eschmann, K. H. 1966. Alimenta 5(2), 43-45. Thomas,
organism (mm) surface colour other S. B. 1969. J. Appl. Bact. 32, 269-296.
E. coli 0.1-2.0 CV.E.D. Red Red ppt around Mossel, D. A. A., Krol, B. and Moerman, P.C. 1972. Alimenta 11(2),
colonies 51-60.
(No growth)
K. aerogenes 0.1-3.0 CV.E.G. Pink-Red (No growth)
Proteus spp. 1.0-2.0 CV.E.G. Yellow (grey centre)
(fishy odour)
Ps. aeruginosa 0.1-1.0 CV.CR.D. Pink (Green pigment)
Yellow
Salmonella spp. 2.0-3.0 CV.E.G. Yellow (black centre)
Shigella spp. 0.5-2.0 CV.E.G. Pink-
Yellow
Gram positive organisms – no growth.

83
 T.C.B.S. Cholera Medium Tetrathionate Broth Base A.P.H.A.
(Thiosulphate Citrate Bile Salts Sucrose Agar) LAB 97
LAB 96 Description
A selective enrichment broth for the growth of Salmonella typhi and
Description other Salmonella spp. from faeces, foods etc. It conforms to the
T.C.B.S. is designed for the selective isolation of Vibrio species, formulation recommended by the American Public Health
particularly V. cholerae. The formulation was developed by Association for use in the examination of dairy products and foods
Kobayashi, Enomoto, Sakazaki and Kuwahara and inhibits most of for salmonellae. Organisms which reduce tetrathionate, such as
the Enterobacteriaceae for at least 24 hours. Therefore heavy salmonellae, proliferate in the medium, whilst most enteric organisms
inoculation of the medium is possible. are inhibited. Certain members of the Proteus group will also reduce
tetrathionate thereby impairing the performance of the medium in
Formula g/litre some cases. To overcome this, Novobiocin may be added to the
medium at a level of 40 microgram/ml before addition of the iodine.
Yeast Extract 5.5 Gram-positive organisms are inhibited by the inclusion of bile salts.
Peptone Mix 10.0
Formula g/litre
Sodium thiosulphate 10.0
Balanced Peptone No. 1 5.0
Sodium citrate 10.0
Bile Salts 1.0
Bile salts 9.0
Calcium carbonate 10.0
Sucrose 17.0
Sodium thiosulphate 30.0
Sodium chloride 10.0
Ferric citrate 1.0 Method for reconstitution
Weigh 46 grams of powder and add to 1 litre of deionised water.
Bromothymol blue 0.04
Bring to the boil with frequent swirling to fully dissolve the medium.
Thymol blue 0.04 Cool to 45˚C and add 20ml of iodine solution prepared as indicated
below. Mix well before dispensing into bottles and continue swirling
Agar No. 1 15.0 whilst dispensing to avoid the calcium carbonate sedimenting. For
the best results the medium should be used the same day as prepared.
Method of reconstitution Iodine solution: Dissolve 5g of potassium iodide and 6g of iodine
Weigh 88 grams of powder and add to 1 litre of deionised water. crystals in 20mls of distilled water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil. Cool
to 47˚C and pour into petri dishes. DO NOT AUTOCLAVE OR Appearance: Turbid white.
OVERHEAT THIS MEDIUM. pH: 8.4 ± 0.2
Appearance: Dark green clear agar.
Minimum Q.C. organisms: Salmonella sp. NCIMB 50076
pH: 8.6 ± 0.2 E. coli (inhibition) NCIMB 50034
Minimum Q.C. organisms: V. cholerae (type F) Storage of Prepared Medium: Capped containers – up to 7 days at
E. coli (inhibition) NCIMB 50034 4˚C in the dark (without iodine solution).
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the Inoculation: Add 1 part of sample suspension or inoculated
dark. Capped containers – up to 1 month at 15-20˚C in the dark. pre-enrichment medium to 9 parts of Tetrathionate Broth.
Inoculation: Surface plating with a heavy inoculum, streak out to Incubation: 12-24 hours at 37˚C.
single colonies. Subculture: Onto LAB 34 Brilliant Green Agar and either LAB 32
Incubation: 37˚C aerobically for 18-24 hours. XLD or LAB 110 Hektoen Enteric or other Salmonella selective
media.
Growth Characteristics References
colony size shape & Standard methods for the Examination of Dairy products, 10th
organism (mm) surface colour other Edition. APHA, 1953.
Vibrio. cholerae 2.0-3.0 CV.E.G. Yellow may revert to
green at R.T.
V. parahaemolyticus 3.0-5.0 CV.E.G. Blue or
Green
V. alginolyticus 3.0-5.0 CV.E.G. Yellow
V. metschnikovii 2.0-4.0 CV.E.G. Yellow
V. fluvialis 2.0-3.0 CV.E.G. Yellow
V. vulnificus 2.0-3.0 CV.E.G. Yellow
V. mimicus 2.0-3.0 CV.E.G. Green
Enterococci 1.0 CV.E.G. Yellow
Proteus spp. 1.0 F.CR.G. Green/
Yellow
Ples. shigelloides P.P. Green

References
Kobayashi, T., Enomoto, S., Sakazaki, R. and Kuwahara, S. 1963.
Jap. Bacteriol 18: 10-11, 387-391.
Furniss, A. L., Lee J. V. and Donovan, T. J. 1978. The Vibrios, PHLS
Monograph No. 11.
84
Thioglycollate Medium (Brewer) Todd Hewitt Broth
LAB 64 LAB075
Description Description
This is the original formula introduced by Brewer in 1940 as a clear A nutritious broth medium formulated by Todd and Hewitt for the
medium for the cultivation of anaerobes. It has found applications as production of antigenic streptococcai haemolysin. Todd Hewitt Broth
a sterility test medium and as a blood culture medium although it has is also used to cultivate streptococci prior to serological grouping.
been superseded by Fluid Thioglycollate LAB 25 and Fastidious The use of a fermentable sugar in the formulation leads to the
Anaerobe Broth LAB 71 for these purposes. production of acid which would normally inactivate the haemolysin.
This is prevented by the inclusion of buffers to maintain the pH of the
The agar makes the medium viscous slowing down the permeation of
medium thus preserving the haemolysin, as well as promoting the
oxygen and any convection currents. Sodium thioglycollate acts as a
growth of pneumococci.
reducing agent and also neutralises the bacteriostatic properties of
mercurial compounds. Methylene blue is a redox indicator which is
colourless at low Eh but turns blue on exposure to oxygen. Formula g/litre
Infusion from fat-free minced meat 10.0
Formula g/litre
Tryptone 20.0
Beef Extract 1.0
Dextrose 2.0
Yeast Extract 2.0
Sodium bicarbonate 2.0
Balanced Peptone No. 1 5.0
Sodium chloride 2.0
Dextrose 5.0
Disodium phosphate anhydrous 0.4
Sodium chloride 5.0
Sodium thioglycollate 1.1 Method for Reconstitution
Weight 36.4grams of powder and disperse in 1 litre of deionised
Methylene blue 0.002 water. Allow to soak for 10 minutes, swirl to mix and warm to
Agar No. 1 1.0 dissolve. Dispense into 10ml volumes in screw capped containers and
sterilise by autoclaving at 121˚C for 15 minutes.
Method for reconstitution Appearance: Pale straw, clear broth
Weigh 20 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes then bring to the boil with gentle pH: 7.8 ± 0.2
agitation to dissolve the solids. Distribute into screw top containers
leaving minimal headspace. Sterilise by autoclaving at 121˚C for 15 Inoculation: Pick a well isolated colony for subculture into Todd
minutes. Tighten caps as soon as possible after autoclaving. Hewitt Broth.
Appearance: Straw coloured, translucent, viscous liquid which may Incubation: 37˚C for 18-48hrs, aerobically.
have a blue surface due to contact with oxygen. If the medium has a Storage: Capped containers - up to 3 months at 15-20oC in the dark.
diffuse blue tinge it should not be used until the oxygen has been
driven off by holding in a boiling water bath for 5 minutes. Do not Minimum Q.C. Organisms Streptococcus pyogenes
reheat more than once. ATCC 19615.
pH: 7.2 ± 0.2

Minimum Q.C. organisms: C. perfringens NCIMB 50027 Reference:

Todd E.W., and Hewitt L.F., (1932) A New Culture Medium for the
Storage of Prepared Medium: Capped container – up to 3 months Production of Antigenic Streptococcal Haemolysin. J. Path. Bact. 35
at 15-20˚C in the dark. (1) 973-974.

Inoculation: Ensure adequate dispersal of the inoculum in the broth. Updyke E., L., and Nickle M.I. (1954) A Dehydrated Medium for the
Preparation of Type Specific Extracts of Group A Streptococci. Appl.
Incubation: 37˚C for 24-72 hours. Microbiol. 2 117-118.
Growth Indicators: A diffuse turbidity or individual colonies.

References
Brewer, J. H. 1940. Clear liquid mediums for the culture of
anaerobes. J. Amer. Med. Ass. 115: 598-600.

85
 Triple Sugar Iron Agar Interpretation
Slant/butt Colour Utilisation
LAB 53 Alkaline/acid Red/yellow Glucose only fermented
Peptones utilised
Description
This is a modification of the Krumwiede and Kohn medium of 1917 Acid/acid Yellow/yellow Glucose fermented Lactose
which differentiates some of the Enterobacteriaceae on the basis of + or sucrose fermented
four reactions; fermentation of lactose, glucose and sucrose and H2S Alkaline/alkaline Red/Red Neither glucose, lactose,
production. This medium should be used in conjunction with a urease nor sucrose fermented
test to eliminate Proteus spp. when screening for Salmonella spp. Peptones utilised

Formula g/litre Organism Butt Slant Sulphide

production
Beef Extract 3.0
Shigella dysenteriae NC
Yeast Extract 3.0 S. sonnei Acid or -
Balanced Peptone No. 1 20.0 S. flexneri Alk.
Sodium chloride 5.0 Salmonella typhi Acid NC +
Lactose 10.0 S. paratyphi Acid NC -
S. cholerasuis Gas
Sucrose 10.0
S. typhimurium Acid
Glucose 1.0 S. enteritidis Gas NC +
S. pullorum
Ferric citrate 0.3
S. gallinarum Acid NC +
Sodium thiosulphate 0.3
E. coli
Phenol red 0.025 Enterobacter Acid Acid -
aerogenes Gas
Agar No. 2 12.0 E. cloacae
Proteus mirabilis Acid Acid +
Method for reconstitution Gas
Weigh 65 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to the boil with Providencia rettgeri Acid NC -
frequent swirling to dissolve the solids. Distribute into tubes and NC = No Change
sterilise at 121˚C for 15 minutes. Allow to set as a slope ensuring that
the slant is over a butt approximately 3cm deep.
References
Appearance: Reddish-orange gel.
American Public Health Association 1963. Diagnostic Procedures
pH: 7.4 ± 0.2 and Reagents, 4th edn., A.P.H.A., New York.
American Public Health Association 1966. Recommended Methods
Minimum Q.C. organisms: E. coli NCIMB 50034
for the Microbiological Examination of Foods. 2nd edn., A.P.H.A.,
Ps. aeruginosa NCIMB 50067 New York.
Storage of Prepared Medium: Capped containers – up to 3 months Edwards, P. R. and Ewing, W. H. 1962. Identification of
at 15-20˚C in the dark. Enterobacteriaceae. Burgess Publishing Co., Minneapolis.
Inoculation: A heavy inoculum is streaked over the surface of the
slope and stabbed into the butt.
Incubation: 37˚C aerobically for 24 hours.

