The document discusses various biochemical tests including IMViC tests which are used to identify and differentiate members of the Enterobacteriaceae family. The IMViC tests examine indole production, methyl red reaction, Voges-Proskauer reaction, and citrate utilization. The document provides information on the principles, procedures, and expected results of each biochemical test as well as the microorganisms that typically demonstrate positive or negative reactions.

1. SHITAL TRIVEDI
ASSISTANT PROFESSOR,
Department of pharmaceutical technology,
L.J.INSTITUTE OF PHARMACY
Subject: Pharmaceutical Microbiology
SEMESTER 5
IMViC-BIOCHEMICAL TESTS

2. BIOCHEMICAL TESTS
• Biochemical tests are the tests to identify chemical reaction/metabolic
reaction occurring in biological organisms.
• Biochemical tests are based on the various metabolic pathways
involving specific enzyme systems in biological organisms.
• Biochemical tests are the important methods for the microbial
identification.

3. IMViC Tests
• IMViC is an acronym that stands for four different tests.
• Each of the letters in “IMViC” stands for one of these tests.
I- Indole Test,
M - Methyl red,
V - Voges-Proskauer, and
C – Citrate
Lowercase “i” is added for the ease of pronunciation.
• IMViC tests are employed in the identification/differentiation of members
of family enterobacteriaceae.

4. Indole Test
Principle:
+ Kovac’s or Ehrlich’s reagent
(4 (p)-dimethylamino benzaldehyde)
Red-colored compound
Test Organism

5. Indole Test
Procedure:
• Two methods are in use:
1. Conventional tube method requiring overnight incubation, which
identifies weak indole producing organisms.
2. Spot indole test, which detects rapid indole producing organisms

6. Procedure of Conventional Tube method for Indole Test
(1) (2) (3)
Test
organism
(Inoculation) Kevok’s reagent, 0.5 ml
Incubation
(At 37°C for 24-48 hours
in ambient air.)
Tryptone broth Culture broth Observation

7. Spot Indole Test
Principle:
Indole + Cinnamaldehyde Blue-green compound.
Procedure of Spot Indole Test:
1% paradimethylaminocinnamaldehyde Inoculation of test organism
30 seconds
Piece of filter paper
Observation &
Interpretation.

8. Indole positive organisms:
Most strains of E.coli, Proteus vulgaris, Morganella morganii and
Providenicia species.
• Morphological characteristics, gram stain result, colony
characteristics and biochemical tests can assist in the rapid
identification of test organisms.
• Example:
• Gram negative, a flat, dry lactose fermenting (pink) colony on
MacConkey agar that is also spot indole positive and oxidase
negative can be reported presumptively as E.coli.

9. In Diagnostic laboratories, Indole test can is performed
using multitest agar. :
Three most commonly used agar medium are:
•Sulfide-indole-motility (SIM) medium: The SIM medium is a multitest agar used
to test for indole production while simultaneously determining motility and
hydrogen sulfide producing abilities of the isolate.
•Motility-indole-urease (MIU) medium: MIU medium is used to test for indole
and urease producing characteristics of the organism along with a test for
motility.
•Motility-indole-ornithine (MIO) medium: In addition to testing for indole
production, MIO agar is used to test for motility and ornithine decarboxylase.

10. Methyl red Test
Principle:
• Methyl Red (MR) test determines whether the microbe performs
mixed acids fermentation when supplied with glucose.
Embden–Meyerhof–Parnas (EMP
pathway)/ Glycolysis
Glucose
Pyruvic acid
Mixed acids pH
≤ pH 4.4
Methyl red
Yellow Red (+ve test)

11. Methyl red test
Media and Reagents:
• Methyl red-Voges-Proskauer (MR/VP) broth (formulated by Clark and
Lubs) is used in this test. The composition of MR/VP broth is as
follows:
Ingredient MR/VP broth (g/L)
Polypeptone 7 g
Glucose 5 g
Dipotassium phosphate 5 g
Distilled water q.s.. 1 L
Final pH 6.9
• Methyl Red pH Indicator:
Methyl red: 0.1 g in 300 mL of 95% ethyl alcohol.
Distilled water: 200 mL

12. Quality Control
• Positive and negative controls should be run after the preparation of
each lot of medium and after making each batch of reagent.
• Suggested controls include the followings:
• Positive control: Escherichia coli
• Negative control: Klebsiella (formerly Enterobacter) aerogenes

13. Methyl red Test
Procedure:
(1) (2) (3)
Test
organism
(Inoculation) Methyl red indicator, 5 drops
Few minutes
Incubation
(At 35 °C for 48 hours.)
MR-VP broth Culture broth Observation

