Preparation dispensing & sterilization of media

for cultivation of micro organisms

K.S.L.PERERA
GS/MSc/FOOD/3631/08
2008/2010
20/03/2010
5.0 IMViC Test
Title: IMViC Test
Date: 20.03.2010
Experiment No :05
5.1 Introduction:
The IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae).
This family includes
a. Pathogens- Salmonella, Shigella
b. Occasional pathogens- Proteus, Kiebsiella
c. Normal Intestinal Flora- Escherichia, Enterobactor

These are the Indole test (tryptone broth), the Methyl Red and Voges-Proskauer tests
(MRVP broth) and the Citrate test (Citrate agar slants).

5.2 Indol Test

5.2.1 Introduction

• Indole test

Tryptophan hydrolysis -Some bacteria split tryptophan into indole and pyruvic acid
using the hydrolase called tryptophanase. Indole can be detected with Kovac's reagent
(Indole reagent). This test is very important in differentiating E. coli (indole positive)
from some closely related enteric bacteria. It also differentiates Proteus mirabilis
(indole negative) from all other Proteus species (indole positive). Tryptone broth is
used for this test as it contains a large amount of tryptophan.

Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent. Indole reacts with
the aldehyde in the reagent to give a red color. An alcoholic layer concentrates the red color
as a ring at the top.

5.2.2 Materials
• Cultures: 24 to 48 hour bacterial cultures of Escherichia coli and
Staphylococcus aureus
 • Media: SIM agar/Peptone water
o Tubes with tryptone /peptone broth
• Reagent: Kovac’s reagent(p-dimethyl amino benzaldehyde , HCl and
butanol)
• Equipment: Bunsen burner
• Inoculating loop
• Glass ware marking pencil

5.2.3 Procedure
 The tubes of Tryptone/Peptone were inoculated using sterile technique
with given bacterial culture- E.coli and Staphylococcus aureus
 One tube was served as control
 Incubate at 37 0C for 24-48 hours
 Few drops of Kovac’s reagent was added to the culture medium.

5.2.4 Observations.
A deep red colour developed in presence of indole which separate out on the top
layer.

The cultures we used to perform this test was E.coli & Staphylococcus aureus.

Cultures Test results for Indol test

E.coli Positive
Staphylococcus aureus Negative
5.2.5 Discussion

Indole is generated by reductive deamination from tryptophan via the intermediate

molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction,
during which the amine (-NH2) group of the tryptophan molecule is removed. Final
products of the reaction are indole, pyruvic acid, ammonia (NH3) and energy.
Pyridoxal phosphate is required as a coenzyme.

Indole-Positive Bacteria

Bacteria that test positive for cleaving indole from tryptophan include: Aeromonas
hydrophilia, Aeromonas punctata, Bacillus alvei, most Citrobacter sp., Edwardsiella
sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, most Proteus sp.
(not P. mirabilis), Plesiomonas shigelloides, Pasturella multocida, Pasturella
pneumotropica, Streptococcus faecalis, and Vibrio sp.

Indole-Negative Bacteria

Bacteria which give negative results for the indole test include: Actinobacillus spp.,
Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordtella sp.,
Enterobacter sp., Lactobasillus spp., most Haemophilus sp., most Klebsiella sp.,
Neisseria sp., Pasturella haemolytica, Pasturella ureae, Proteus mirabilis,
Pseudomonas sp., Salmonella sp., Serratia sp., Yersinia sp.

5.3 Methyl Red Test

 6 Methyl Red test

Mixed acid fermentation - Many gram-negative intestinal bacteria can be differentiated

based on the products produced when they ferment the glucose in MR-VP medium.
Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic,
succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids
produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red
when added to the medium if the organism was a mixed acid fermenter. Many of these
organisms also produce gas.