86
Tryptone Bile Agar Tryptone Bile Glucuronide Agar
LAB 72 LAB 162
Description Description
First introduced by Delaney, McCarthy and Grasso in 1962 as a A medium developed for the simple enumeration of E.coli without the
method for detecting faecal coliforms in water supplies based on the need for membranes, or pre-incubation on Minerals Modified
production of indole on a bile medium at 44˚C. The idea was applied Glutamate Medium. Based upon the formulation of Tryptone Bile
to foodstuffs by Anderson and Baird-Parker in 1975. The inoculum is Agar, LAB 072, the medium has been modified by the addition of a
placed onto the membrane on a resuscitation agar and incubated at chromogenic substrate to detect the B-glucuronidase enzyme, which
37˚C for 4 hours. The membrane is then transferred to a Tryptone is highly specific for E.coli*, and is detected by the MUG reagent in
Bile Agar plate and incubated at 44˚C: after incubation the membrane other formulations. The advantage of the chromogenic substrate is that
is flooded with indole reagent. Indole positive colonies produce a red it requires no UV lamp to visualise the reaction, and it is concentrated
colour on the membrane and are easily counted. within the colony, facilitating easier enumeration in the presence of
other organisms, or when large numbers are present on the plate.
Formula g/litre
Formula g/litre
Tryptone 20.0
Tryptone 20.0
Bile Salts No. 3 1.5
Bile Salts No.3 1.5
Agar No. 2 15.0
X-glucuronide 0.075
Method for reconstitution Agar 15.0
Weigh 36.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by
Method for reconstitution
autoclaving at 121˚C for 15 minutes. Cool to 47˚C and pour into petri
dishes. Weigh 36.5 grams of powder, disperse in 1 litre of deionised water
and allow to soak for 10 minutes. Swirl to mix and sterilise at 121˚C
Appearance: Straw coloured, clear gel. for 15 minutes. Cool to 47˚C and pour into petri dishes. Dry the
pH: 7.2 ± 0.2 surface prior to inoculation.
Appearance: Straw, clear gel.
Minimum Q.C. organisms: E. coli NCIMB 50034
pH: 7.2 + 0.2
Storage of Prepared Medium: Plates – up to 4 days at 2-8˚C in the
dark. Capped container – up to 1 month at 2-8˚C in the dark. Minimum QC organisms: E. coli (blue). NCIMB 50034
Inoculation: 1ml of a 1:10 dilution of homogenised sample onto a Enterobacter sp (cream).
membrane. The recommended membranes are 85mm in diameter NCIMB 50029
with a 0.45 micron pore size manufactured from cellulose esters.
Incubation: 30˚C for 4 hours, followed by 18 hours at 44˚C.
Incubation: 4 hours at 37˚C on Nutrient Agar LAB 8 or Minerals
Modified Glutamate Medium LAB 80A and 80B plus agar – then 18- Interpretation: Count all blue colonies as presumptive E.coli,
24 hours on Tryptone Bile Agar at 44˚C. calculate the cfu/g in the original material. A simple indole test may
be performed by placing 1 drop of Kovac’s reagent onto a colony. If
Indole reagent: 5% p-dimethylaminobenzaldehyde in N-HC1 indole positive a red halo will appear in the medium around the
(Vracko & Sherris 1963). colony. If negative then the halo will be white.
Indole reaction: Pipette 1-2ml of reagent into petri dish lid, remove Storage: Plates – up to 7 days at 2-8˚C in the dark.
membrane with forceps and place on reagent. Allow to stand for 5 Capped container – up to 3 months at 15-20˚C in the dark
minutes for reaction to develop, then dry in sunlight to ‘fix’ the
colour. Count all pink colonies as E. coli.
References
Dibb W.L. and Bottolfsen K.L. (1984) Evaluation of Rosco
Growth characteristics Diagnostic B-glucuronidase Tablets in the Identification of Urinary
colony size shape & indole reaction on Isolates of Escherichia coli. Acta Path. Microbiol. Immunol. Scand.
organism (mm) surface membrane Sect. B.92 261-264.
E. coli 1.0-3.0 CV.E. positive – Hansen W. and Yourassowsky E. (1984) Detection of B-
pink colour glucuronidase in Lactose Fermenting Members of the Family
Enterobacteriaceae and its Presence in Bacterial Urine Cultures. J.
other Clin. Micro. 20 (6) 1177-1179.
Enterobacteriaceae 0.5-2.0 negative – no colour
Robinson B.J. (1984) Evaluation of a Fluorogenic Assay for
Klebsiella spp. no growth Detection of E.coli. App. & Env. Microbiol. 48 (2) 285-288
Pseudomonas spp. no growth Perez J.L., Berrocal C.I., and Berrocal L. (1986) Evaluation of a
Staphylococcus Commercial B-glucuronidase Test for the Rapid and Economical
spp. no growth Identification of Escherichia coli. J. App. Bacteriol. 61 541-545.
Bacillus spp. no growth Raghubeer E. and Matches J.R. (1990) Temperature Range for
Growth of Escherichia coli Serotype 0157:H7 and Selected
Coliforms in E.coli Medium. J. Clin. Micro. 28 (4) 803-805.
References
Anderson, J. M., Baird-Parker, A. C. 1975. A rapid and direct method Bolton F.J. (1995) Personal Communication.
for enumerating Eschericia coli biotype I in food. J. Appl. Bact. 39: *96-97% of E.coli strains positive. A notable exception is E.coli 0157 H7.
111-117.
Delaney, J. E., McCarthy, J. A. & Grasso, R. J. 1962. Measurement
of E. coli type I by the membrane filter technique Wat. Sewage Wks.
109, 289.
Baird, R. M., Corry, J. E. L., Curtis, G. D. U. 1988. Pharmacopoeia
of culture media for food microbiology. Int. J. Food Microbiol. 276-
277.

87
 Tryptone Glucose Extract Agar Tryptone Soy Agar (U.S.P.)
LAB 63 LAB 11
Description Description
A plate count agar suggested by the American Public Health A general purpose agar which will support the growth of a wide range
Association (A.P.H.A.) for estimation of total viable counts in food of micro organisms. The formula conforms with that laid down by the
and dairy products. This medium is also recommended by the United States Pharmacopeia for sterility testing. The medium can be
Association of Official Analytical Chemists (A.O.A.C.). used for phage typing, colicine typing and for testing the X and V
factor requirements of Haemophilus spp.
Formula g/litre
Formula g/litre
Beef Extract 3.0
Tryptone 15.0
Tryptone 5.0
Soy Peptone 5.0
Glucose 1.0
Sodium chloride 5.0
Agar No. 1 15.0
Agar No. 2 12.0
Method for reconstitution
Weigh 24 grams of powder, disperse in 1 litre of deionised water. Method for reconstitution
Allow to soak for 10 minutes, swirl to mix then boil to dissolve Weigh 37 grams of powder, disperse in 1 litre of deionised water,
before distributing into tubes or bottles. Sterilise at 121˚C for 15 allow to soak for 10 minutes, swirl to mix then sterilise for 15
minutes. minutes at 121˚C. Cool to 47˚C, mix well and then pour plates.
Appearance: Pale straw colour, clear. Appearance: Pale straw coloured, clear gel.
pH: 7.0 ± 0.2 pH: 7.3 ± 0.2

Minimum Q.C. organisms: S. epidermidis NCIMB 50082 Minimum Q.C. organisms: E. coli NCIMB 50034
E. coli NCIMB 50034 S. epidermidis NCIMB 50082

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark. Capped containers – up to 3 months at 15-20˚C in the dark. dark. Capped containers – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique. Inoculation: Surface plating.
Incubation: 30˚C aerobically for 48 hours for aerobic mesophile Incubation: Time and temperature to suit organisms, usually
count. 6˚C aerobically for 10 days for aerobic psychrotroph count. aerobic.
55˚C aerobically for 48 hours for aerobic thermophile count.
Growth Characteristics
References
Association of Official Analytical Chemists (AOAC). 1978. colony size shape &
Bacteriological Analytical Manual, 5th ed.: Association of Official organism (mm) surface colour other
Analytical Chemists. S. aureus 1.0-1.5 CV.E.G. White-
Hausler, W. J. (Ed.) 1976. Standard Methods for the Examination of Yellow
Dairy Products, 14th edn.: American Public Health Association. Other
Speck, M. J. (Ed.) 1976. Compendium of Methods for the staphylococci 1.0-1.5 CV.E.G. White- strain-dependent
Microbiological Examination of Foods.: American Public Health Yellow
Association. Strep. pyogenes PP-0.5 CV.E.G. Transp.
S. pneumoniae PP CV.E.G. Transp. (CO2)
Ent. faecalis 0.5-1.0 CV.E.G. Grey-
White
Klebsiella spp. 2.0-3.0 CV.E.G. White mucoid
Ps. aeruginosa 0.5-3.0 F.CR.D. Grey- (marked strain
Green variation)

References
United States Pharmacopeia 21st edition. 1985.
Blair, J. E. and Carr, M. 1953. The bacteriophage typing of
staphylococci. J. Infect. Dis. 93: 1-13.
Examination of Dairy Products. A.P.H.A., New York.

88
Tryptone Soy Broth U.S.P. Tryptone Water
(Soybean Casein Digest Medium U.S.P.) LAB 129
LAB 4 Description
A substrate for the testing of an organism’s ability to produce indole
Description from tryptophane. The indole test is frequently used in the
A general purpose nutritious broth capable of growing a wide range classification of coliform organisms. This product is preferable to
of bacteria and fungi. The medium is recommended by the United peptone water LAB 104 because it has a higher content of tryptophane.
States Pharmacopeia for the sterility testing of a wide range of
pharmaceutical products. The medium is also widely used for blood
Formula g/litre
cultures although the high carbohydrate level may cause rapid growth
and subsequent death of acid-producing organisms. Tryptone 10.0
Sodium chloride 5.0
Formula g/litre
Tryptone (Casein Digest USP) 17.0 Method for reconstitution
Soy Peptone 3.0 Weigh 15 grams of powder, disperse in 1 litre of deionised water.
Heat to dissolve then distribute into screw cap bottles. Sterilise by
Sodium chloride 5.0 autoclaving at 121˚C for 15 minutes.
Dipotassium phosphate 2.5 Appearance: Colourless, clear.
Dextrose 2.5 pH: 7.5 ± 0.2

Minimum Q.C. organisms: E. coli NCIMB 50034

Method for reconstitution
Weigh 30 grams of powder, disperse in 1 litre of deionised water.
Swirl to mix and warm if necessary to dissolve. Dispense into tubes
or flasks and sterilise at 121˚C for 15 minutes. Do not exceed
temperature.
Appearance: Straw coloured, clear.
pH: 7.3 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
S. aureus NCIMB 50080
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Incubation: 20-25˚C aerobically for 14 days, for sterility tests. 37˚C
aerobically for 14 days for blood cultures.
Growth indicators: Turbidity or precipitate.
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark.
Inoculation: From pure culture.
Incubation: 37˚C for 24-48 hours.
Interpretation: Indole positive organisms will give a distinct colour
change when either Kovac’s or Ehrlich’s indole reagent is added.
References
American Public Health Association. 1955. American Water Works
Association 10th edn. 391-392.
MacFaddin, J. 1983. Biochemical tests for the identification of
medical bacteria. 2nd edn. Williams & Wilkins, Baltimore.

References
United States Pharmacopeia 21st edition 1985.
Tryptose Phosphate Broth
LAB 62
Description
This is a versatile nutritionally rich buffered glucose broth. The
medium is a general purpose broth that has been used as a blood
culture medium, a supplement for animal cell culture, and with the
addition of sodium azide 0.025% as a selective medium for
streptococci in dairy products.

