3.17.23.

1
3.17.23 Indole Test
I. PRINCIPLE The ability of an organism to split indole
from the amino acid tryptophan is due to
the presence of tryptophanase. Indole, if
present, combines with the aldehyde in the
reagent to produce a pink to red-violet qui-
noidal compound (benzaldehyde reagent)
or a blue to green color (cinnamaldehyde
reagent). In the rapid spot test, indole is
detected directly from a colony growing
on a medium rich in tryptophan.
II. MICROORGANISMS TESTED A. Fresh growth of a gram-negative rod on medium that does not contain dyes and
contains tryptophan, e.g., BAP or CHOC
B. Anaerobic gram-positive rods
C. Anaerobic gram-negative rods
III. MEDIA, REAGENTS, AND
SUPPLIES
A. Reagents
Obtain indole reagents commercially,
or prepare in-house.
1. Rapid spot indole: see Appendix
3.17.231.
a. Prepare either 5% p-dimeth-
ylaminobenzaldehyde or 1%
paradimethylaminocinnamalde-
hyde in 10% (vol/vol) concen-
trated HCl (1, 6)
b. Do not use the spot benzalde-
hyde reagent for anaerobes.
2. Tube indole
a. Kovacs method for aerobically
growing organisms (3)
(1) Reagent: see Appendix
3.17.231.
(2) Medium: broth that contains
tryptophan (containing pep-
tone, tryptone, or casein).
Semisolid agar, such as mo-
tility-indole-ornithine agar
or sulde-indole-motility
agar, can be used.
b. Ehrlichs method for anaerobes
and weak indole producers (2)
(1) Reagent: see Appendix
3.17.231.
(2) Medium: heart infusion or
anaerobic medium with
tryptophan
(3) Xylene
3. Clearly label indole reagents, indi-
cating preparation and expiration
dates.
a. Record the expiration date in a
work record (in-house prepara-
tion) or on a receipt record
(commercial record).
b. Store indole reagents in a dark
bottle at 4C.
B. Supplies
1. Sterile loop, swab, or stick for har-
vesting
2. Filter paper (optional)
Indole Test 3.17.23.2
IV. QUALITY CONTROL
A. Do not use benzaldehyde reagents (including Ehrlichs and Kovacs) if color is
not pale yellow.
B. Perform QC
1. On each new lot or shipment of reagent prior to putting it into use
2. Although not required, QC in-house-prepared reagents weekly, as they can
deteriorate, especially if not stored at 4C. Discard if reactions become weak
(4).
C. Organisms
1. Escherichia coli ATCC 25922indole positive
2. Pseudomonas aeruginosa ATCC 27853indole negative
3. For Ehrlichs reagent for use with anaerobic microorganisms
a. Porphyromonas asaccharolytica ATCC 25260indole positive
b. Bacteroides fragilis ATCC 25285indole negative
V. PROCEDURE
A. Rapid spot indole
Use one of the methods below.
1. Moisten lter paper with reagent. Using a wooden stick, rub portion of col-
ony onto paper.
2. Sweep the colony onto a swab. Add drop of indole reagent to the colony
swab.
3. Add reagent directly to the colony growing on the agar surface.
B. Tube test
1. Inoculate liquid tube medium or stab agar medium with colony.
2. Incubate for 18 to 24 h. If broth is used for indole production, decant a
portion of the medium to a second tube before testing.
3. For Ehrlichs method
a. Add 0.5 ml of xylene to tube and invert to mix. Let settle.
b. Add 6 drops of Ehrlichs indole reagent down the side of the tube and
observe color below the xylene layer.
4. For Kovacs method, add 3 drops of Kovacs reagent down the side of the
tube and observe color change at meniscus.
5. If test is negative, repeat test after an additional 24 h of incubation, if desired.
VI. INTERPRETATION
A. The development of a brown-red to purple-red color (benzaldehyde reagents)
or blue color (cinnamaldehyde reagent) within 20 s indicates the presence of
indole.
B. A negative test is colorless or slightly yellow.
VII. REPORTING RESULTS
A. E. coli is indole positive, as are many other Enterobacteriaceae, Vibrio, Aero-
monas, Plesiomonas, and Pasteurella.
B. Several fastidious gram-negative rods are indole positive, such as Cardiobac-
terium hominis and Pasteurella bettyae. See Table 3.18.23 for others.
C. Propionibacterium acnes is indole positive.
D. Delftia (Comamonas) acidovorans produces a pumpkin orange reaction from
the production of anthranilic acid, rather than indole, from tryptophan with
Kovacs reagent (5).