5.3.2 Materials
• Cultures: 24 to 48 hour bacterial cultures of Escherichia coli and
Staphylococcus aureus
• Media:
o Tubes with MR- VP broth
• Reagent: Methy red indicator
• Equipment: Bunsen burner
• Inoculating loop
• Glass ware marking pencil

5.3.4 Procedure
 The tubes of MR-VP broth were inoculated using sterile technique with
given bacterial culture
 One tube was served as control
 Incubate at 370C for 24-48 hours
 Test the presense of mixed acids by the addition of methy red indicaror.

5.3.5 Observations

Enterics that subsequently metabolize pyruvic acid to other acids lower the pH of the
medium to 4.2. At this pH, methyl red turns red. A red color represents a positive test.
Enterics that subsequently metabolize pyruvic acid to neutral end-products lower the
pH of the medium to only 6.0. At this pH, methyl red is yellow. A yellow color
represents a negative test.

The cultures we used to perform this test was E.coli & Staphylococcus aureus.

Cultures Test results for Methyl Red test

E.coli Positive
Staphylococcus aureus Positive

5.3.6 Discussion

The methyl red test is used to identify enteric bacteria based on their pattern of
glucose metabolism. All enterics initially produce pyruvic acid from glucose
metabolism. Some enteric subsequently use the mixed acid pathway to metabolize
pyruvic acid to other acids, such as lactic, acetic, and formic acids. These bacteria are
called methyl-red positive and include Escherichia coli and Proteus vulgaris.

5.4 Voges Proskaur Test

5.4.1 Introduction
Organisms that are negative in the methyl red test may be producing 2, 3 butanediol
and ethanol instead of acids. These non-acid products do not lower the pH as much as
acids do. Enterobacter, Serratia and some species of Bacillus produce these
substances. There is no satisfactory test for determining production of 2, 3 butanediol.
A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.
5.4.2 Materials
• Cultures: 24 to 48 hour bacterial cultures of Escherichia coli and
Staphylococcus aureus
• Media:
o Tubes with MR- VP broth
• Reagent: Barrit’s reagent-(Napthol solution and 16% KOH solution)
• Equipment: Bunsen burner
 Inoculating loop
 Glass ware marking pencil

5.4.3 Procedure
 The tubes of MR-VP broth were inoculated using sterile technique with
given bacterial culture
 One tube was served as control
 Incubate at 370C for 24-48 hours
 Test the presense of acetyl-methyl carbinol by the addition of Berrit’s
reagent.
 Shaked well and allowed for 15 minutes. The presence of rose colouration
is a positive result.
5.4.4 Observations

Tube 1: Negative -- no maroon band formed in tube after addition of Barritt's

Reagents A and B

Tube 2: Positive -- maroon band formed after addition of Barritt's Reagents A and B

The cultures we used to perform this test was E.coli & Staphylococcus aureus.
Cultures Test results for Voges Proskaur test
E.coli Negative
Staphylococcus aureus Positive

5.4.5 Discussion

MRVP broth contains peptone, glucose, and phosphate buffer. In this broth, some
bacteria will ferment the glucose to produce a mixture of fermentation acids (lactic,
acetic, and formic acids), while others will produce only acetic acid, and some will
not ferment the glucose at all.

The Voges-Proskauer test is an assay for organisms which ferment glucose to form
only one fermentation product, usually acetic acid. The acetic acid is not strong
enough to overcome the phosphate buffer in the MRVP broth. Instead, the acetic acid
is converted to acetylmethylcarbinol, which leads to a pH of approximately 6.2. After
incubation of the organism in the MRVP broth, Barritt’s Reagent A (a-napthol) and B
(40% KOH) are added. The reagents will react with acetylmethylcarbinol, and a
positive reaction will show a maroon band at the top of the broth in the tube which
will diffuse over time into the rest of the media.

The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B
(potassium hydroxide). When these reagents are added to a broth in which acetyl
methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This
color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl
carbinol, but Enterobacter and Klebsiella do.

5.5 Citrate test(Simmon’s Citrate slant)

Introduction

Simmon's citrate agar tests for the ability of an organism to use citrate as its sole
source of carbon. This media contains a pH indicator called bromthymol blue. The
agar media changes from green to blue at an alkaline pH.