Formula g/litre
Tryptose 20.0
Dextrose 2.0
Sodium chloride 5.0
Disodium phosphate 2.5

Method for reconstitution

Weigh 29.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, heat to dissolve solids then distribute
into final containers. Sterilise by autoclaving at 121˚C for 15 minutes.
Appearance: Very pale straw, clear.
pH: 7.3 ± 0.2

89
 Minimum Q.C. organisms: E. coli NCIMB 50034 Growth Characteristics
S. aureus NCIMB 50080 colony size shape &
organism (mm) surface colour other
Storage of Prepared Medium: Capped containers – up to 3 months
at 15-20˚C in the dark. Strep. mutans 1.0-3.0 ‘heaped’ Yellow Crumbles when
Type A colony touched with wire
Inoculation: For blood culture work dilute sample at least 1:10 granular
in broth. surface
Incubation: Dependent on application. irregular
edge
References Type B 1.0-3.0 As Type A Grey white soft
American Public Health Association 1948. Standard Method for the consistency,
Examination of Dairy Products, 10th edn. A.P.H.A., New York. white precip in agar
American Public Health Association, 1950. Diagnostic Procedures (glistening drop)
and Reagents, 3rd edn., A.P.H.A., New York. S. sanguis 1.0-3.0 Convex White very rubbery
glossy (glistening drop)
crenated

T.Y.C. Medium References

de Stoppelaar, J. D. van de Houte, J. and de Moor, C. E. 1967. The
(Tryptone Yeast Cystine) presence of dextran forming bacteria resembling Streptococcus bovis
and Streptococcus sanguis in dental plaque. Arch. Oral Biol. 12:
LAB 35 1199-1201. de Stoppelaar, J. D. 1971. Streptococcus mutans,
Streptococcus sanguis and dental caries. Thesis, Rijksuniversiteit,
Description Utrecht.
A medium designed by J. D. de Stoppelaar in 1967 to differentiate Emilson, C. G., Bratthall, D. 1976. Growth of Streptococcus mutans
Streptococcus sanguis (frequently found in dental plaque) from on various selective media. Journal of Clinical Microbiology 4: 95-
Streptococcus mutans (implicated in dental caries). The medium uses 98.
a high sucrose content to promote the formation of specific glucans
by S. mutans thus forming distinctive colonies. It can be made Gold, O. G., Jordon, H. V., Van Houte, J. 1973. A selective medium
selective by the addition of 0.2 units per ml of Bacitracin. for Streptococcus mutans. Archives of Oral Biology 19: 1357-1364.
Ikeda, T., Sandham, H. J. 1972a. A high-sucrose medium for the
Formula g/litre identification of Streptococcus mutans. Archives of Oral Biol. 4: 781-
783.
Tryptone 15.0
Wade, W. G., Alldred, M. J., Walker, D. M. (1986. J. Med. Microbiol.
Yeast Extract 5.0 22: 319-323. An improved medium for isolation of Streptococcus
L-Cystine 0.2 mutans.

Sodium sulphite 0.1

Sodium chloride 1.0
Disodium phosphate anhydrous 0.8 Urea Agar Base
Sodium bicarbonate 2.0 (Christensen)
Sodium acetate anhydrous 12.0 LAB 130
Sucrose 50.0
Description
Agar No. 2 12.0 This is a modification of Christensen’s urea base for the detection of
rapid urease production by Proteus spp. Other enterobacteria will
Method for reconstitution split the urea, but this will be delayed. This delay is achieved by the
Weigh 98 grams of powder, disperse in 1 litre of deionised water. incorporation of glucose and the introduction of a buffering system
Allow to soak for 10 minutes, swirl to mix, then sterilise for 15 into the medium. The indicator for ammonia production is phenol
minutes at 121˚C. Cool to 47˚C, mix and pour plates. red.
Appearance: White, translucent gel.
Formula g/litre
pH: 7.3 ± 0.2
Peptone 1.0
Minimum Q.C. organisms: S. mutans ATCC 25175 Glucose 1.0
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in Sodium chloride 5.0
the dark.
Disodium phosphate 1.2
Inoculation: Surface, streaking out for single colonies.
Potassium dihydrogen phosphate 0.8
Incubation: 37˚C for 4-5 days in an atmosphere of 90% H2, 10%
CO2. Phenol red 0.012
Agar No. 1 12.0

Method for reconstitution

Weigh 2.1 grams of powder, disperse in 95ml of deionised water.
Allow to soak for 10 minutes, swirl to mix, then sterilise at 121˚C for
15 minutes. Allow to cool to 47˚C, add aseptically 5ml sterile urea
solution X130. Distribute into sterile bottles and slopes, allow to set
in the sloped position.
Appearance: Yellow/pale pink, translucent.
pH: 6.8 ± 0.2

90
 Minimum Q.C. organisms: Proteus spp. Organism Growth Characteristics
E. coli NCIMB 50034 Proteus spp. Red colour 4-6 hours
Corynebacterium hoffmani Red colour 18-24 hours
Storage of Prepared Medium: Capped container – up to 1 month at
2-8˚C in the dark. C. ulcerans Red colour 18-24 hours
Inoculation: Pure culture using straight wire for stab/streak Some strains Citrobacter Red colour 18-24 hours
technique. Klebsiella ,, ,,
Incubation: 37˚C for 4-6 hours or overnight, aerobically. Escherichia ,, ,,
Interpretation: Production of red colour in under 6 hours is positive Yersinia ,, ,,
for rapid urease production.
Staphylococcus ,, ,,
Organism Growth Characteristics Pasteurella multocida Red colour 18-24 hours
Proteus spp. Red colour 4-6 hours
Citrobacter spp. Red colour 18-24 hours
References
Maslen L.G.C. 1952. Routine use of liquid urea medium
Klebsiella spp. Red colour 18-24 hours for identifying Salmonella and Shigella organisms. J. Brit. Med. 2:
Staphylococcus spp. Red colour 24-48 Hours 545-546.
Helicobacter pylori Red colour 30 minutes

References
Christensen, W. B. 1946. Urea decomposition as a means of UVM Base
differentiating Proteus and paracolon cultures from each other and
from Salmonella and Shigella types. J. Bacteriol. 52: 461-466. LAB 155
Description
UVM (University of Vermont Medium) Base is a two stage selective
enrichment broth for the isolation of Listeria from meat products and
Urea Broth Base environmental swabs, and forms the basis of the USDA method. The
original method has been modified to replace the second stage broth
(Christensen) (UVM II) with Fraser broth LAB164 (McClain & Lee 1989)

LAB 131 Formula g/litre

Description Tryptone 5.0

This is a liquid version of Christensen’s medium (LAB 130) Meat Peptone 5.0
introduced by Maslen in 1952. This modification allows inoculation
by Pasteur pipette, and it is easier to detect contamination in a fluid Beef Extract 5.0
rather than in a slope. Maslen also claimed that it is easier to detect Yeast Extract 5.0
positive results.
Sodium chloride 20.0
Formula g/litre Disodium hydrogen phosphate 9.6
Peptone 1.0 Potassium dihydrogen phosphate 1.35
Glucose 1.0 Aesculin 1.0
Disodium phosphate 1.2
pH: 7.4 ± 0.2
Potassium dihydrogen phosphate 0.8
Appearance: Straw opalescent broth
Sodium chloride 5.0
Phenol red 0.004 Method for reconstitution
Weigh 52 grams of powder and add to 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix and sterilise at 121˚C for
Method for reconstitution 15 minutes. Cool to 47˚C and add 2 vials of UVM I supplement
Weigh 0.9 grams of powder, add to 95ml of deionised water. Swirl to (X155) or UVM II supplement (X156) as required. Mix well and
mix then sterilise by autoclaving at 121˚C for 15 minutes. Allow to distribute into sterile tubes or bottles.
cool to 47˚C then add aseptically 5ml of X130 sterile urea solution. Inoculation: UVM I – Add 25g sample to 225ml of UVM I and
Distribute into sterile screw cap bijou bottles. homogenise. UVM II – Subculture 0.1ml of enriched UVM I into
Appearance: Yellow, clear. 10ml UVM II
pH: 6.8 ± 0.2 Incubation: UVM I – 30˚C aerobically for 24 hrs. Subculture onto
selective agars and into UVM II. UVM II – 30˚C for 24 and 48 hrs.
Minimum Q.C. organisms: Proteus sp. Subculture onto selective agars at 24 and 48 hrs. Incubate selective
E. coli NCIMB 50034 agars for 24 and 48 hrs.

Minimum QC organism: Listeria sp. NCIMB 50007

Storage of Prepared Medium: Capped container – up to 1 month at
2-8˚C in the dark.
E.coli (inhibition) NCIMB 50034

Inoculation: Fluid culture by pasteur pipette or straight wire from

pure growth. References
McClain D., and Lee W.H. (1989) FSIS method for isolation of
Incubation: 37˚C for 4-6 hours – preferably in a water bath for most
L.monocytogenes from processed meat and poultry products.
rapid growth, aerobically.
Lab.Comm.No.57, Revised May 24, 1989. US Dept of Agric.FSIS,
Interpretation: The production of a red colour in under six hours is Microbiol. Div.
a positive result for rapid urease.
Warburton D.W. et al (1991) A Canadian comparative study of
modified versions of the FDA and USDA methods for the detection
of L.monocytogenes. J.Food Protection 54 (9) 669-676

91
 Violet Red Bile Agar (V.R.B.A.) Violet Red Bile Glucose Agar
LAB 31 (V.R.B.G.A.)
Description LAB 88
A medium for the enumeration of coliform organisms in food and
dairy products. The selectivity of the medium is due to the presence Description
of bile salts and crystal violet. Lactose fermenters produce red/purple A modification of Violet Red Bile Agar LAB 31 introduced by
colonies often surrounded by a halo of the same colour. Non lactose Mossel in 1978. V.R.B.A. LAB 31 contains lactose which is
fermenters produce pale colonies. Selectivity can be increased by fermented by members of the coli/aerogenes group, this medium
incubation at 42-44˚C. gives a ‘coliform’ count. V.R.B.G.A. LAB 88 has substituted lactose
with glucose. Glucose is fermented by all members of the
Enterobacteriaceae thus V.R.B.G.A. gives a presumptive
Formula g/litre
Enterobacteriaceae count. Bile salts and crystal violet are used to
Yeast Extract 3.0 inhibit Gram positive and non-enteric organisms. The overlay
procedure ensures anaerobic conditions and suppresses the growth of
Balanced Peptone No. 1 7.0 non-fermentative Gram negative bacteria.
Sodium chloride 5.0
Formula g/litre
Bile Salts No. 3 1.5
Yeast Extract 3.0
Lactose 10.0
Balanced Peptone No. 1 7.0
Neutral red 0.03
Sodium chloride 5.0
Crystal violet 0.002
Bile Salts No. 3 1.5
Agar No. 2 12.0
Glucose 10.0
Method for reconstitution Neutral red 0.03
Weigh 38.5 grams of powder, disperse in 1 litre of deionised water.
Dissolve by bringing to the boil with frequent swirling of the flask to Crystal violet 0.002
prevent overheating. Autoclaving is not necessary. Cool to 45˚C and Agar No. 2 12.0
distribute into bottles or tubes. If held molten in a water bath, use
within 3 hr.
Method for reconstitution
Appearance: Light purple-violet, clear gel. Weigh 38.5 grams of powder, disperse in 1 litre of deionised water.
pH: 7.4 ± 0.2 Allow to soak for 10 minutes then swirl to mix. Bring to the boil with
frequent swirling to prevent overheating. Further sterilisation is not
Minimum Q.C. organisms: E. coli NCIMB 50034 required. Cool to 45˚C, mix well and dispense into tubes or bottles.
S. epidermidis (inhibition) If held molten in a water bath, use within 3 hr.
NCIMB 50082 Appearance: Light purple-violet, clear.

