BACHELOR OF SCIENCE IN MEDICAL LABORATORY

PROGRAMME NAME: BACHELOR OF SCIENCE IN MEDICAL LABORATORY

SCIENCE

COURSE : MEDICAL BACTERIOLOGY

COURSE CODE : MLSC 353

LECTURER’S NAME : MR. J. KATUAH

ASSIGNMENT TOPIC : IMViC TEST

STUDENT NAME : VERONICA GRECIAN

STUDENT ID : BMLS/21/386

DUE DATE : 07TH NOVEMBER, 2023

 IMViC TEST

IMViC test is a series of four useful tests that are commonly employed in the identification
of members of enterobacteriaceae. These include; Indole test, Methyl Red test (MR), Voges
Proskauer (VP) test and Citrate utilization test. The letter ´´i´´ is only for rhyming purpose
(Rao, 2006).

PRINCIPLE OF IMViC TEST

The IMViC test is based on the variations in the metabolic requirements and properties of
different genera and species of bacteria. The Indole test and citrate utilization test, detect
the ability of bacteria to produce specific enzymes and utilize specific nutrients. On the other
hand the MR test and VP test detect the final metabolic products produced by bacteria
utilizing specific nutrients. For this purpose, the test bacteria is cultured in specific culture
media; different media for the different tests (expect MR and VP which need the same culture
media; MR-VP media) (Dahal, 2022).

HOW TO PERFORM IMViC TEST

Three test tubes must be inoculated to obtain the results of these four tests: indole test, methyl
red – Voges Proskauer broth (MR-VP broth), and citrate. IMViC tests are employed in the
identification/differentiation of members of the family Enterobacteriaceae.

INDOLE TEST

Principle: some bacteria can produce indole from an amino acid tryptophan using the
enzyme tryptophanase.

The production of indole can be detected using Ehrlich’s reagent or Kovac’s reagent. Indole
reacts with the aldehyde in the reagent to give a red or pink color. An alcoholic layer
concentrates the red color as a ring at the top. The bacteria to be tested must be inoculated in
peptone water, which contains amino acid tryptophan and incubated overnight at 37 degree
celcius. The bacteria Escherichia coli is positive with this test (Rao, 2006).

METHYL RED (MR) TEST:

Principle: This is to detect the ability of an organism to produce and maintain stable
acid end products from glucose fermentation. Some bacteria produce large amounts of acids
from glucose fermentation that they overcome the buffering action of the system. Methyl Red
is a pH indicator, which remains red in color at a pH of 4.4 or less. The bacterium to be tested
is inoculated into glucose phosphate broth, which contains glucose and a phosphate buffer
and incubated at 37 ºC for 48 hours. Development of red color is taken as positive. MR
negative organism produce yellow color. Escherichia coli, Proteus, Serratia, Citrobacter,
salmonela and Yersinia are positive.

VOGES PROSKAUER (VP) TEST

Principle: while MR test is useful in detecting mixed acid producers, VP test detect butylene
glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of
butylene glycol. In this test two reagents, 40% potassium hydroxide (KOH) and alpha-
naphthol are added to test broth after incubation and exposed to atmospheric oxygen.
If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then
reacts with guanidine components of peptone, in the presence of alpha-naphthol to
produce red color. Role of alpha-naphthol is that of a catalyst and a color intensifier.
Bacterium to be tested is inoculated into glucose phosphate broth and incubated for at least
48 hours. Klebsiella, Serratia, and Enterobacter are positive (Rao, 2006).

CITRATE UTILIZATION TEST

Principle: this test detects the ability of an organism to utilize citrate as the sole source of
carbon and energy. Bacteria are inoculated on a medium containing sodium citrate and a pH
indicator bromothymol blue. The medium also contains inorganic ammonium salts, which is
utilized as sole source of nitrogen. Utilization of citrate involves the enzyme citritase,
which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken

down to pyruvate and CO₂. Production of Na₂CO₃ as well as NH₃ from utilization of sodium

citrate and ammonium salt respectively results in alkaline pH. This results in change of
medium’s color from green to blue. Bacterial colonies are picked up from a straight wire and
inoculated into slope of Simmon’s citrate agar and incubated overnight at 37ºC. If the
organism has the ability to utilize citrate, the medium changes its color from green to blue.
Klebsiella, Proteus, and Serratia are positive (Rao, 2006).

Citrate Oxaloacetate + Acetate

Oxaloacetate pyruvate +CO₂

CO₂ +Na + H₂O Na₂CO₃

 REFERENCES

Dahal, P., 2022. microbe notes. [Online]

Available at: htpps://microbenotes.com
[Accessed 10 July 2022].

Rao, S., 2006. microrao. [Online]

Available at: https://www.microrao.com
[Accessed 12 June 2006].