IMVIC REACTIONS

LAKSILU PEIRIS
GS/MSc/FOOD/3630/08
2008/2010
20/03/2010
5.0 IMViC Test
Title: IMViC Test
Date: 20.03.2010
Experiment No :05
5.1 Introduction:
There four test to differentiate between principle of microorganisms of
enterobacteriaceae. This group can be found in the intestinal tract of human and lower
mammals.
This family includes
a. Pathogens- Salmonella, Shigella
b. Occasional pathogens- Proteus, Kiebsiella
c. Normal Intestinal Flora- Escherichia, Enterobactor
IMViC reactions are a set of four useful reactions that are commonly employed in the
identification of members of family enterobacteriaceae. The four reactions are: Indole
test, Methyl Red test, Voges Proskauer test and Citrate utilization test. The letter “i” is
only for rhyming purpose.

This test uses Simmon's citrate agar to determine the ability of a microorganism to use
citrate as its sole carbon source. The citrate agar is green before inoculation, and turns
blue as a positive test indicator.

These IMViC tests are useful for differentiating the family Enterobacteriaceae,
especially when used alongside the Urease test.
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.

5.2 Indol Test

5.2.1 Introduction

• Indole test

Some bacteria can produce indole from amino acid tryptophan using the enzyme
typtophanase.

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

• Methyl Red test

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.

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

Principle: While MR test is useful in detecting mixed acid producers, VP test detects butylene
glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of
butylene glycol. In this test two reagents,40% 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 guanidinecomponents 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.

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

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.
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