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in pH: 7.4 ± 0.2

the dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Minimum Q.C. organisms: E. coli NCIMB 50034
Inoculation: Pour plate (with or without overlay) or surface spread. S. epidermidis (inhibition)
Incubation: 37˚C for 18-24 hours for ‘coliforms’; 4˚C for 10 days NCIMB 50082
for psychrotrophs; 32˚C for 24-48 hours for mesotrophs; 42˚C for 18
hours for thermotrophs. Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
dark. Capped containers – up to 1 month at 15-20˚C in the dark.
Interpretation: Count all red/purple colonies > 0.5mm in diameter.
Calculate the number of coliforms in original sample. Inoculation: Pour plate method with overlay.
Incubation: 37˚C aerobically for 18-24 hours.
References
Interpretation: Count all red/purple colonies > 0.5mm in diameter.
American Public Health Association 1972, Standard Methods for the Calculate the number of Enterobacteriaceae in original sample.
Examination of Dairy Products. 13th edn. (ed. W. H. Hausler),
A.P.H.A., Washington.
References
American Public Health Association 1966. Recommended Methods Pharmacopoeia of Culture Medium for Food Microbiology 1987. Int.
for the Micro biological Examination of Foods. 2nd end. (ed. J. M. J. Food Microbiol. 5: 3: 280-81.
Sharf) A.P.H.A., Washington.
Mossel, D. A. A., Mengerink, W. H. J. and Scholts, H. H. 1962. Use
Davis, J. G. 1951. Milk Testing Dairy Industries, London. of a modified MacConkey agar medium for the selective growth and
Mossel, D. A. A., Eelderink I. and Sutherland, J. P. 1977. enumeration of Enterobacteriacaea. J. Bacteriol. 84: 381.
Development and use of single, ‘polytropic’ diagnostic tubes for the
approximate taxonomic grouping of bacteria, isolated from foods,
water and medicinal preparations. Zbl. Bakt. Hyg. I., Orig., A 278,
66-79.
Mossel, D. A. A., Eelderink, I. Koopmans M. and van Rossem, F.
1979. Influence of carbon source, bile salts and incubation
temperature on the recovery of Enterobacteriaceae from foods using
MacConkey type agars. J. Food Protec. 42, 470-475.
Mossel, D. A. A., van der Zee, H. Hardon, A. P. and van Netten, P.
1986. The enumeration of thermotropic types amongst the
Enterobacteriaceae colonizing perishable foods. J. Appl. Bacteriol.
60, 289-295.

92
W. L. Nutrient Agar Wort Agar
(Wallerstein Laboratory) LAB 38
LAB 79 Description
A medium for the enumeration of yeasts and moulds in butter,
Description developed by Parfitt in 1933. The medium can be modified to enable
This medium was developed by Green and Gray in 1950 for the it to isolate osmophilic yeasts from soft drinks and sugar products by
isolation and enumeration of yeasts, moulds and bacteria in the the addition of high concentrations of sucrose and glucose.
brewing process. The medium has a pH of 5.5 which is optimum for
Brewers yeast and will allow the growth of a wide range of organisms
Formula g/litre
including Enterobacteriaceae, Flavobacterium, Lactobacillus and
Pediococcus spp. as well as yeasts and moulds. If a process involving Malt Extract 15.0
bakers or distillers yeast is under examination the pH should be
adjusted to 6.5. The medium may be adapted to detect bacteria only Peptone 0.78
by the addition of 0.004 g/litre of Actidione to suppress the yeasts.
Maltose 12.75
Formula g/litre Dextrin 2.75
Yeast Extract 4.0 Dipotassium phosphate 1.0
Tryptone 5.0 Ammonium chloride 1.0
Dextrose 50.0 Agar No. 2 15.0
Potassium dihydrogen phosphate 0.55
Method for reconstitution
Potassium chloride 0.425 Weigh 48.3 grams of powder and disperse in 1 litre of deionised
water. Add 2.35mls of glycerol. Allow to soak for 10 minutes, swirl
Calcium chloride 0.125 to mix then sterilise for 15 minutes at 121 C. Use 60 grams per litre
Magnesium sulphate 0.125 if required for inoculation by plate streaking with a wire loop. Do not
exceed time or temperature of sterilisation. If osmophilic
Ferric chloride 0.0025 modification is required add 48.3 grams of powder to 1 litre of a
solution containing 35% w/v sucrose and 10% w/v glucose then
Manganese sulphate 0.0025 sterilise at 108˚C (5 p.s.i.) for 20 minutes.
Bromocresol green 0.022 Appearance: Light Brown, translucent.
Agar No. 2 15.0 pH: 5.0 ± 0.2

Method for reconstitution Minimum Q.C. organisms: S. cerevisiae.

Weigh 75 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then sterilise by Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in the
autoclaving at 121˚C for 15 minutes. If adjustment of pH to 6.5 is dark. Capped container – up to 1 month at 15-20˚C in the dark.
required used 1% sodium bicarbonate. Inoculation: Pour plate or surface spread.
Appearance: Green, clear. Incubation: 25˚C aerobically for 5 days.
pH: 5.5 ± 0.2
Growth Characteristics
Minimum Q.C. organisms: S. cerevisiae.
colony size shape &
Storage of Prepared Medium: Plates – up to 7 days at 4˚C in the organism (mm) surface colour other
dark. Capped container – up to 1 month at 4˚C in the dark. Candida spp. 4.0 CV.E.G White
Inoculation: Surface plating or pour plate. Fungi Varies with
Incubation: 30˚C aerobically for 48 hours (bacteria); 20˚C species
aerobically for 48 hours (yeasts). S. cerevisiae 2.0-3.0 Varies with Cream
Interpretation: Count all colonies. Calculate organisms per ml in strain
original sample.
References
References Parfitt, E. H. 1933. The influence of media upon the yeast and mould
Green, S. R. and Greay, P. P. 1950. Differential Procedure Applicable count of butter. J. Dairy Sci. 16: 141-147.
to Investigation in Brewing. Wallerstein Lab. Comm. 13,357. Scarr, M. P. 1959. Selective media used in the microbiological
Hall, Jean F. 1971. Detection of Wild Yeasts in the Brewery. J. Inst. examination of sugar products. J. Sci. Fd. Agric. 10: 678-681.
Brewing, 77: 513-516.

93
 Wort Broth X.L.D. Agar
LAB 99 (Xylose Lysine Decarboxylase Agar)
Description LAB 32
A broth version of the medium LAB 38 Wort Agar developed by
Parfitt for the enumeration of yeasts and moulds, in butter. The Description
medium can be modified for the isolation of osmophilic yeasts from This medium was introduced by Taylor in 1965 to improve the
soft drinks and sugar products by the addition of high concentrations recovery and recognition of Shigella spp, and has proved to be an
of sucrose and glucose. excellent medium for Salmonella spp. The medium is low in nutrients
and relies on a small amount of sodium desoxycholate for selectivity.
The indicator system is novel and complex. Most enteric organisms
Formula g/litre
except Shigella, will ferment xylose to produce acid. However the
Malt Extract 15.0 salmonellae will also decarboxylate the lysine to keep the pH neutral.
At near neutral pH Salmononella can produce H2S from the reduction
Peptone 0.78 of thiosulphate producing black or black centred colonies.
Citrobacter spp. can also decarboxylate lysine, however, the acid
Maltose 12.75
produced by fermentation of both lactose and sucrose will keep the
Dextrin 2.75 pH too acid for H2S to be produced.
Dipotassium phosphate 1.0
Formula g/litre
Ammonium chloride 1.0
Xylose 3.75
Method for reconstitution L-Lysine 5.0
Weigh 33.3 grams of powder and disperse in 1 litre of deionised Lactose 7.5
water, add 2.35mls. of glycerol. Allow to soak for 10 minutes, swirl
to mix then sterilise by autoclaving at 121˚C for 15 minutes. If Sucrose 7.5
osmophilic version is required disperse 33.3 grams of powder in 1
litre of a solution of 35% w/v sucrose and 10% w/v glucose then Sodium chloride 5.0
sterilise at 108˚C (5 p.s.i.) for 20 minutes. Yeast Extract 3.0
Appearance: Light Brown, translucent. Phenol red 0.08
pH: 4.8 ± 0.2
Agar No. 2 13.0
Minimum Q.C. organisms: S. cerevisiae. Sodium desoxycholate 1.0

Storage of Prepared Medium: Capped container – up to 1 month at Sodium thiosulphate 6.8

15-20˚C in the dark. Ferric ammonium citrate 0.8
Incubation: 25˚C aerobically for 5 days.
Method for reconstitution
Weigh 53.5 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix. Bring rapidly to the boil
with frequent stirring, and transfer immediately to a 47˚C water bath.
Pour into plates as soon as the medium has cooled. Protracted boiling
or prolonged holding at elevated temperature induces precipitation.
Appearance: Light rose, clear gel.
pH: 7.4 ± 0.2

Minimum Q.C. organisms: Salmonella sp. NCIMB 50076

E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 37˚C for 18-24 hours aerobically.

Growth Characteristics
colony size shape &
organism (mm) surface colour other
Salmonella spp. 1.0-2.5 CV.E.G. Trans. (clearing of acid
black ppt of coliforms)
centre
Shigella sonnei 1.5-2.5 CV.E.G. Pink
S. flexneri 1.0-2.0 CV.E.G. Pink
S. dysenteriae 0.5-1.5 CV.E.G. Pink
E. coli 0.5-1.5 CV.E.G.(D) Yellow inhibited (ppt
around colony)
Citrobacter spp. 1.0-1.5 CV.E.G.(D) Yellow (black centre)
Proteus spp 1.0-2.5 CV.E.G. Trans. Pink fishy odour
(black
centre)

94
References
Taylor, W. I. 1965. Isolation of shigellae. I. Xylose Lysine Agars:
New media for the isolation of enteric pathogens. Am. J. Clin.
Pathol., 44: 471-475.
Taylor, W. I., and Harris, B. 1965. Isolation of shigellae. II.
Comparison of plating media and enrichment broths. Am. J. Clin.
Pathol., 44(4), 476-479.
Taylor, W. I., and Harris, B. 1967. Isolation of shigellae. III.
Comparison of new and traditional media with stool specimens. Am.
J. Clin. Pathol., 48: 350-355. Taylor, W. I., and Schelhart, D. 1967.
Isolation of shigellae. IV. Comparison of plating media with stools.
Am. J. Clin. Pathol., 48: 356-362.
Yeast Extract Agar
(Yeastrel Milk Agar)
LAB 18
Description
A nutrient agar corresponding to the Standard Formulation for the
plate count of micro-organisms in water and dairy products. This
medium is also useful for teaching and demonstration purposes using
non-fastidious organisms.
Formula g/litre
Yeast Extract 3.0
Balanced Peptone No. 1 5.0
Agar No. 1 15.0
Method for reconstitution
Weigh 23 grams of powder, disperse in 1 litre of deionised water.
Free steam or boil to dissolve. Mix well, and dispense into containers.
Sterilise for 15 minutes at 121˚C.
To prepare Yeastrel Milk Agar add 10mls of fresh milk before
autoclaving.
Appearance: Pale straw, clear gel.
pH: 7.2 ± 0.2
Minimum Q.C. organisms: E. coli NCIMB 50034
S. epidermidis NCIMB 50082
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark. Capped container – up to 3 months at 15-20˚C in the dark.
Inoculation: Pour plate technique or surface spreading.
Incubation: 30˚C aerobically for 48 hours for aerobic mesophil
count. 6˚C aerobically for 10 days for aerobic psychrotroph count.
55˚C aerobically for 48 hours for aerobic thermophil count.
References
Ministry of Health, Public Health Laboratory Service Water
Committee 1969. The Bacteriological Examination of Water
Supplies, 4th Edn. report No. 71. H.M.S.O., London.
British Standard 4285: Methods of Microbiological Examination for
Dairy Purposes.
Yeast Extract Dextrose
Chloramphenicol Agar
LAB 119
Description
A selective medium for the enumeration of yeasts and moulds in milk
and other dairy products. The formulation meets the requirements of
the International Milk Union (1980), the International Organisation
for Standardisation (I.S.O.) and the British Standards Institute
(B.S.I.). The medium is said to have superior storage properties to
O.G.Y.E. and also has the advantage of incorporating an autoclavable
supplement.
Formula g/litre
Yeast Extract 5.0
Dextrose 20.0
Agar No. 1 15.0
Method for reconstitution
Weigh 40 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, swirl to mix then bring to boil.
Add 2 vials of X009 which have been dissolved in ethanol and
autoclave at 121˚C for 10 minutes. Allow to cool to 45˚C before using
with poured plate technique. THIS MEDIUM MUST NOT BE
RE-AUTOCLAVED.
Appearance: Pale yellow, clear.
pH: 6.6 ± 0.2
Minimum Q.C. organisms: Aspergillus sp. NCIMB 50097
S. cerevisiae
E. coli (inhibition) NCIMB 50034
Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in
the dark.
Incubation: 25˚C for 5 days, aerobically.
Inoculation: Pour plate technique.
Interpretation: Count all colonies.
References
Engel, G. 1982. Verglich verschieden Nährböden zum quantitativen
Nachweis von Hefen und Schimmelpilzen in Milch und
Milchprodukten. Milchwiss. 37: 727-730.
International Organisation for Standardization (ISO): Milk and milk
products – enumeration of yeasts and moulds – colony count
technique at 25˚C – standard method ISO/DIS 6611.
International Milchwirtschaftsverband: Milch und Milchprodukten –
Zählung von Hefen und Schimmelpilzen (Kolonieählung bel 25 C). –
International IMV Standard 94: 1980 in Milchwiss. 36: 220-222.
Normenausschufl Lebensmittel und landwirtshaft. Produkte in DIN
Deutsches Institut für Normung e.V. Mikrobiologische
Milchuntersuchung. Bestimmung der Anzahl von Hefen und
Schimmelpilzen. Reference method DIN 10186.
British Standards Institute. B.S. 4285. Section 3.6: 1986.
95
 Yersinia Selective Agar
(Schiemann’s C.I.N. Agar)
LAB 120
Description
This medium is based on the work of Schiemann. It is used for the
isolation and enumeration of Yersinia spp. from clinical samples and
from food. The selective components are sodium desoxycholate,
crystal violet, cefsulodin, irgasan and novobiocin. Yersiniae ferment
mannitol with an intense, localised, acid production in the centre of
the colony which produces a red ‘bull’s eye’ appearance. The ratio of
transparent border to red centre varies with serotype and
environmental strains may appear rough with an irregular edge. Most
other enteric bacteria, if they grow, produce a larger colony with a
diffuse pinkish centre and opaque outer zone.

