IMVIC test

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 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. b. Occas Occasio iona nall path pathog ogen enss- Proteus, Kiebsiella c. Norm Normal al Inte Intest stin inal al Flor Floraa- 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, Enterobacteriaceae, especially when used alongside the Urease test. 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 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 Escherichia coli and

Staphylococcus aureus •

Media: SIM agar/Peptone water o

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Tubes with tryptone /peptone broth

Reagent: Kovac’s reagent(p-dimethyl amino benzaldehyde , HCl and butanol)

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Equipment: Bunsen burner

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Inoculating loop

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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

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One tube was served as control

5.2 5.2.4

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Incubate at 37 0C for 24-48 hours

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Few drops of Kovac’s reagent was added to the culture medium.

Obsservat Ob rvatio ions ns..

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 E.coli Staphylococcus aureus 5.2.5

Test results for Indol test Positive Negative

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 indole, pyruvic acid, 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 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 •



Media: o

Tubes with MR- VP broth

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Reagent: Methy red indicator

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Inoculating loop

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5.3.4 Procedure 

The tubes of MR-VP broth were inoculated using sterile technique with given bacterial culture

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Incubate at 370C for 24-48 hours

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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.


Cultures E.coli Staphylococcus aureus

Test results for Methyl Red test Positive 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 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 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 guanidinecomponen guanidinecomponents ts 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 •



Media: o

Tubes with MR- VP broth

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Reagent: Barrit’s reagent-(Napthol solution and 16% KOH solution)

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Equipment: Bunsen burner 

Inoculating loop

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5.4.3 Procedure 

The tubes of MR-VP broth were inoculated using sterile technique with given bacterial culture

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Test the presense of acetyl-methyl carbinol by the addition of Berrit’s reagent.

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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 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.


Test results for Voges Proskaur test Negative Positive

5.4.5 Discussion

MRVP broth contains peptone, glucose, 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, product, usually acetic acid. The acetic acid is not strong enough to overcome overcome the phosphate buffer buffer in the MRVP broth. broth. Instead, the acetic acid is converted to acetylmethylcarbinol, which 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, 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 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 t he enzyme citritase, which breaks down citrate to oxaloacetate oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO 2. 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 Escherichia coli and Enterobacter

aerogenes •

Media:Simmon’s citrate agar slant

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Reagent: Bromthymol blue

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Inoculating loop

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5.5.3 Procedure 

The indicator was added prior to autoclave.

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The tubes of Simmon’s Citrate agar slant were inoculated using sterile sterile technique with given bacterial culture

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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 Observations

Negative Citrate- Blue color Positive Citrate- Prussian blue color



Test results for Citrate test Negative 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 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 and then methyl red is added. A red broth is a positive test result, whereas a yellow or 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 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 produce a red color. color. A positive VP test is red on top of the 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 organism to use citrate as its sole carbon source. 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 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

IMVIC test

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