3.17.23.3 Aerobic Bacteriology
VIII. LIMITATIONS
A. Detectable indole will diffuse to colonies within 5 mm of a 2- to 3-mm colony,
giving false-positive results.
B. Do not use media that contain dyes (e.g., EMB, MAC).
C. Growth medium must contain an adequate amount of tryptophan. Do not use
Mueller-Hinton agar for test, because tryptophan is destroyed during the acid
hydrolysis of casein.
D. Only the cinnamaldehyde reagent can be used for spot testing of anaerobic
microorganisms. It is the more sensitive reagent, but it is less stable.
E. Do not use a plate with a nitrate disk to perform the indole test, as nitrate can
interfere with the spot indole test by inducing false-negative results.
F. If the rapid indole test is negative, the isolate could be positive in the more
sensitive tube test. Extraction with xylene is the most sensitive test. Xylene
substitutes are less sensitive.
G. For fastidious gram-negative rods, such as C. hominis, a heavy inoculum and
extraction are necessary.
REFERENCES
1. Bale, M. J., S. M. McLaws, and J. Matsen.
1984. The spot indole test for identication of
swarming Proteus. Am. J. Clin. Pathol. 83:87
90.
2. Bohme, A. 1905. Die Anwendung der Ehr-
lichschen Indolreaktion fur bakteriologische
Zwecke. Zentralbl. Bakteriol. Parasitenkd. In-
fektionskr. Hyg. I Abt. Orig. 40:129133.
3. Kovacs, N. 1928. Eine vereinfachte Methode
zum Nachweis der Indolbildung durch Bak-
terien. S. Immunitaetsforsch. 55:311315.
4. MacFaddin, J. F. 2000. Biochemical Tests for
the Identication of Medical Bacteria, 3rd ed.,
p. 221232. The Lippincott, Williams & Wil-
kins Co., Philadelphia, Pa.
5. Marraro, R. V., J. L. Mitchell, and C. R.
Payet. 1977. A chromogenic characteristic of
an aerobic pseudomonad species in 2% tryp-
tone (indole) broth. Am. Med. Technol. 39:13
19.
6. Vracko, R., and J. C. Sherris. 1963. Indole-
spot test in bacteriology. Am. J. Clin. Pathol.
39:429432.
APPENDIX 3.17.231 Reagent Preparation
Caution: HCl is toxic and burns. Make indole reagents in a fume hood. Add acid to water;
do not add water to acid.
A. Ehrlichs reagent
p-dimethylaminobenzaldehyde . . . . . . . . . . . . . . . . 1 g
ethyl alcohol, 95% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95 ml
hydrochloric acid, concentrated . . . . . . . . . . . . . .20 ml
1. Dissolve the aldehyde in the alcohol (this may require gentle heating).
2. Working under a fume hood, slowly add the acid (never add alcohol to acid). Mix
constantly.
3. The reagent should be pale yellow and stable for 1 year.
B. Kovacs reagent
p-dimethylaminobenzaldehyde . . . . . . . . . . . . . . .10 g
isobutyl or isoamyl alcohol (absolute) . . . . 150 ml
hydrochloric acid, concentrated . . . . . . . . . . . . . .50 ml
1. Dissolve the aldehyde in the alcohol (this may require gentle heating).
2. Working under a fume hood, slowly add the acid (never add alcohol to acid). Mix
constantly.
3. The reagent should be pale yellow and stable for 1 year.
Include QC information on
reagent container and in
QC records.
C. 5% p-Dimethylaminobenzaldehyde
p-dimethylaminobenzaldehyde . . . . . . . . . . . . . . . . 5 g
10% (vol/vol) hydrochloric acid . . . . . . . . . . . 100 ml
1. Add 10 ml of concentrated HCl to 90 ml of water.
2. Dissolve the aldehyde in the acid (use a fume hood).
3. Shelf life is 4 months.
D. 1% p-Dimethylaminocinnamaldehyde
p-dimethylaminocinnamaldehyde . . . . . . . . . . . . . 1 g
10% (vol/vol) hydrochloric acid . . . . . . . . . . . 100 ml
1. Add 10 ml of HCl to 90 ml of water.
2. Dissolve the aldehyde in the acid (use a fume hood).
3. Shelf life is 2 months (1).
Indole Test 3.17.23.4
Reference
1. MacFaddin, J. F. 2000. Biochemical Tests for
the Identication of Medical Bacteria, 3rd ed.,
P. 221232. The Lippincott, Williams & Wil-
kins Co., Philadelphia, Pa.
APPENDIX 3.17.231 (continued)