Formula g/litre
Peptone Mixture 22.5
Mannitol 20.0
Sodium chloride 1.0
Magnesium sulphate 0.01
Sodium pyruvate 2.0
Sodium desoxycholate 0.5
Neutral red 0.03
Crystal violet 0.001
Agar No. 2 12.0

Method for reconstitution

Weigh 58 grams of powder, disperse in 1 litre of deionised water.
Allow to soak for 10 minutes, then bring to the boil for 1 minute only.
DO NOT AUTOCLAVE. Allow to cool to 47˚C add 2 ampoules
C.I.N. supplement X120. Mix well, pour plates.
Appearance: Red, clear.
pH: 7.4 ± 0.2

Minimum Q.C. organisms: Y. enterocolitica

E. coli (inhibition) NCIMB 50034

Storage of Prepared Medium: Plates – up to 7 days at 2-8˚C in

the dark.
Inoculation: Surface, streaking out for single colonies.
Incubation: 30˚C aerobically for 24 hours.

Growth Characteristics
colony size shape &
organism (mm) surface colour other
Y. enterocolitica 1.0-2.5 CV.E.G. Red centre Colony varies
with strain,
may be
rough &
irregular
Citrobacter
spp. 2.5-3.0 CV.E.G. Pale pink (may
not grow)
colony
Gram +ve
organisms no growth

References
Schiemann, D. A. 1979. Synthesis of a selective agar medium for
Yersinia enterocolitica. Can. J. Microbiol. 25: 1298-1304.
Schiemann, D. A. 1982. Development of a two step enrichment
procedure for recovery of Yersinia enterocolitica from food. Appl.
Eniviron. Microbiol. 43: 14-27.
Mossel, D. A. A. 1987. Cefsulodin Irgasan Novobiocin (C.I.N.) agar.
Int. J. Food. Microbiol. 5: 208, 209.

96
 X091, X291
Lyophilised Selective Agents
NALIDIXIC ACID for the isolation of non-sporing anaerobes
from clinical material.
Presentation and Shelf Life
Suitable for use with LAB 90 Fastidious Anaerobe Agar. When used
LAB M lyophilised supplements are presented in packs of 10 vials, with other blood agar bases, e.g. LAB 1 Columbia Agar, further
and for the majority of the supplements each vial is sufficient for enrichment of the medium with haemin, menadione and sodium
500mls of medium. Larger and smaller volumes are indicated for pyruvate is beneficial. The addition of Tween 80, which, enhances the
relevant products. growth of anaerobic cocci, to the medium is required for N.A.T.
The shelf life of freeze-dried supplements is 2-3 years provided they medium. The Tween 80 may be added before sterilisation at a
are stored in a refrigerator at 2-8˚C. Once rehydrated the stability of concentration of 0.1%.
antibiotics varies greatly and will determine the shelf life of the
prepared agars and broths. For this reason any unused, rehydrated, Final Concentration mg/litre
supplement should be discarded, as even deep-freezing may not
prevent the rapid degradation of the antibiotics. To ensure the correct Nalidixic acid 10
level of selective supplements the entire vial contents must be added Add 1 vial X091 to 500mls medium
to the stated volume of cooled, molten medium.
Add 1 vial X291 to 1 litre medium
Rehydration
Vials should be rehydrated aseptically using a sterile needle and Rehydrate contents of each vial with 5mls sterile deionised water.
syringe charged with 5mls of the specified diluent for the particular Add aseptically to sterilised medium cooled to 47˚C together with
supplement being added. The supplement should be rehydrated, other additives, mix gently and pour.
withdrawn and added to the medium in a single process, followed by
immediate disposal of the syringe into an approved container. Under Reference:
no circumstances attempt to re-sheath an exposed needle. Wren, M. W. D., 1980. J. Clin. Path. 33: 61-65. Multiple Selective
If sterile needles and syringes are not readily available, the rubber Media for the isolation of anaerobic bacteria.
stopper may be completely removed and, using careful aseptic X092
technique, rehydrate the supplement using a sterile pipette.
METRONIDAZOLE, NALIDIXIC ACID for the isolation of
Addition Actinomyces spp. from clinical material.
Most antibiotics are heat labile, and so to prevent a reduction of Suitable for use with LAB 90 Fastidious Anaerobe Agar. The
potency the medium should be cooled to 47˚C by holding in a water metronidazole will suppress the growth of most other anaerobes.
bath set at this temperature.
Once the supplement has been added the medium must be gently but Final Concentration mg/litre
thoroughly mixed to ensure that the selective agents are evenly
distributed. Failure to do this will result in a range of concentrations Metronidazole 10
in the plates/bottles and consequent inconsistency in results. The Nalidixic acid 30
shelf life of supplemented media is governed by the stability of the
added components, and is generally shorter than unsupplemented Add 1 vial X092 to 500mls. medium.
agars and broths. For information on the shelf life of prepared media
consult the individual product listings in the previous section of the Rehydrate contents of each vial with 5mls sterile deionised water.
manual. Add aseptically to sterilised medium cooled to 47˚C together with
other additives, mix gently and pour.

Reference:
Anaerobes Stannard, A. E., National Hospital for Nervous Diseases, London.
Personal Communication.
X090, X290 X015, X215
NALIDIXIC ACID, VANCOMYCIN for the isolation of Gram NEOMYCIN 75 for the isolation of Clostridium spp. and other
negative anaerobes from clinical material. anaerobes.
Suitable for use with LAB 90 Fastidious Anaerobe Agar. When used When added to blood agar the resulting medium will allow the
with other blood agar bases, e.g. LAB 1 Columbia Agar, further growth of clostridia, most Bacteriodes fragilis strains and some
enrichment of the medium with haemin and menadione is beneficial. anaerobic cocci.

Final Concentration mg/litre Final Concentration mg/litre

Nalidixic acid 10 Neomycin 75
Vancomycin 2.5 Add 1 vial X015 to 500mls medium
Add 1 vial X090 to 500mls medium Add 1 vial X215 to 1 litre medium
Add 1 vial X290 to 1 litre medium
Reconstitute each vial by the addition of 5mls of sterile deionised
Rehydrate contents of vial with 5mls sterile deionised water. Add water. Add aseptically to sterilised medium cooled to 47˚C, mix
aseptically to sterilised medium cooled to 47˚C together with other gently and pour.
additives, mix gently and pour.

Reference:
Wren, M. W. D., 1980. J. Clin. Path. 33: 61-65. Multiple Selective
Media for the isolation of anaerobic bacteria.

97
 Reference:
X016
Micro-organisms in Food. Ed. Thatcher, F. S., Clarke, D. F. published
NEOMYCIN 100 for the selective isolation of Clostridium spp. by Univ. of Toronto Press.
When added to egg yolk medium this supplement will allow the
growth of clostridia whilst inhibiting other lecithinase producing
organisms.

Final Concentration mg/litre

Campylobacter species
Neomycin 100 X112, X212
Add 1 vial X016 to 500mls medium CEFOPERAZONE, AMPHOTERACIN for the isolation of
Campylobacter spp. from clinical, environmental and food
samples.
Reconstitution as X015.
Suitable for use with LAB 112 Campylobacter Selective Medium
X018 (blood free) or with blood agar media. Incubation at 37˚C gives better
results than at 42˚C and is generally more convenient.
KANAMYCIN 75 for the selective isolation of Clostridium spp.
and other anaerobes.
Final Concentration mg/litre
An alternative to X015. Kanamycin is more inhibitory to anaerobic
cocci. Cefoperazone 32
Amphotericin 10
Final Concentration mg/litre
Add 1 vial X112 to 500mls medium
Kanamycin 75
Add 1 vial X212 to 1 litre medium
Add 1 vial X018 to 500mls medium
Rehydrate contents of vial with 5mls of sterile deionised water. Add
Reconstitution as X015, X016. aseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:
Lowbury, C. J. L., Lilly, H. A. 1955. A selective plate medium for Cl.
welchi. J. Path. & Bact. 70: 105.
Collee, J. G., Watt, B. 1971. Changing approaches to the sporing
anaerobes in medical microbiology. Spore Research ed. A. N.
Barkeer.
Sutter, V. L., Citron, D. M., Edelstein, M. A. C., Finegold, S. M.
1985. Wadsworth Anaerobic Bacteriology Manual 4 ed. Star
publishers, Belmont, California.
Wren, M. W. D. 1980. Multiple selective media for the isolation of
anaerobic bacteria from clinical specimens. J. Clin. Path. 33: 61-65.
Reference:
Bolton, F. J., Hutchinson, D. N., Parker, G. 1988. Reassessment of
Selective Agars and Filtration Techniques for Isolation of
Campylobacter Species from Faeces. Eur. J. Clin. Microbiol. Infect.
Dis. 7: 155-160.
X214
VANCOMYCIN, POLYMYXIN, TRIMETHOPRIM, to make
Skirrow’s medium for the isolation of Campylobacter spp.
Suitable for use with LAB001 Columbia Agar or other blood agar
bases, supplemented with lysed horse blood.

