IMViC Tests and Catalase Test

The aim of this experiment is to determine kind of Enterobacteriaceae bacteria that are found in the tubes labelled as α and β by using IMViC tests.
In microbiology, kind of bacteria in Enterobacteriaceae of determination is done by using some methods. The rapid and useful test is IMViC. For checking danger or safety of water or food, this test is so important. (1)
Enterobacteriaceae is a huge family in bacteria. They have Gram-Negative bacteria including harmless and pathogenic kinds. Coliforms are found in this family. Coliforms are separated according to fermenting lactose with gases and acid. Coliforms are rod-shaped, Gram-Negative, non-spore forming bacteria. They mostly live in aquatic environment and fecal contamination is considered with coliforms. Because of that, they are so important as indicator for these purpose. They are used for pollutions or determining pathogenic organisms. For example, Escherichia coli is one of the most known coliform like Klebsiella pneumonia and Enterobacter aerogenes. Coliforms are obtained by using IMViC test. IMViC include some serial test to determine species of coliform. Each letter except “i” refers to first letter of tests. (1, 2 and 3)
Indole test is first test and used for obtaining present of indole amino acid. Some coliforms use tryptophan as substrate and they produce indole after reaction. Kovac’s reagent, so dangerous with air, is used for that. The reagent reacts with presence of indole and gives pink/red colour on top layer of the tube. E.coli is one of bacteria giving positive result for indole test. Many Bacillus gives negative result like this experiment. (4, 5 and 6)
Methyl-red or shortly MR test is based on pH value in solution. It acts like an indicator giving red colour with acidic solution. Glycoses is first step for generating energy for living organisms. At end of glycoses, pyruvates or pyruvic acid are produced from glucose. Naturally pyruvic acid makes acidic. Bacteria convert them to more stable and safer acidic forms such as formic acid, acetic acid or lactic acid. Eventually acidic media causes change of colour with reagent. E.coli forms acidic media and it causes positive or red colour at MR test. B.subtilis demonstrates negative result or yellow-brown colour. (4, 5 and 6)
VP test or Voger-Proskauer test is the other step of IMViC. VP is for determined existence of acetone in bacterial culture. Some bacteria live by using glucose and turning into acetylmethylcarbinol, shortly acetone. Two solutions are added for that. First one is alpha-naphthol. It binds to acetone and the second solution, potassium hydroxide forms cherry red colour if it is positive. Negative is to see yellow-brown colour for this test. B.subtilis is in positive group; however same test gives negative with E.coli. (4, 5 and 6)
Citrate Utilization test is a test to control bacteria use whether citrate or not as energy-carbon source. When bacteria are added into a media that have sodium citrate and pH indicator like bromothymol blue as basic test materials, citrase enzyme in bacteria breaks down citrate to oxaloacetate and acetate. Oxaloacetate becomes to pyruvate and CO2. On the other hand, after some reaction with acetate, sodium citrate and ammonium salts cause changes in alkaline pH value. These changes forms colour media to blue from green if it is positive like in B.subtilis. Negative result doesn’t demonstrate any growth on media. E.coli can be given an example for negative. (4, 5 and 6)
Catalyst test is one of simple tests. Some bacteria can break down H2O2 to water for producing energy. In this test, H2O2 is dropped onto cells directly and if bubble is observed, it means test is positive. For E.coli, it is variable, but for B.subtilis, it is positive. (4, 5 and 6)
Material and Methods:
Indole Production Test,
0.3 ml of Kovac’s reagent was taken and transmitted into the sample tubes α and β with Tryptone in hood. After a while, colours of samples were observed.
Methyl-Red Test,
1 ml of methyl-red indicator was added into the samples with glucose phosphate broth and the samples were waited for colour change.
Voges-Proskauer Test,
0.9 ml of solution A and 0.3 ml of solution B of Barrit’s reagent were added into samples and they were observed.
Citrate Utilization Test,
Tubes with Simmon’s Citrate Agar were transmitted into test samples and they were waited for 48 hours at 37°C in incubator. After that, they could be observed.
Catalase Test,
