Identification of Bacteria: Biochemical Tests, (IMViC Test).

Introduction:

• Living microbes can be differentiated by enzyme catalyzed metabolic reactions.

• Presence or absence of particular enzymes, intermediary metabolite, end products give valuable information in identification and classification of microbes.

• IMViC is an acronym used for four tests as,

• I= Indole Test.

• M= Methyl Red Test.

• V= Voges-Proskauer test.

• i= Added for ease of pronunciation.

• C= Citrate utilization test.

• IMViC tests are employed in the identification/differentiation of members of family enterobacteriaceae.

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 or Ehrlich’s reagent which contains 4 (p)-dimethylamino benzaldehyde, which reacts with indole to produce a red-colored compound.

• Indole test is a commonly used biochemical test (e.g. in IMVIC test, SIM test, etc). Indole test helps to differentiate Enterobacteriaceae and other genera.

Procedure of  Conventional Tube method:

1. Inoculate the tryptophan broth with broth culture.

2. Incubate at  37°C for 24-28 hours in ambient air.

3. Add 0.5 ml of Kovac’s reagent to the broth culture.

Result:

• Positive: Pink colored ring after the addition of an appropriate reagent

• Negative: No color change even after the addition of an appropriate reagent. e.g. Klebsiella pneumoniae.

Methyl Red (MR) test:

• This test is utilized to detect presence of acids during fermentation of glucose.

Principle:

• In acid fermentation, three acids (acetic, lactic and succinic) are formed in significant amounts per mol of glucose fermented.

• These large amounts of acid result in a significant decrease in the pH of the medium below 4.4. 

• This is visualized by using a pH indicator, methyl red, which is yellow above pH 5.1 and red at pH 4.4.

Procedure:

1. Glucose Phosphate Broth is used in this test.

2. Inoculate two tubes containing Broth with a pure culture of the microorganisms under investigation.

3. Incubate at 35 °C for up to 4 days.

4. Add about 5 drops of the methyl red indicator solution to the tube.

5. A positive reaction is indicated if the color of the medium changes to red within a few minutes.

Result:

• The development of a stable red color on the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test. 

Voges-Proskauer (VP) test:

Principle:

• Pyruvic Acid the major metabolite of glucose fermentation is further metabolized by the bacterias to produce acetoin (acetyl methyl carbinol), a neutral-reacting end product.

• 

• In the presence of atmospheric oxygen and 40% potassium hydroxide, acetoin is converted to diacetyl, and alpha-naphthol serves as a catalyst to bring out a red complex.

Procedure:

1. Inoculate a tube of Glucose phosphate  broth with a pure culture of the test organism.

2. Incubate for 24 hours at 35°C

3. At the end of this time, aliquot 1 mL of broth to clean the test tube.

4. Add 0.6mL of 5% α-naphthol, followed by 0.2 mL of 40% KOH. (Note: It is essential that the reagents be added in this order.)

5. Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

Results:

• 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.

Citrate utilization test:

• Citrate utilization test is used to determine the ability of bacteria to utilize sodium citrate. 

• When an organic acid such as citrate is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced ultimately. 

• The carbon dioxide that is released will subsequently react with water and the sodium ion in the medium to produce sodium carbonate, an alkaline compound that will raise the pH.  

• Growth usually results in the bromothymol blue indicator, turning from green to blue. The bromothymol blue pH indicator is a deep forest green at neutral pH.  With an increase in medium pH to above 7.6, bromothymol blue changes to blue.

Result:

• Citrate positive:  growth will be visible on the slant surface and the medium will be an intense blue. 

• The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.