Final Concentration mg/litre

Vancomycin 10
Bacillus cereus Polymyxin 2500 iu/litre
X073 Trimethoprim 5
EGG YOLK EMULSION. Add 1 vial of X214 to 1 litre of medium
A concentrated sterile emulsion of top quality egg yolks which may
Rehydrate contents of vial with 5ml sterile deionised water. Add
be incorporated into several media formulations for the detection of
aseptically to sterilised medium cooled to 47˚C, along with other
lecithinase production by micro organisms.
additives, mix well and pour.
The most common use is in Bacillus Cereus Medium LAB073, but it
can also be used in media for staphylococci and with Fildes extract Reference:
and serum to prepare Nagler plates for clostridia. Skirrow, M.B. (1977) British Medical Journal 2 11-9.
Presented in 100ml bottles, add 100ml to 900ml of Bacillus cereus
medium, or 50ml to Blood Agar Base LAB028 containing Fildes X131
extract and serum.
C.V.T.C. For the isolation of Campylobacter spp. from food and
X074 environmental samples by the enrichment broth technique.
Developed for use with LAB 135 Campylobacter Enrichment Broth.
POLYMYXIN for the isolation of B. cereus from foods. Gives higher isolation rates than Preston broth and does not require
Suitable for the preparation of LAB 73 Bacillus cereus Medium modified atmosphere incubation.
(P.R.E.P.). The addition of X073 sterile egg yolk emulsion is also
required. Final Concentration mg/litre
Cefoperazone 20
Final Concentration
Vancomycin 20
Polymyxin B 8mg/litre = 64,000i.u/litre
Trimethoprim 20
Add 1 vial X074 to 500mls medium
Cycloheximide 50
Rehydrate contents of vial with 5mls of sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C together with egg Add 1 vial X131 to 500mls medium
yolk emulsion, mix gently and pour.
Rehydrate contents of vial with 5mls of sterile 50% alcohol. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and
dispense into sterile containers.
98
Reference:
X161
Bolton, F. J., Preston., P. H. L. S. Personal communication, 1989.
CEFIXIME TELLURITE supplement for the isolation of E.coli
O157:H7 from food, environmental and clinical samples.
For the addition to LAB161 Sorbitol MacConkey Agar (SMAC)
Clostridium difficile Final concentration mg/litre
X093 Cefixime 0.05
CYCLOSERINE, CEFOXITIN for the isolation of Clostridium Potassium tellurite 2.5
difficile from clinical materials.
Suitable for use with LAB 90 Fastidious Anaerobe Agar. Rehydrate contents of vial with 5ml sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Final Concentration mg/litre Add 1 vial of X161 to 500ml of Sorbitol MacConkey Agar (SMAC).
D-Cycloserine 250
Cefoxitin 8 X546
Add 1 vial X093 to 500mls medium
V.C.C. Supplement for the selective enrichment of E.coli 0157:H7
from food and other samples.
Rehydrate contents of vial with 5mls of water. Add aseptically to
sterilised medium cooled to 47˚C together with other additives, mix For use with Buffered Peptone Water LAB046
gently and pour.
Final Concentration mg/litre
Reference:
Vancomycin 8.0
George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. 1976.
Selective and differential medium for isolation of Clostridium Cefixime 0.05
difficile.
Cefsulodin 10.0
Add 1 vial of X546 to 2.25 litres of LAB046

Clostridium perfringens Rehydrate the contents of one vial with 20mL of sterile deionised
water. Add aseptically to sterilised medium cooled to 47˚C. Mix well
and dispense into 225mL aliquots.
X109, X110
SULPHADIAZINE (X109). OLEANDOMYCIN PHOSPHATE,
POLYMIXIN (X110).
For use with LAB 109 Perfringens agar to prepare O.P.S.P. for the
selective isolation of Clostridium perfringens from foodstuffs.
Gardnerella vaginalis
X011
Final Concentration mg/litre
COLISTIN, NALIDIXIC ACID for the isolation of G. vaginalis
Sulphadiazine 100 from clinical material.
Oleandomycin 0.5 Suitable for addition to LAB 1 Columbia Agar or LAB 15 Blood
Agar Base No. 2 to produce a selective isolation medium.
Polymyxin 10,000 i.u./litre
Add 1 vial X109 and 1 vial X110 to 500mls medium Final Concentration mg/litre
Colistin 10
Rehydrate contents of vials with 5mls of sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and pour. Nalidixic acid 15
Add 1 vial X011 to 500mls medium
Reference:
Handford, P. M. 1974. J. Appl. Bact. 37, 559-570.
Rehydrate contents of vial with 5mls sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, together with any
other additives, mix gently and pour.

Escherichia coli Reference:

X150
NOVOBIOCIN for the enrichment of E.coli O157:H7 from food,
environmental and clinical samples.
For the addition to LAB165 O157 Broth MTSB
Goldberg, R. L., Washington, J. A. II 1976. “Comparison of Isolation
of Haemophilus vaginalis (Corynebacterium vaginalae) from
Peptone-Starch-Dextrose Agar and Columbia, Colistin, Nalidixic
Acid Agar. J. Clin. Microbiol. 4(3): 245.

Final concentration mg/litre

Novobiocin 20
Rehydrate contents of vial with 5ml sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X150 to 500ml of O157 Broth MTSB.

99
 Gram Positive Cocci Impedance Microbiology
X012 X137
COLISTIN, NALIDIXIC ACID for the preparation of Columbia T.M.A.O. Selenite for inclusion in Easter and Gibson Salmonella
C.N.A. medium. Detection Medium LAB 137.
A medium selective for Gram positive cocci is obtained when this The growth of Salmonella in the medium reduces T.M.A.O. to
antibiotic mixture is added to LAB 1 Columbia Agar. T.M.A. and in so doing, significantly increases the conductivity of the
medium. The incorporation of sodium biselenite makes the medium
Final Concentration mg/litre selective for salmonellae.

Colistin 10 Final Concentration g/litre

Nalidixic acid 10
Add 1 vial X012 to 500mls medium
Rehydrate contents of vial with 5mls sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, together with any
other additives, mix gently and pour.
Reference:
Ellner, P. D., Stossel, C. I. Drakeford, E. Vasi, F. 1966. “A new culture
medium for medical bacteriology.” Amer. J. Clin. Path. 45: 502.
Haemophilus influenzae
X260
BACITRACIN for the isolation of Haemophilus influenzae.
Suitable for use with Columbia blood agar base and other blood agars
supplemented with heated (“chocolated”) blood.
T.M.A.O. (Trimethylamine-N-oxide) 5.0
Sodium biselenite 4.0
Add 1 vial X137 to 100mls medium
Reconstitute contents with 5mls of sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C. Swirl to mix then
dispense into sterile containers.
References:
Easter, M. C., Gibson, D. M. 1985. Rapid and automated detection of
Salmonella by electrical measurements, J. Hyg. 94: 245-262.
Gibson, D. M. 1987. Some modifications to the media for rapid
automated detection of salmonellas by conductance. H. Appl.
Bacteriol. 63: 299-304.
Odgen, I. D., Cann, D. C. 1987. A modified conductance medium for
the detection of Salmonella spp. J. Appl. Bacteriol. 63: 359-464.

Final Concentration mg/litre

Listeria monocytogenes
Bacitracin 75 X122
Add 1 vial of X260 to 1 litre of medium. C.C.C.A.F. Cefotetan, Cycloheximide, Colistin, Acriflavine,
Fosfomycin, for the isolation of Listeria monocytogenes from
Rehydrate contents of vial with 5ml sterile deionised water. Add environmental, clinical and food samples.
aseptically to sterilised medium with heated blood cooled to 47˚C, For addition to LAB 122 Listeria Isolation Medium.
mix well and pour.
Final Concentration mg/litre
Cefotetan 2

Helicobacter pylori Cycloheximide 400

Colistin 20
X040
Fosfomycin 10
VANCOMYCIN, CEFSULODIN, AMPHOTERICIN, for the
isolation of Helicobacter pylori. Acriflavine 5
For addition to Helicobacter pylori medium LAB140 Add 1 vial X122 to 500mls medium

Final Concentration mg/litre Reconstitute contents of vial by the addition of sterile 50% ethanol in
water. Add aseptically to sterilised medium cooled to 47˚C, mix
Cefsulodin 10 gently then pour.
Vancomycin 10
Reference:
Amphotericin 20 Curtis, et al. 1989. A selective differential medium for the isolation of
Add 1 vial to 500ml medium Listeria monocytogenes. Lett. in Appl. Microbiol. 8: 95-98.

Rehydrate contents of vial with 5ml sterile deionised water. Add

aseptically to sterilised medium cooled to 47˚C, along with other
additives, mix well and pour.

100
 X138 X155, X555
N.A.C. Nalidixic Acid, Acriflavine, Cycloheximide for the UVMI. Supplement for the primary enrichment of Listeria spp
selective enrichment broth culture of Listeria monocytogenes. from food and environmental samples.
For addition to LAB 138 Listeria Enrichment Broth recommended by For addition to LAB155 UVM Broth Base
the F.D.A. for Listeria isolation from food and environmental
samples. Final Concentration mg/litre

Final Concentration mg/litre Nalidixic acid 20

Nalidixic acid 40 Acriflavine 12

Cycloheximide 50 Rehydrate contents of vial with 5ml sterile deionised water (10ml for
X555). Add aseptically to sterilised medium cooled to 47˚C, mix well
Acriflavine 15
and pour.
Add 1 vial of X138 to 500mls medium Add 1 vial of X155 to 500ml of UVM Broth Base
Reconstitute contents of vial by the addition of sterile 50% ethanol in Add 1 vial of X555 to 2.25 litres of UVM Broth Base
water. Add aseptically to sterilised medium cooled to 47˚C, mix
gently then pour.
X156
UVMII. Supplement for the secondary enrichment of Listeria spp
Reference: from food and environmental samples
Lovett et al. 1987. Listeria monocytogenes in raw milk: detection For the addition to LAB155 UVM Broth Base
incidence and pathogenicity. J. Food Protect. 50: 188-192.
X144 Final Concentration mg/litre

P.A.C. supplement for the enrichment and isolation of Listeria Nalidixic acid 20
spp from food and environmental samples. Acriflavine 25
For the addition to LAB144 Palcam Broth and Lab148 Palcam Agar
Rehydrate contents of vial with 5ml sterile deionised water. Add
Final concentration aseptically to sterilised medium cooled to 47˚C, mix well and pour.

Polymixin 10mg/litre Add 1 vial of X156 to 500ml of UVM Broth Base

Acriflavine 5mg/litre
Ceftazidime 20mg/litre

Rehydrate contents of vial with 5ml sterile deionised water. Add

Neisseria gonorrhoeae
aseptically to sterilised medium cooled to 47˚C, along with other X070, X270
additives, mix well and pour.
Add 1 vial of X144 to 500ml of Palcam Broth or Palcam Agar. L.C.A.T. Lincomycin, Colistin, Amphotericin, Trimethoprim for the
isolation of Neisseria spp. from clinical material.
X164, X564 L.C.A.T. is often preferred to X068 V.C.N.T. for the isolation of
N. gonorrhoeae because of the emergence of vancomycin sensitive
1/2 FRASER supplement for the primary enrichment of Listeria strains. The antifungal agent amphotericin is more readily soluble and
spp from food and environmental samples. therefore a more active antifungal than nystatin. L.C.A.T. is quoted as
For addition to LAB164 Fraser Broth Base the selective agent for New York City G.C. agar but can readily be
substituted for V.C.N. or V.C.N.T. in Thayer Martin G.C. agar.
Final Concentration mg/litre
Final Concentration mg/litre
Ferric ammonium citrate 500
Lincomycin 1
Acriflavine 12.5
Colistin 6
Nalidixic acid 10
Amphotericin 1
Rehydrate contents of vial with 2ml 50% methanol (5ml for X564). Trimethoprim 6.5
Add aseptically to sterilised medium cooled to 47˚C, mix well
and pour. Add 1 vial X070 to 500mls medium
Add 1 vial of X164 to 450ml of Fraser Broth Base Add 1 vial X270 to 1 litre medium
Add 1 vial of X564 to 2.25 litres of Fraser Broth Base
Rehydrate contents of vial with 5mls sterile 25% alcohol in water.
X165 Add aseptically to sterilised medium cooled to 47˚C together with
other additives, mix gently and pour.
FRASER supplement for the secondary enrichment of Listeria
spp from food and environmental samples.
Reference:
For addition to LAB164 Fraser Broth Base Young, H. 1978. Cultural Diagnosis of Gonorrhoea with modified
N.Y.C. Medium. Brit. Journ. Ven. Dis. 54: 36-40.
Final Concentration mg/litre
Ferric ammonium citrate 500
Acriflavine 25
Nalidixic acid 20

Rehydrate contents of vial with 2ml 50% methanol. Add aseptically

to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X165 to 500ml of Fraser Broth Base
101
 X069, X269 Pseudomonas species
L.C.T. Lincomycin, Colistin, Trimethoprim. A variant of X108
L.C.A.T. with the amphotericin omitted to permit the growth of
yeasts. MODIFIED C.F.C. – Cephalothin, Fucidin, Cetrimide for the
selective isolation of Pseudomonas spp.
Concentrations and rehydration as L.C.A.T. When added to LAB 108 Pseudomonas Agar, to prepare C.F.C.
Add 1 vial X069 to 500mls medium medium this supplement can be used to select pseudomonads from
food and environmental samples.
Add 1 vial X269 to 1 litre medium
Final Concentration mg/litre
X068, X268 Cephalothin 50
V.C.N.T. Vancomycin, Colistin, Nystatin, Trimethoprim for Fucidin 10
Thayer Martin Medium.
Cetrimide 10
The addition of trimethoprim in V.C.N.T. inhibits the swarming of
Proteus spp. which occasionally make interpretation difficult. Add 1 vial X108 to 500mls medium

Final Concentration mg/litre Rehydrate contents of vial with 5mls of sterile 50% alcohol. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
Vancomycin 3
Colistin 7.5 Reference:
Mead, G. C. and Adams, B. W. 1977. Br. Poult. Sci. 18: 661-667.
Nystatin 12.5
X107
Trimethoprim 5
C.N. Cetrimide, Nalidixic acid for the isolation of Pseudomonas
Add 1 vial X068 to 500mls medium aeruginosa.
Add 1 vial X268 to 1 litre medium Suitable for use with LAB 108 Pseudomonas Agar to make the
medium selective for Ps. aeruginosa.
Rehydration as for X067.
Final Concentration mg/litre
Reference:
Thayer, J. D. and Martin, J. E. 1966. Improved medium selective for Cetrimide 200
the cultivation of N. gonorrhoeae and N. meningitidis. Public Health Nalidixic acid 15
rep. 81: 559-562.
Add 1 vial X107 to 500mls medium
X271
Rehydrate contents of vial with 5mls of sterile deionised water. Add
GROWTH SUPPLEMENT, to improve the isolation of Neisseria
aseptically to sterilised medium cooled to 47˚C, mix gently and pour.
spp from selective media.
For addition to GC agar base LAB067.

