Indole Test

This test demonstrate the ability of certain bacteria to decompose the amino
acid tryptophane to indole, which accumulates in the medium. Indole production
test is important in the identification of Enterobacteria. Most strains of E. coli, P.
vulgaris, P. rettgeri, M. morgani and Providencia species break down the amino
acid tryptophan with the release of indole. This is performed by a chain of a
number of different intracellular enzymes, a system generally referred to as
“tryptophanase.” It is used as part of the IMViC procedures,a tests designed to
distinguish among members of the family Enterobacteriaceae.
A variation on this test using Ehrlich’s reagent (using ethyl alcohol in place of
isoamyl alcohol, developed by Paul Ehrlich) is used when performing the test
on non-fermenters and anaerobes.
Procedure of Indole Test

1. Take a sterilized test tubes containing 4 ml of tryptophan broth.

2. Inoculate the tube aseptically by taking the growth from 18 to 24 hrs
culture.
3. Incubate the tube at 37°C for 24-28 hours.
4. Add 0.5 ml of Kovac’s reagent to the broth culture.
5. Observe for the presence or absence of ring.
Indole Spot Reagent (DMACA) Procedure

1. Place several drops of Indole Spot Reagent on a piece of filter paper.

2. With an inoculating loop or wooden applicator stick, pick a portion of
an 18-24 hour isolated colony from a non-selective media and rub it
onto the reagent saturated area of the filter paper.
3. Examine immediately
Result Interpretation of Indole Test

Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the
medium within seconds of adding the reagent.

Examples:  Aeromonas hydrophila, Aeromonas punctata, Bacillus

alvei,Edwardsiella sp., Escherichia coli, Flavobacterium sp., Haemophilus
influenzae, Klebsiella oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Plesiomonas
shigelloides,Pasteurella multocida, Pasteurella pneumotropica, Enterococcus faecalis,
and Vibrio sp.

Negative: No color change even after the addition of appropriate reagent.

Examples: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp.,
most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp.,
most Klebsiella sp., Neisseria sp., Pasteurella haemolytica, Pasteurella ureae, Proteus
mirabilis, P. penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.
Indole Spot Reagent Result

Positive reaction: The development of a blue color within 3 minutes.

Negative reaction: The development of a pink color within 3 minutes.
Spot Indole Test

Method

1. Place several drops of 1% p-dimethylaminocinnamaldehyde reagent on a piece of

filter paper until saturation.
2. With an inoculating loop or wooden applicator stick, pick a portion of an 18-24
hour isolated colony from a non-selective media and rub it onto the reagent
saturated area of the filter paper.
3. Observe for color development within 1 to 3 minutes.
Expected Results

 Positive: A positive reaction is denoted by the appearance of a blue to blue-green

color change on the bacterial smear within 2-3 minutes.
 Negative: Negative reactions remain colorless or light pink.
Note: Positive reaction is Red-violet in the case of Providencia alcalifaciens.

Uses of Indole Test

1. To differentiate Proteus
mirabilis (indole negative) from all
other Proteus species (indole
positive).
2. To differentiate Klebssiella
pneumoniae (indole negative)
from Klebsiella oxytoca (indole
positive).
3. To differentiate Citrobacter
freundii (indole negative)
from Citrobacter koseri (indole
positive).
Quality Control for Indole Test

Positive Control: Escherichia coli NCTC 10418

Negative Control: Proteus mirabilis NCTC 10975
Limitations of Indole Test

1. Indole tests may be used as an aid in the identification and differentiation of

gram-positive and gram-negative organisms. Additional biochemical testing using
pure cultures is recommended for complete identification.
2. The tube test is a more sensitive method of detecting indole than the spot test.
3. When performing a spot test, Kovacs Indole Reagent may be used as a substitute
for the spot test reagent. However, Kovacs Indole Reagent, when used as the spot
test reagent, is less sensitive in detecting indole than the Indole Spot Reagent
(DMACA).
4. Kovacs Indole Reagent is not recommended for use with anaerobic bacteria. The
Indole Spot Reagent (DMACA) is suitable for anaerobe use.
5. Since peptones have been shown to vary with regard to their suitability for use
with indole testing, media selected for indole determination should be tested with
known positive and negative organisms to insure suitability.
6. Media containing glucose should not be used for indole testing due to the
formation of acid end products which have been shown to reduce indole
production. Mueller Hinton Agar should also not be used for this test because
tryptophan is destroyed during acid hydrolysis of casein.
7. Media containing dye, such as MacConkey and EMB, are unsuitable sources of
inoculum due to possible carryover of dye and subsequent interference of indole
color interpretation.
8. Indole-positive colonies have been reported to cause adjacent indole-negative
colonies to appear false-positive due to diffusion of indole into the media. To
avoid false-positives, select colonies of different morphologies that are separated
by at least 5mm for indole testing.
 \