5.5.2 Materials
• Cultures: 24 to 48 hour bacterial cultures of Escherichia coli and Enterobacter
aerogenes
• Media:Simmon’s citrate agar slant
• Reagent: Bromthymol blue
• Equipment: Bunsen burner
• Inoculating loop
• Glass ware marking pencil

5.5.3 Procedure
 The indicator was added prior to autoclave.
 The tubes of Simmon’s Citrate agar slant were inoculated using sterile
technique with given bacterial culture
 One tube was served as control
 Incubate at 370C for 24-48 hours
 Observed and recorded results. Utilization of citrate is an alkaline
reaction.The colour of medium changes from green to Prussian blue
recorded positive (Bromothymol blue indicator is incorporated in the
medium.

5.5.4 Observations

Negative Citrate- Blue color

Positive Citrate- Prussian blue color
The cultures we used to perform this test was E.coli & Staphylococcus aureus.

Cultures Test results for Citrate test

E.coli Negative
Staphylococcus aureus Negative

5.5.5 Discussion
The citrate test utilizes Simmon's citrate media to determine if a bacterium can
grow utilizing citrate as its sole carbon and energy source. Simmon's media
contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol
blue is yellow at acidic pH's (around 6), and gradually changes to blue at more
alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so
it is an intermediate green color. Growth of bacteria in the media leads to
development of a Prussian blue color (positive citrate). Enterobacter and
Klebsiella are citrate positive while E.coli is negative.

5.6 Conclusion
Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and
Klebsiella give the reverse: --++.The culture we used Staphylococcus aureus gives
-++- results for IMVic test.

5.7 Discussion

The IMViC tests are used to differentiate the enterics (Family Enterobacteriaceae).
The IMViC tests are useful for differentiating the Enterobacteriaceae, especially when
used alongside the urease test. When used alone, the IMViC tests are particularly
useful for differentiating Escherichia coli, Enterobacter aerogenes, Enterobacter
cloacae, and Klebsiella pneumoniae (although colonial morphology and the presence
of capsules can also be used to differentiate Klebsiella).

Except for the lowercase "i", which is added for ease of pronunciation, each of the
letters in "IMViC" stands for one of these tests. "I" is for indole; "M" is for methyl
red; "V" is for Voges-Proskauer, and "C" is for citrate.

These are the Indole test (tryptone broth), the Methyl Red and Voges-Proskauer tests
(MRVP broth) and the Citrate test (Citrate agar slants).

Methyl red (MR) is a test used to detect organisms capable of overcoming an added
phosphate buffer in the medium to lower the pH of the broth. The test was performed
only on gram (-) bacteria, as it mostly tests for enterics who can do this by performing
mixed acid fermentation. After inoculation, the broth is incubated for 5 days and
then methyl red is added. A red broth is a positive test result, whereas a yellow or
orange broth is a negative test result.
Voges-Proskauer (VP) is a test used to detect organisms that ferment but
quickly convert their acid products to acetoin. Addition of the VP reagents (KOH and
α-napththylamine) oxidizes acetoin to diacetyl, which in turn reacts with guanidine
nuclei to produce a red color. A positive VP test is red on top of the medium. No
color change is negative.
The citrate test was performed only on the gram (-) bacteria and was used to
determine the ability of an organism to use citrate as its sole carbon source. Bacteria
that possess citrate-permease are able to do this. The medium used for this test
contains bromthymol blue dye, which is green at neutral pH, but blue at a basic pH.
Bacteria that are able to survive and utilize the citrate, convert ammonium phosphate
to ammonia and ammonium hydroxide, which alkalinize the agar, turning it blue.
Thus, the conversion of the medium to blue is a positive citrate test. No color change
is negative.

5.8 References

Analytical Microbiology by Frederick Kavanagh

• Web sites- http://en.wikipedia.org/wiki/IMViC