Final Concentration mg/litre Reference:

Goto, S., Enomoto, S. 1970. Jap. J. Microbiol. 14: 65-72.
L-cystine 11
L-cysteine 259
X140
Thiamine HCl 0.03 TICARCILLIN, POLYMYXIN, for the isolation of Burkholderia
(Pseudomonas) cepacia
Ferric nitrate 0.2 Suitable for use with LAB108 pseudomonas selective agar, or
Co-Carboxylase 1 specific selective bases such as that described by Gilligan et al.
NAD 1.0 Final Concentration mg/litre
Guanine HCl 0.3 Ticarcillin 100
Adenine 10 Polymyxin 300,000 iu/litre
L-glutamine 100 Add 1 vial to 500ml of medium
PABA 0.13
Rehydrate contents of vial with 5ml sterile deionised water. Add
Vitamin B12 0.1 aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial to 1 litre of medium
Reference:
Rehydrate contents of vial with 5ml sterile deionised water. Add Gilligan P.H., Gage P.A., Bradshaw L.M., Schidlow D.V., DeCicco
aseptically to sterilised medium cooled to 47˚C, along with other B.T. (1985) Isolation medium for the recovery of Pseudomonas
additives, mix well and pour. cepacia from respiratory secretions of patients with cystic fibrosis.
J.Clin.Microbiol. 22 (1) 5-8.

102
Pre-Incubation Test (P-INC) Salmonella
X019, X219 X150
PENICILLIN, NISIN, CRYSTAL VIOLET, for accelerated shelf NOVOBIOCIN, for the isolation of Salmonella using semi-solid
life determination of dairy products. technology
The Pre-incubation test uses a selective mixture to inhibit Gram For addition to LAB150 MSRV and LAB537 Diassalm
positive organisms whilst allowing the growth of Gram negative
bacteria, the main cause of post-pasteurisation contamination and a Final Concentration mg/litre
major factor in determining the shelf life of the product.
The technique is also useful for monitoring plant hygiene. Novobiocin 20 (MSRV)
Novobiocin 10 (Diassalm)
Final Concentration mg/litre
Add 1 vial to 500ml (MSRV)
Penicillin 20,000iu/litre
Add 1 vial to 1 litre (Diassalm)
Nisin 40,000iu/litre
Crystal violet 2.0 Rehydrate contents of vial with 5ml sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix well and pour.
Add 1 vial of X019 to 200ml of Milk Agar LAB019
Add 1 vial of X219 to 1 litre of Milk Agar LAB019 References:
De Smedt J.M., and Bolderdijk R.F., (1986) Dynamics of salmonella
Rehydrate contents of 1 vial with 5mls sterile deionised water. Add isolation with modified semi-solid Rappaport Vassiliadis medium.
aseptically to sterilised medium cooled to 47˚C, mix thoroughly and J.Food Protection 50 658-661
pour plates. Van Netten P, Van Der Zee H., and Van Der Moosdijk A., (1991) The
use of diagnostic selective semi-solid medium for the isolation of
Method A Salsmonella enteritidis from poultry. Proceedings of the 10th
Pre-incubate test material at 21˚C for 24hr. Prepare suitable dilution symposium on the quality of poultry meat. Spelderholt Beckbergen
series, and inoculate Milk Agar plates containing P-INC supplement. 56-67.
Incubate at 21˚C for 24hr, and count all colonies (some may be small,
use of a hand lens is recommended). Calculate the CFU/ml and using
the tables of Griffith’s et al the shelf life can be determined.

Method B
Rehydrate X219 with 1ml of deionised water only, add 0.1ml to the
test material and incubate at 20˚C for 24hr. Prepare suitable dilution
series, and inoculate Milk Agar plates. Proceed as for Method A
above.
References:
Griffiths M.W., and Phillips J.D. (1985) J.Appl.Bact. 57, 107.
Staphylococci
X085
EGG YOLK TELLURITE
A sterile emulsion of egg yolk and potassium tellurite for use as a
selective and differential agent in Baird Parker Medium Base
LAB085. The complete medium is selective for S.aureus, and the
addition of egg yolk tellurite aids differentiation of this organism
from others capable of growing on the agar.

Griffiths M.W., and Phillips J.D., and Muir D.D. (1980) J. Soc. Dairy
Technol. 33, 8.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1981) J. Soc. Dairy
Technol. 34, 142.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) J. Soc. Dairy
Technol. 37, 22.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Rapid
detection of post-pasteurised contamination. Hannah Research Inst.
Bulletin No.10.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Dairy Ind.
Int. 50 (3) 25
Griffiths M.W., and Phillips J.D., and Muir D.D. (1984) Post-
pasteurisation contamination - the major cause of failure of fresh
dairy products. Hannah Research Inst.
Griffiths M.W., and Phillips J.D., and Muir D.D. (1986) Aust. J.
Dairy Technol. 41, 77-79.
Presented in 100ml bottles with a tellurite concentration of 0.2% to
give a final concentration in the complete medium of 0.01% (w/v).
Add 50ml to 1 litre of Baird Parker Medium Base.
X207
METHICILLIN, for the isolation of Methicillin Resistant
S.aureus (MRSA)
Suitable for use with LAB007 Mannitol salt agar.
Final Concentration mg/litre
Methicillin 4
Add 1 vial of X207 to 1 litre of medium
Rehydrate contents of vial with 5ml sterile deionised water. Add
aseptically to sterilised medium cooled to 47˚C, mix well and pour.

103
 Streptococci References:
Mossel, D. A. A., et al. 1970. O.G.Y.E. for the selective enumeration
X013 of moulds and yeasts in food and clinical material. J. Appl. Bact. 35:
454-457.
COLISTIN, OXOLINIC ACID for the selective isolation of
streptococci from clinical material.
When added to LAB 1 Columbia agar or LAB 15 Blood Agar Base
No. 2, X013 renders the medium selective for streptococci. Alteration
in haemolysis patterns may occur when azide or crystal violet are Yersinia
employed as selective agents but this does not occur with X013.
X120
Final Concentration mg/litre C.I.N. - Cefsulodin, Irgasan, Novobiocin for the isolation of
Yersinia spp. from clinical and environmental material.
Colistin 10
For addition to LAB 120 Yersinia C.I.N. Agar Base used in the
Oxolinic acid 5 selective isolation of Y. enterocolitica.
Add 1 vial X013 to 500mls medium
Final Concentration mg/litre
Rehydrate contents of vial with 5mls of sterile deionised water. Add Cefsulodin 15
aseptically to sterilised medium cooled to 47˚C together with other
additives, mix gently and pour. Irgasan 4
Novobiocin 2.5
Reference:
Petts, D. 1984. Colistin - Oxolinic Acid - Blood Agar: a new selective Add 1 vial X120 to 500mls medium
medium for streptococci. J. Clin. Microbiol. 19: 4-7.
Rehydrate contents of vial with 5mls of 30% sterile alcohol. Add
aseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:
Yeasts and Moulds Schiemann, D. A. 1979. Synthesis of a selective medium of Yersinia
enterocolitica. Can. J. Microbiol. 25 (2): 1298.
X009, X209
Schiemann, D. A. 1980. Isolation of toxigenic Yersinia enterocolitica
CHLORAMPHENICOL for the selective isolation of yeasts and from retail pork products. J. Food Prot. 43: 360.
moulds from food, environmental and clinical specimens. Schiemann, D. A. 1982. Development of a two-step enrichment
Chloramphenicol’s broad antibiotic spectrum suppresses most procedure for recovery of Yersinia enterocolitica from food. Appl.
contaminating bacteria allowing the yeasts and moulds to grow. It can Microbiol. 43 (1): 14.
be added to such media as LAB 95 Sabouraud Dextrose Agar, LAB
36 Rose Bengal Chloramphenicol Agar, LAB 37 Malt Extract Agar
and LAB 117 Dermatophyte Test Medium to increase their selectivity
whilst not lowering the pH. Reduction of pH will increase the
selectivity of a yeast and mould medium but will also inhibit some
yeasts as well as having a deleterious effect on the agar gel.

Final Concentration mg/litre

Chloramphenicol 100
Add 1 vial X009 to 500mls medium
Add 1 vial X209 to 1 litre medium

Rehydrate contents of vial with 5mls of Ethyl or Methyl alcohol. Add

aseptically to sterilised medium cooled to 47˚C, mix gently and pour.

References:
Jervis, B. 1973. Rose Bengal Chlortetracycline agar with other media
for the selective isolation and enumeration of moulds and yeasts in
foods. J. Appl. Bact. 36 Pages 723-727.
X089
OXYTETRACYCLINE for O.G.Y.E. medium.
For use with LAB 89 Oxytetracycline Glucose Yeast Extract Agar for
the enumeration of yeasts and moulds from foodstuffs. Highly
proteinaceous foods and incubation above 30˚C will inactivate
oxytetracycline.

Final Concentration mg/litre

Oxytetracycline 100
Add 1 vial X089 to 500mls medium

Rehydrate contents of vial with 5mls sterile deionised water. Add

aseptically to sterilised medium cooled to 47˚C, mix gently and pour.

104
 Agars, Peptones, Extracts & Typical Analysis
Gel strength (Nikan) 650-1000g/m2
Other Media Constituents Colourimetry (1.5% soln at 65˚C) > 0.28 at 340nm
> 0.02 at 525nm
Sourcing
The Lab M range of media constituents are selected on the basis of Melting point > 85˚C
quality and performance from the world’s leading suppliers. It is a
Setting point 32-35˚C
deliberate policy not to invest in our own peptone manufacturing
facility in order to allow our microbiologist freedom to choose the pH 6.5-7.4
best ingredients available on the international market.
Moisture < 10%
Agars Total ash < 3%
A range of agars are offered to suit all microbiological applications.
Koch originally used gelatin to solidify culture media, but the Calcium < 0.02%
superior properties of agar resulted in its universal adoption as the Magnesium < 0.02%
gelling agent of choice. Careful selection of agars is vital as they can
interact with nutrient components in a beneficial or deleterious Sodium chloride < 1.0%
manner.
Iron < 0.01%
Peptones and Extracts Insoluble ash < 0.1%
Like agars, peptones and extracts are biologically variable products
requiring careful selection. They provide the amino acids and Sulphate 1.5%
peptides required by micro-organisms for growth as well as other Salmonella Absent
vital growth factors such as minerals, vitamins and nucleic acid
fractions. TVC < 10 3/g
To ensure we use only the best available peptones and extracts these Spores < 2/g
materials are exhaustively tested. Growth parameters are obtained by
classical microbiological techniques and by automated growth rate
analysis. Chemical and physical properties are also closely
monitored. LAB M can select specific peptones for special purposes
such as vaccine production and fermentation processes. If more
information is required on special services please contact LAB M or
Agar No. 2
your local agent. MC 6
A bacteriological agar which gives a firm gel at working
concentrations of 1.0 to 1.5% which is reasonably clear. This agar is
recommended for all culture media except sensitivity testing media
Acid Hydrolysed Casein and those where absolute clarity is advantageous.