Methyl Red (MR)

Procedure of Methyl Red (MR) Test

1. Prior to inoculation, allow medium to equilibrate to room
temperature.
2. Using organisms taken from an 18-24 hour pure culture, lightly
inoculate the medium.
3. Incubate aerobically at 37 degrees C. for 24 hours.
4. Following 24 hours of incubation, aliquot 1ml of the broth to a
clean test tube.
5. Reincubate the remaining broth for an additional 24 hours.
6. Add 2 to 3 drops of methyl red indicator to aliquot.
7. Observe for red color immediately.

Positive Reaction: A distinct red color (A)

Examples: E. coli, Yersinia sps, etc.
Negative Reaction: A yellow color (B)
Examples: Enterobacter aerogenes, Klebsiella pneumoniae,  etc.
A weak positive is red-orange. If an orange color is seen, incubate the
remainder of the broth for up to 4  days and repeat the test after further
The methyl red (MR) test detects the production of sufficient acid during the
fermentation of glucose and the maintenance of conditions such that the pH
of an old culture is sustained below a value of about 4.5, as shown by a
change in the color of the methyl red indicator which is added at the end of
the period of incubation. 

Clark and Lubs developed MR-VP Broth which allowed both the MR and VP
tests to be performed from the same inoculated medium by aliquoting
portions to different tubes.

Principle of Methyl Red (MR) Test

Some bacteria have the ability to utilize glucose and convert it to a stable acid
like lactic acid, acetic acid or formic acid as the end product.

These bacteria initially metabolise glucose to pyruvic acid, which is further

metabolized through the ‘mixed acid pathway to produce the stable acid.
The type of acid produced differs from species to species and depends on the
specific enzymatic pathways present in the bacteria. The acid so produced
decreases the pH to 4.5 or below, which is indicated by a change in the color
of methyl red from yellow to red.
In the methyl red test (MR test), the test bacteria is grown in a broth medium
containing glucose. If the bacteria has the ability to utilise glucose with
production of a stable acid, the color of the methyl red changes from yellow
to red, when added into the broth culture.
The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic
acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol
of H2 per mol of glucose fermented. The large quantity of acids produced causes
a significant decrease in the pH of the culture medium.
Media and Reagents used in Methyl Red (MR) Test
MRVP broth (pH 6.9)
Ingredients per liter of deionized water:
buffered peptone= 7.0 gm
glucose= 5.0 gm
dipotassium phosphate= 5.0 gm
Methyl red solution, 0.02%
a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
c. Store at 4 to 8 degree C in a brown bottle. Solution is stable for 1 year.
Procedure of Methyl Red (MR) Test
8. Prior to inoculation, allow medium to equilibrate to room
temperature.
9. Using organisms taken from an 18-24 hour pure culture, lightly
inoculate the medium.
10.Incubate aerobically at 37 degrees C. for 24 hours.
11.Following 24 hours of incubation, aliquot 1ml of the broth to a
clean test tube.
12.Reincubate the remaining broth for an additional 24 hours.
13.Add 2 to 3 drops of methyl red indicator to aliquot.
14.Observe for red color immediately.

Result Interpretation of Methyl Red (MR) test

Positive Reaction: A distinct red color (A)

Examples: E. coli, Yersinia sps, etc.
Negative Reaction: A yellow color (B)
Examples: Enterobacter aerogenes, Klebsiella pneumoniae,  etc.
A weak positive is red-orange. If an orange color is seen, incubate the
remainder of the broth for up to 4  days and repeat the test after further
incubation. In this case it may also be  helpful to set up a duplicate broth at 25C.

Uses of Methyl Red (MR) Test

Originally the paired MR-VP tests were used to distinguish between members
of the family Enterobacteriaceae, but now they are used to characterize other
groups of bacteria including Actinobacteria.

Quality Control of Methyl Red (MR) Test

Klebsiella pneumoniae ATCC 13883—MR negative (yellow)
Escherichia coli ATCC 25922—MR positive (red)

Limitations of Methyl Red (MR) Test

1. It is recommended that further biochemical tests be performed on
pure cultures for complete identification.
2. The methyl red test must not be performed unless the medium
has been incubated for a minimum of 48 hours. Tests that are run
too early may result in false-positive interpretation.
3. It is important that a light inoculum be used. If an inoculum is too
heavy, bacterial growth may be inhibited and result in invalid test
results.
4. Incubation periods up to 5 days may be necessary for the methyl
red test.