3% H2O2 solution was dropped onto bacterial colonies of the test samples that were incubated at small amounts onto a plate before. The samples form bubble was observed.
Samples Indole Methyl-red Voger-Proskauer Citrate Utilization
α – + – +
β + – – –
Table 1- the results of IMViC tests for 2 strains (α and β)
Catalase Test
α +
β +
Table 2- Catalase Test for 2 strains (α and β)
α and β refer to E.coli and B.subtilis as unknown samples.
In this experiment, it is aimed to determine kinds of the samples in coliforms by using some serial tests according to metabolic activities.
According to the results of this experiment, it can be seen that α and β have different metabolic activity. For first test for IMViC, indole test was done for each sample. Indole test is bacteria use tryptophan and turns it into indole amino acid. At presence of indole in media, Kovac’s reagent gives a pink layer on top of tube by forming a complex with indole. The sample β gave positive for indole test and the bacteria in sample β uses tryptophan for its metabolism. E.coli is one of kinds of bacteria that are positive at indole test. (5,6)
At second test, methyl-red test, reagent is used for obtaining pH value in sample basically. Methyl red acts as indicator to shows colour change according to pH value. Some microorganism metabolizes glucose to pyruvic acid and it makes environment acidic, but they convert pyruvic acid to some forms such as lactic acid, acetic acid or formic acid for preventing accumulation of pyruvic acid. However environment is still acidic and methyl red can give a reaction. Positive test that bacteria produce low pH value about 4 forms red colour from yellow. Negative gives yellow with more basic environment. α and β indicate different and unexpected result for MR test. The sample α may be B.subtilis and the sample β may be E.coli by comparing with the other tests, however α and β show unexpected results. There may be experimental error. The tubes that have been labelled as α and β could have been confused before experimental process. α and β may have been confused again at observation part. These kind of experimental errors may be and also contaminated culture with the other bacterial cultures or wrong aseptic techniques may give these results. (5, 6)
VP test or Voger-Proskauer is used for identified presence of acetone in bacterial culture. At VP is applied with two solutions for reacting with acetone and giving colour. These are alpha-naphthol and potassium hydroxide. If bacteria turns glucose into acetylmethylcarbinol at digestion, alpha-naphthol reacts and potassium hydroxide gives cherry red colour for positive result. At negative result, yellow-brown or copper colour indicates. In this experiment, there is no positive result. Both cell cultures don’t give acetone after their metabolic activity as expected. E.coli and B.subtilis must have negative result after VP test. (5, 6)
Citrate utilization test is to detect use of citrate by bacteria. Some bacteria use citrate for their metabolic activity. Citrate utilization test uses reagent that can change their colour after reaction in pH value. If test is positive, a change in colour forms from green to blue. B.subtilis can cause this kind of change on media. The sample α can be seen a B.subtilis culture. E.coli culture doesn’t look like that like at β culture. (5, 6)
Catalyse test is the easiest test than the other tests. Drops of H2O2 were poured on two cultures that were found on different places of same slide. Bubbles were observed at both samples. Already forming bubbles were expected at B.subtilis. The sample α that is thought it is B.subtilis shows same results with catalyse test. On the other hand, E.coli bacteria were variable about forming bubbles. In this experiment, β sample is E.coli that can form bubble with H2O2. (5, 6)
Except MR results, samples can be identified which kind of bacteria exist in tubes. According to Table 1 and Table 2, α sample is B.subtilis and β sample is E.coli. (5,6)

[1] Received on 24 May,
MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
[2] Received on 24 May,
Don J. Brenner; Noel R. Krieg; James T. Staley (July 26, 2005) [1984 (Williams & Wilkins)]. George M. Garrity, ed. The Gammaproteobacteria. Bergey’s Manual of Systematic Bacteriology.  2nd ed. New York: Springer.
[3] Received on 24 May,
[4] Received on 24 May,
MacFaddin, Jean F. “Biochemical Tests for Identification of Medical Bacteria.” Williams & Wilkins, 1980, pp 173 – 183.
[5] Received on 24 May,
[6] Received on 24 May,

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