14. • Methyl-Red (MR) positive organisms
• Escherichia coli
• Shigella species
• Salmonella species
• Citrobacter species
• Proteus species
• Yersinia species
• Methyl-Red (MR) negative organisms
• Enterobacter species
• Hafnia species
• Serratia marcescens
• Klebsiella pneumoniae

15. Voges-Proskauer Test
• Voges and Proskauer, in 1898, first observed the production of a red color
after the addition of potassium hydroxide to cultures grown on specific
media.
• Harden later revealed that the development of the red color was a result of
acetyl-methyl carbinol production.
• In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the
medium before adding potassium hydroxide.
The Voges-Proskauer test identifies bacteria that are able to metabolize
pyruvic acid to form acetyl-methyl carbinol (acetoin).
Reference:
https://catalog.hardydiagnostics.com/cp_prod/content/hugo/rapidanginosusi
dkit.html

16. Voges-Proskauer Test
Principle:

17. Voges-Proskauer Test
Media and Reagents:
• MR-VP broth is used for both MR Test and VP test. Only the addition of
reagent differs, and both tests are carried out consecutively.
• Reagents:
• 1. Barritt's A reagent: Alpha-naphthol, 5% color intensifier
• α – naphthol 5g
• Absolute ethyl alcohol q.s. to 100 mL
• 2. Barritt's B reagent: Potassium Hydroxide, 40%, oxidizing agent
• Potassium hydroxide 40g
• Distilled water q.s. to 100 mL

18. Quality Control
• Positive and negative controls should be run after preparation of each
lot of medium and after making each batch of reagent.
• Suggested controls include the following:
• Positive Control: Klebsiella (formerly Enterobacter) aerogenes
• Negative Control: Escherichia coli

19. Voges-Proskauer Test:-Procedure:
(1) (2) (3) (4)
First add 0.6mL of 5% α-naphthol,
(Inoculation) followed by 0.2 mL of 40% KOH.
Test Organism 1 ml aliquot
Incubation
(At 37 °C for 24 - 48 hours.) Shake the tube
gently, then undisturbed for 10-30 min.
Clean test tube
MR-VP broth Culture broth Observation
(The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a
copper like color(reaction between KOH & α-naphthol ), potentially resulting in a false positive interpretation.)

20. Voges-Proskauer (VP) Positive Organisms of
Enterobacteriaceae family are:
• Klebsiella species
• Enterobacter species
• Hafnia species
• Serratia species
• Voges-Proskauer (VP) Positive Organisms
• Citrobacter species
• Shigella species
• Yersinia species
• Salmonella species
• E.coli

21. Citrate utilization test:- Principle:
• Citrate utilization test is used to determine the ability of bacteria to utilize..
Sodium citrate as its only carbon source and
 Inorganic ammonium dihydrogen phosphate as the sole fixed nitrogen
source.
CO2 + water + Sodium ions
Sodium bicarbonate and sodium carbonate
pH
≥ 7.6
Deep forest green to blue color change

22. Citrate utilization test
Media:
• The composition of Simmons citrate agar is as follows: The medium is
poured into a tube on a slant.
Ingredient Simmons Citrate Agar (g/L)
Ammonium dihydrogen phosphate 1 g
Dipotassium phosphate 1 g
Sodium chloride 5 g
Sodium citrate 2 g
Magnesium sulfate 0.20 g
Agar 15 g
Bromothymol blue 0.08 g
Distilled water 1 L
Final pH 6.9

23. Citrate utilization test
Procedure:
(1) (2)
Test organism
(Inoculation by streaking/stabbing) No growth Growth
Incubation
(At 35°C to 37°C for 18 to 24 hours)
or up to 7 days
Simmons citrate agar slant Observation
Bromothymol blue

24. Quality Control
• Positive control: Klebsiella pneumoniae
• Negative control : Escherichia coli

25.  List of Bacteria which gives positive citrate utilization test
• Klebsiella pneumoniae
• Enterobacter species (minority of strains gives negative result)
• Citrobacter freundii
• Salmonella other than Typhi and Paratyphi A
• Serratia marcescens
• Proteus mirabilis (minority of strains gives negative result)
Citrate Test: variable (different strains give different results)
• Proteus vulgaris
• Vibrio cholerae
• Vibrio parahaemolyticus

26.  Citrate test: negative
• Escherichia coli
• Shigella spp
• Salmonella Typhi
• Salmonella Paratyphi A
• Morganella morganii
• Yersinia enterocolitica
Although uncommon, natural E. coli variants that are citrate positive have
been isolated. Citrate-negative strains of E. aerogenes have also been
found.
Content and images used in this presentation may be subject to copyright

27. References:
• https://microbeonline.com/imvic-tests-principle-procedure-and-
results/
• https://catalog.hardydiagnostics.com/cp_prod/content/hugo/rapidan
ginosusidkit.html