Biochemical characterization of bacteria by IMViV, Catalase, Oxidase, TSI, Carbohydrate
fermentation test
1. INDOLE TEST -
Principle:
Indole test is used to determine the ability of an organism to split amino acid tryptophan to form
the compound indole. Tryptophan is hydrolyzed by tryptophanase to produce three possible end
products – one of which is indole. Indole production is detected by Kovac’s reagent which
contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured
compound. Indole test helps to differentiate Enterobacteriaceae and other genera.
Procedure:
Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism
in tryptophan broth.
Incubate at 37°C for 24-28 hours in ambient air.
Add 0.5 ml of Kovac’s reagent to the broth culture.
Result -
Positive: Pink colored rink after addition of appropriate reagent
Negative: No color change even after the addition of appropriate reagent.
2. METHYL RED TEST –
Principle-
Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation when
supplied glucose. Types and proportion of fermentation products produced by anaerobic
fermentation of glucose is one of the key taxonomic characteristics which help to differentiate
various genera of enteric bacteria. In mixed acid fermentation, three acids (acetic, lactic and
succinic) are formed in significant amounts. 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. These large amounts of acid results significant
decrease in the pH of the medium below 4.4. This is visualized by using pH indicator, methyl red
(p-dimethylaminoaeobenzene-O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4.
The pH at which methyl red detects acid is considerably lower than the pH for other indicators
used in bacteriologic culture media. Thus, to produce a color change, the test organism must
produce large quantities of acid from carbohydrate substrate being used.
, Procedure:
Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under
investigation.
Incubate at 35 °C for up to 4 days.
Add about 5 drops of the methyl red indicator solution.
A positive reaction is indicated, if the colour of the medium changes to red within a few minutes.
Result -
MR Positive: When the culture medium turns red after addition of methyl red, because of a pH at
or below 4.4 from the fermentation of glucose.
MR Negative: When the culture medium remains yellow, which occurs when less acid is produced
(pH is higher) from the fermentation of glucose.
3. VOGES-PROSKAUER TEST
Principle:
In Voges-Proskauer pyruvic acid, the pivotal compound in the fermentative degradation of
glucose, is further metabolized through various metabolic pathways, depending on the enzyme
systems possessed by different bacteria. One such pathways result in the production of acetion
(acetyl methyl carbinol), a neutral-reacting end product.
Procedure:
Inoculate a tube of MR/VP broth with a pure culture of the test organism.
Incubate for 24 hours at 35oC
After incubation, aliquot 1 mL of broth to clean test tube.
Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH.
Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 minutes.
Result and Interpretation:
A positive test is represented by the development of a red color 15 minutes or more after the
addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin. The
test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures
may produce a copper like color, potentially resulting in a false positive interpretation.
fermentation test
1. INDOLE TEST -
Principle:
Indole test is used to determine the ability of an organism to split amino acid tryptophan to form
the compound indole. Tryptophan is hydrolyzed by tryptophanase to produce three possible end
products – one of which is indole. Indole production is detected by Kovac’s reagent which
contains 4 (p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured
compound. Indole test helps to differentiate Enterobacteriaceae and other genera.
Procedure:
Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism
in tryptophan broth.
Incubate at 37°C for 24-28 hours in ambient air.
Add 0.5 ml of Kovac’s reagent to the broth culture.
Result -
Positive: Pink colored rink after addition of appropriate reagent
Negative: No color change even after the addition of appropriate reagent.
2. METHYL RED TEST –
Principle-
Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation when
supplied glucose. Types and proportion of fermentation products produced by anaerobic
fermentation of glucose is one of the key taxonomic characteristics which help to differentiate
various genera of enteric bacteria. In mixed acid fermentation, three acids (acetic, lactic and
succinic) are formed in significant amounts. 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. These large amounts of acid results significant
decrease in the pH of the medium below 4.4. This is visualized by using pH indicator, methyl red
(p-dimethylaminoaeobenzene-O-carboxylic acid), which is yellow above pH 5.1 and red at pH 4.4.
The pH at which methyl red detects acid is considerably lower than the pH for other indicators
used in bacteriologic culture media. Thus, to produce a color change, the test organism must
produce large quantities of acid from carbohydrate substrate being used.
, Procedure:
Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under
investigation.
Incubate at 35 °C for up to 4 days.
Add about 5 drops of the methyl red indicator solution.
A positive reaction is indicated, if the colour of the medium changes to red within a few minutes.
Result -
MR Positive: When the culture medium turns red after addition of methyl red, because of a pH at
or below 4.4 from the fermentation of glucose.
MR Negative: When the culture medium remains yellow, which occurs when less acid is produced
(pH is higher) from the fermentation of glucose.
3. VOGES-PROSKAUER TEST
Principle:
In Voges-Proskauer pyruvic acid, the pivotal compound in the fermentative degradation of
glucose, is further metabolized through various metabolic pathways, depending on the enzyme
systems possessed by different bacteria. One such pathways result in the production of acetion
(acetyl methyl carbinol), a neutral-reacting end product.
Procedure:
Inoculate a tube of MR/VP broth with a pure culture of the test organism.
Incubate for 24 hours at 35oC
After incubation, aliquot 1 mL of broth to clean test tube.
Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH.
Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 minutes.
Result and Interpretation:
A positive test is represented by the development of a red color 15 minutes or more after the
addition of the reagents indicating the presence of diacetyl, the oxidation product of acetoin. The
test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures
may produce a copper like color, potentially resulting in a false positive interpretation.
