BIOCHEMICAL TESTS

Submitted by

NIKITA JAIN

Scholar No: 122117115

RABINDRA KUMAR

Scholar No: 122117118

Under the guidance of

Dr. MAMTA VERMA

DEPARTEMENT OF CHEMICAL ENGINEERING & BIOTECHNOLOGY

MAULANA AZAD NATIONAL INSTITUTE OF TECHNOLOGY (MANIT)

BHOPAL-462051

CONTENTS
1) INTRODUCTION…………………………………………………..

2) INDOLE TEST……………………………………………………….

3) METHYL AND VOGES PROSKAUER TEST…………………….

4) CITRATE UTILIZATION TESTS………………………………….

5) OXIDASE TEST………………………………………………………

6) APPLICATIONS………………………………………………………

7) LIMITATIONS…………………………………………………………

8) REFERENCES…………………………………………………………
1) INTRODUCTION

Biochemical testes terms meaning tests used to determine physiological characteristic of

microorganism particularly in terms of bacterial enzymes and chemistry of bio oxidation.

Biochemical tests term meaning tests that show biochemical properties and reaction of
bacteria to achieve identification of microorganism.

The IMViCTests consists of four different tests:-

A. Indole production
B. Methyl Red
C. Voges- Proskauer
D. Citrate Utilization

The name IMViC stands for the first letter of the name of each test in the series ,with the
lower case “i” included just for the ease of pronunciation.

These tests were designed to differentiate gram negative intestinal bacilli (family
Enterobacteriaceae) particularly E.coli and Enterobacter- Klebsiella group, on the basis of
their biochemical properties and enzymatic reactions in the presence of specific substrates.

2) INDOLE PRODUCTION TESTS

Tryptophan, an essential amino acid, is oxidised by some bacteria by an enzyme

tryptophanase resulting in the formation of indole ,pyruvic acid and ammonia. It is
performed by inoculating a bacterium into tryptophan broth ,indole produced during the
reaction is detected by adding Kovac’s reagent (dimethylaminobenzaldehyde) which
produces a cherry –red reagent layer as illustrated:-

Indole + Kovac’s reagent HCl Rosindole + H2O

If we perform the experiments taking cultures of E.coli and E.aerogenes thus E.coli is an
indole positive while E.aerogenes is an indole negative bacterium.

3) METHYL RED AND VOGES –PROSKAUER TESTS

The Methyl red (MR) and the Voges –Proskauer test (V-P) are used to differentiate two
major types of facultatively anaerobic enteric bacteria that produces large amounts of acid
and those that produce the neutral product acetoxin as end product. Both these are performed
simultaneously because they are physiologically related and are performed on the same
medium MR-VP broth. Opposites are usually obtained for the methyl red and Voges –
Proskauer tests,i.e. ,MR+,VP- or MR-,VP+.

In these tests ,if an organisms produces large amounts of organic acids :formic, acetic ,lactic
and succinic (end products )from glucose,the medium will remain red (a positive test) after
the addition of methyl red a pH indicator(i.e. pH remaining below 4.4).In other
organisms,methyl red will turn yellow(a negative test) due to the elevation of pH above 6.0
because of enzymatic conversions of the organic acids(produced during the glucose
fermentation ) to nonacidic end products such as ethanol and acetoxin (acetylmethylcarbinol).

MRVP tests are of value in the separation of E.coli and E.aerogenes(both coliform bacteria)
which appear virtually identical except for certain physiological differences that are used as
indicators of the sanitary quality of water, foods, food-production and eating establishments.

In the MR test, the methyl red indicator in the pH range of 4 will remain red throughout tube
which is indicative of a positive test ,while turning of methyl red to yellow is a negative test.

In the V-P tests, the development of a crimson to ruby pink color,may be most intense of the
surface ,is indicative of positive VP tests while no change in coloration is a negative tests.

E.coli shows a positive MR tests and Eneterobacter aerogenes a negative MR tests while
E.aerogenes shows a positive V-P tests and E.coli a negative V-P tests.

4) CITRATE UTILIZATION TESTS

Citrate tests is used to differentiate among enteric bacteria on the basis of their ability to
ferment citrate as the sole carbon source .The utilization of citrate depends on the presence of
an enzyme citrase produced by an organism, that break down the citrate to oxaloacetic acid
and acetic acid.

These products are later converted to pyruvic acid and carbon dioxide enzymatically as
shown below:-

Citric acidcitrase Oxaloacetic acid + Acetic acid Pyruvic Acid +CO2

The citrate test is performed by inoculating the microorganisms into an organic synthetic
medium, Simmon’s citrate agar ,where sodium citrate is the only source of carbon and
energy.Bromothymol blue is used as an indicator.When the citric acid is metabolized,the
CO2 generated combines with sodium and water to form sodium carbonate an alkaline
product,which changes the colour of the indicator from green to blue and this constitutes a
positive test:

CO2 + 2Na+ +H2O Na2CO3

(blue colour)

Bromothymol blue is green when acidic (pH 6.8 )and blue when alkaline (pH 7.6)

 E.aerogenes is citrate positive where colour of the medium changes from green to blue
while E.coli is citrate negative where medium changes green.

5) OXIDASE TEST

This test depends on the presence of cytochrome oxidase in bacteria that will catalyze the
transport of electrons between electron donors and redox dye. Tetramethyl-p-phenylene
diamine dihydrochloride in the reagent is reduced to deep purple color. This test is used for
the screening of Pseudomonas, Vibrio, Neisseria, Brucella and Pasteurella, which give
positive test. Enterobacteriaceae are oxidase negative.

Oxidase reagent is specially prepared as 10g/l or 1% solution of tetramethyl-p-phenylene

diamine dihydrochloride.

Filter Paper Method

Place a piece of filter paper in petri dish and add 3 drops of freshly prepared oxidase reagent.
Using a sterile glass rod, remove a colony of test organisms from a culture plate and smear it
on the filter paper.

Oxidase positive organisms give blue colour within 5-10 seconds, and in oxidase negative
organisms, colour does not change

6) APPLICATIONS

 Catalase test:- This test determines bacterial production of catalase enzyme.

 Decarboxylase test (lysine & Ornithine):-A determination of bacterial enzymatic
capability to decarboxylatean amino acids to form an amine with resultant alkalinity.
 Esculin test:- To determine the ability of an organism to hydrolyze the glycoside esculin
to esculetin and glucose in the presence of bile.
 Gelatinase test-A test to determine bacterial production of gelatinase enzyme that liquefy
gelatin.
 Indole test-A test to determine bacterial ability to split indole from tryptophan molecule.
 Malonate test-A method to establish if a bacterial isolate is able to utilize sodium
malonate as it only source of carbon.
 OxidaseTest:-Used to demonstrate the ability of a bacterium to produce the enzyme
cytochrome- coxidase, capable of reducing oxygen. Only Aerobic bacteria have this
enzyme. This test will distinguish Aerobic vs. Anaerobic metabolism.
 IMViC: -A battery of biochemical tests known as IMViC is used in the clinical lab to
distinguish between enteric microorganisms. The acronym IMViC stands for indole,
methyl red, Voges- Proskauer and citrate. The"i" in the acronym is added for
pronunciation purposes.
 MethylRed (“M”)–An indicator of low pH (red below pH of 4.4)–used to show the
mixed acid fermentation ability of bacteria.
 VP-Voges-ProskauerTest(“Vi”)–Used to show bacterial production of acetoin, also
known as 2,3-butanediol.
7) LIMITATIONS

 Temperature
 pH
 Media composition

8) REFERENCES: