Biochemical test

FOR S. Y. B.PHARM

1. Biochemical Test Living micro-organism are differentiated on the basis of various enzyme based catalyzed metabolized reaction. Presence or absence of certain enzyme metabolites or end products give valuable information in identifying and classifying m.o . IMViC stands for “I” is for indole ; “M” is for methyl red; “V” is for Voges-Proskauer , and “C” is for citrate,  lowercase “i” is added for the ease of pronunciation.  IMViC is an acronym that stands for  four different tests Indole test Methyl red test Voges-Proskauer test Citrate utilization test IMViC tests are employed in the identification/differentiation of members of family enterobacteriaceae .

2. General procedure for performing IMViC Tests and their interpretations: Cultures of any members of enterobacteriaceae have to grow for 24 to 48 hours at 37°C and the respective tests can be performed:

3. Methyl Red Test For this test we require M icro-organism - E.coli , Enterobacteraerogenes Indicator- Methyl red Media- Glucose phosphate broth media Principle- Some micro-organism utilise glucose for energy production During these process they release acid which changes pH of broth. These change in pH is indicated by change in colour of broth due presence of methyl red.

4. Procedure- Prepare Glucose phosphate broth & sterlize it. Cool media & inoculate test micro-organism in test tube Incubate these test tube to grow for 24 to 48 hours at 37°C . After growth add the Methyl red indicator into it mix well & observe the result. Result Positive test – development of red color negative test – no color change We can identify specific m.o . by using its biochemical characteristics.

5. Citrate Utilization Test For this test we require M icro-organism - E.coli , Enterobacteraerogenes Indicator- Bromothymol blue Media- Citrate broth media Principle- These test differentiate m.o . which utilise citric acid as source of carbon for growth. But test is depend on amount of enzyme. During utilization of citrate it release turbid substance which make solution alkaline. pH change determine by addition of BTB ( Bromothymol blue)

6. Procedure- Prepare citrate broth & sterlize it. Cool media & inoculate test micro-organism in test tube Incubate these test tube to grow for 24 to 48 hours at 37°C . After growth add the Bromothymol blue indicator into it mix well & observe the result. Result Positive test – development of blue color negative test – no color change We can identify specific m.o . by using its biochemical characteristics.

7. Indole Production Test For this test we require M icro-organism - E.coli , Enterobacteraerogenes Indicator- Kovac’s Reagent Media- Peptone broth media Principle- Some micro-organism consist of enzyme tryptophanase . This enzyme degrade amino acid tryptophan into indole , pyruvic acid and ammonia. Indole production is detected by inoculating test m.o . in peptone water.

8. Procedure- Prepare peptone broth & sterlize it. Cool media & inoculate test micro-organism in test tube Incubate these test tube to grow for 24 to 48 hours at 37°C . After growth add the Kovacs reagent into it mix well & observe the result. Result Positive test – development of red color in alcohol layer ( Idole Produced) negative test – no color change We can identify specific m.o . by using its biochemical characteristics.

9. Voges-Proskauer (VP) test : For this test we require M icro-organism - E.coli , Enterobacteraerogenes Indicator- 1% NaOH + 5% ά - Napthol Media- Glucose Phosphate broth media Principle- Some micro-organism ferment m.o . carbohydrate with production of acetyl methyl carbonyl ( Acetoin ). In presence of alkali and atmospheric oxygen , small amount of acetoin present into the media oxidised to diacetyl . These diacetyl react with peptone of broth to give red colour .

10. Procedure- Prepare glucose phosphate broth & sterlize it. Cool media & inoculate test micro-organism in test tube Incubate these test tube to grow for 24 to 48 hours at 37°C . After growth add 1 ml 1% NaOH + 3 ml 5 % ά - Napthol into it mix well & observe the result. Result Positive test – development of pink color negative test – no color change We can identify specific m.o . by using its biochemical characteristics.

11. Thank You