MC 7 Typical Analysis
A soluble protein hyrolysate obtained by digesting casein with hot Gel strength (Nikan) 650-1000g/m2
acid. It is almost free from growth factors, vitamins and antagonists,
and these qualities make it suitable for use as a protein source in Colourimetry (1.5% soln at 65˚C) > 0.3 at 340nm
media for antibiotic and vitamin assays. >0.04 at 525nm

Typical Analysis Appearance cream/white powder

Appearance cream/white powder Melting point >85˚C

Solubility in water at 5% total Setting point 32-35˚C

Clarity clear and colourless pH 6.5-7.4

pH of 2% solution 6.0 ± 0.5 Moisture <12%

Total Nitrogen 8.3% ± 0.5 Total ash <3.5%

Total Amino Nitrogen 6.1% ± 0.5 Calcium < 0.5%

Magnesium < 0.1%
Sodium chloride <1.0%
Iron < 0.01%
Agar No. 1 Insoluble ash < 0.1%
MC 2 Sulphate < 3.0%
A high clarity agar with good gelling properties and a low Salmonella Absent
concentration of metal ions. This agar is suitable for all
bacteriological purposes including sensitivity testing and pour plate TVC < 10 3/g
techniques. A firm gel is obtained at working concentrations of 1.0 to
1.5%. No significant precipitation is observed on reheating or Spores < 2/g
prolonged holding at 65˚C.

105
 Agar No.4 Balanced Peptone No. 1
Plant Tissue Culture Grade MC 4
MC 29 A rich mixture of tryptone and meat peptones which fulfills the
nutritional demands of a wide variety of micro-organisms. This
Lab M Agar No.4 has been selected specifically for use as a gelling peptone is used in many LAB M culture media formulations.
agent in plant tissue culture techniques. The product is selected
primarily on gel strength, a parameter of particular importance for
Typical Analysis
this application, and then tested to ensure it meets the parameters set
by a major plant producer. The agar contains no nutrients for plant Appearance beige powder
growth and is designed to be incorporated into classical formulations
such as Murashige and Skoog, as well as customer’s own Solubility in water at 5% total
formulations.
Clarity clear, pale straw colour
Typical analysis pH of 2% solution 7.2 ± 0.2
Ash 2.30% Total Nitrogen 12.8% ± 0.5
Acid Insoluble Ash 0.16% Amino Nitrogen 5.1% ± 0.5
Calcium 0.31%
Magnesium 0.12%
Iron 0.018% Beef Extract
Total Nitrogen 0.15%
MC 19
Recommended Concentration 0.75 - 1.5%
This complements the nutritive properties of peptones in culture
Melting Point 88-91˚C media and is often used as an added enrichment. Beef extract can be
used as a direct replacement for meat peptones and, as it contains no
Setting Point 32-33˚C carbohydrates, can be used as a component of media for fermentation
Mesh 80 studies.

pH (1.5% at 20˚C) 7.0 ± 0.2 Typical Analysis

Gel Strength (1.5% W/V) >700g/cm2 Appearance light brown powder
Solubility in water at 5% total
Clarity clear, light brown colour
Bacteriological Peptone pH of 2% solution 7.0 ± 0.2

MC 24 Total Nitrogen 12.0% ± 0.5

Amino Nitrogen 1.6% ± 0.5
An economical source of nutrients provided by a balanced mixture of
meat peptones and tryptone. The growth requirements of most non
fastidious organisms will be fulfilled by the range of amino acids,
peptides and proteoses in this mixture.

Typical Analysis
Bile Salts No. 3
Appearance cream powder MC 25
Solubility in water at 5% total A refined bile salt, comprising mainly sodium cholate and sodium
desoxycholate. It is used as a selective agent in culture media such as
Clarity clear, pale straw colour Violet Red Bile Agar for the enumeration of coliforms, MacConkey
pH of 2% solution 7.2 ± 0.2 Agar No. 3 for the isolation and differentiation of enteric organisms,
and SS Agar for the isolation of enteric pathogens. This fraction of
Total Nitrogen 12% ± 0.5 bile is highly active, allowing maximum selection of organisms of
enteric origin at relatively low concentrations (0.15%).
Amino Nitrogen 5% ± 0.5
Typical Analysis
Appearance white powder
Solubility in water at 2% total
Clarity clear
pH of a 2% solution 8.0 ± 0.5

106
Malt Extract
MC 23
A water soluble extract of malted barley suitable for use in the
cultivation of yeasts and moulds. Malt extract has a very high
carbohydrate content and consequently is very sensitive to over
heating which will cause a darkening of the medium.
Skim Milk Powder
MC 27
A bacteriological grade of thermophile free spray dried skim milk.
Used in Milk Plate Count Agar LAB 115 and in media for diagnostic
tests involving the digestion or coagulation of casein and the
fermentation of lactose. Recommended working concentration 10%.

Typical Analysis Typical Analysis

Appearance yellow/brown powder Appearance white powder
Solubility in water at 5% total Clarity opaque white suspension
Clarity clear, light brown colour Total Nitrogen 5.3% ± 0.5
pH of 2% solution 5.2 ± 0.2 Lactose 48.0% ± 0.5
Maltose 55%
Other Carbohydrates 40%
Protein 5% Sodium Desoxycholate
MC26
Sodium desoxycholate is a specific bile acid, derived from
Mycological Peptone deconjugated bile salts. Leifson showed that desoxycholic acid had
the most inhibitory effect on bacterial growth, and that this could be
MC 9 enhanced by the removal of magnesium ions by chelating with
sodium citrate. These components comprise the selective agents in
A mixture of peptones with a high carbohydrate content suitable for DCA, DCA (Hynes) and DCLS.
the rapid growth and colonial development of yeasts and moulds.
Bacterial growth is inhibited by the low pH of this peptone. Typical Analysis
Appearance white powder
Typical Analysis
pH of 2% solution 8.3 ± 0.5
Appearance beige powder
Solubility in water at 2% total
Solubility in water at 5% total
Moisture <5%
Clarity clear, pale straw colour
Heavy metals <20 ppm
pH of 2% solution 5.4 ± 0.1
Sodium cholate <2%
Total Nitrogen 13% ± 0.5
Amino Nitrogen 1.4% ± 0.5

Soy Peptone
Proteose Peptone A MC 3
MC 11 Prepared using the enzyme papain to digest soyabean meal, this
peptone is a rich source of nutrients with a high carbohydrate content.
An enzymatic digest of meat adapted to encourage the production of Most organisms will grow rapidly in this peptone but some bacteria
toxins by Corynebacterium diphtheriae, staphylococci, Salmonella, will produce high levels of acid leading to auto-sterilisation unless an
and clostridia. This peptone is highly nutritious and suitable for use adequate buffering system is incorporated.
in culture media for fastidious organisms such as Neisseria,
Haemophilus and Pasteurella species. Typical Analysis
Appearance cream powder
Typical Analysis
Solubility in water at 5% total
Appearance cream powder
Clarity clear, straw colour
Solubility in water at 5% total
pH of 2% solution 7.1 ± 0.2
Clarity clear, light straw colour
Total Nitrogen 9.0% ± 0.5
pH of 2% solution 7.0 ± 0.2
Amino Nitrogen 1.6% ± 0.5
Total Nitrogen 12.0% ± 0.5
Amino Nitrogen 5.8% ± 0.5

107
 Tryptone
MC 5
An enzymatic hydrolysate of casein, rich in peptones and amino acids
(including tryptophane). This peptone can be utilised by most
bacteria as a growth substrate. This peptone conforms to the U.S.P.
requirements for a pancreatic digest of casein.

Typical Analysis
Appearance cream powder
Solubility in water at 5% total
Clarity clear, pale straw colour
pH of 2% solution 7.2 ± 0.5
Total Nitrogen 13.0% ± 0.5
Amino Nitrogen 4.9% ± 0.5

Tryptose
MC 8
A blend of peptones suitable for the cultivation of most fastidious
organisms including Neisseria gonorrhoeae, Streptococcus milleri
and Brucella spp. especially where rapid or profuse growth is
required such as in blood culture media and blood agars.

Typical Analysis
Appearance beige powder
Solubility in water at 5% total
Clarity clear, light straw colour
pH of 2% solution 7.2 ± 0.2
Total Nitrogen 12.5% ± 0.5
Amino Nitrogen 4.9% ± 0.5

Yeast Extract Powder

MC 1
Prepared by the autolysis of Saccharomyces cerevisae under
thermostatically controlled conditions to protect the B vitamins and
other heat labile constituents. This extract provides a mixture of
amino acids, peptides, vitamins and carbohydrates making it suitable
for many applications.

Typical Analysis
Appearance yellow powder
Solubility in water at 5% total
Clarity clear, pale yellow
pH of 2% solution 7.0 ± 0.2
Total Nitrogen 10.5% ± 0.5
Amino Nitrogen 5.3% ± 0.5

108
 TYPICAL ANALYSIS

MC 7 MC24 MC 4 MC 9 MC 11 MC 3 MC 5 MC 8 MC 1
Acid Hydrolysed Bacteriological Balanced Mycological Proteose Soy Peptone Tryptone Tryptose Yeast
Casein Peptone Peptone No. 1 Peptone Peptone A Extract

Total Nitrogen 8.3% 12.0% 12.5% 12.6% 12.0% 9.0-9.4% 12.8% 12.7% 10.5%
LAB M Peptones

Total AminoNitrogen 6.1% 5.0% 5.1% 1.4% 5.8% 1.6-1.8% 4.8% 4.9% 5.3%

Amino N/Total N. 73.4% 41.7% 40.8% 11.1% 48.0% 17.7% 37.5% 38.7% 50.4%

Total Amino Acid Assay (mg/g)

Lysine 53.5 62.0 75.3 38.9 48.2 43.1 79.7 77.5 49.0

Histidine 18.7 21.0 26.8 12.7 15.6 16.2 31.2 29.0 14.0

Arginine 22.1 32.0 41.0 55.0 50.6 39.2 37.5 39.2 27.0

Aspartic Acid 44.1 69.0 58.4 69.9 63.9 74.4 62.8 60.6 52.0

Threonine 23.5 40.0 31.1 17.7 27.5 21.7 36.9 34.0 33.0

Serine 30.0 40.0 40.3 26.2 36.2 27.0 50.3 45.3 34.0

Glutamic Acid 130.0 160.0 138.9 103.5 100.2 110.0 184.0 161.5 73.0

Proline 52.2 46.0 61.0 74.5 55.6 28.0 82.1 71.6 26.0

Glycine 11.1 29.0 44.5 105.9 83.38 22.6 15.6 30.0 25.0

Alanine 19.0 39.0 38.1 49.9 52.3 23.1 26.9 32.5 51.0

Cystine 1.1 1.0 1.1 2.7 8.4 5.3 2.2 1.6 6.0

Valine 35.2 45.0 45.3 22.9 35.2 23.7 59.2 52.3 37.0

Methionine 10.4 8.0 19.1 7.0 12.3 6.2 25.0 22.1 9.0

Isoleucine 27.9 33.0 43.8 18.7 23.3 26.8 58.5 51.1 73.0

Leucine 30.9 65.0 65.3 32.0 55.5 38.6 83.5 74.4 73.0

Tyrosine 12.7 12.0 9.8 12.8 13.3 16.8 14.7 12.2 12.0

Phenylalanine 17.0 34.0 31.8 20.2 27.2 22.7 42.4 37.1 25.0

Tryptophane _ 3.0 4.9 1.9 4.6 3.7 6.6 5.7 9.0

109
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 Cover Picture:
Campylobacter jejuni on
LAB M Limited Campylobacter Blood Free

Topley House Selective Medium

(modified CCDA improved).
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Lancashire BL9 6AU . UK
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