How is IMViC Biochemical test performed ?

How is the IMViC Biochemical test performed?

In the IMViC test there are four biochemical tests and this test is carried out individually. IMViC test is carried out to identify members of the Enterobacteriaceae family.

Each letter of IMViC test stand for individual test

  1. I – Indole production test.
  2. M – Methyl red test.
  3. V- Voges- Proskauer test.
  4. C – Citrate utilization.
  5. i – Lower case i is used for ease of pronunciation.

We will go through these tests one by one.

Indole production test.


The aim of this test is to determine the capability of a bacterial culture to produce Indole.


  • Bacterial culture
  • Tryptophan broth
  • Kovac’s reagent or Ehrlich’s reagent.
  • Xylene

The composition of media and reagents

1. Tryptophan broth medium

Tryptone         – 1 gm

Distilled water- 100 ml

2. Kovac’s reagent

p-dimethylamino benzaldehyde -15 gms

Isoamyl alcohol                           – 150 ml

HCL                                                – 75 ml

3. Ehrlich’s reagent

p-dimethylamino benzaldehyde  – 1 gm

95 % ethanol                                – 95 ml

Concentrated HCl                        -20 ml



  1. Tryptone broth is prepared, distributed in the test tube and sterilized by autoclaving.
  2.  The sterile tryptone broth tubes are inoculated with loopful of suspension and incubated at 37° C for 24 hours.
  3. After incubation, add 3-4 drops of xylene in tubes and shake vigorously and keep the tubes still so that two layers get separated.
  4. After separation of two layers add 1 ml of Kovac’s reagent or Ehrlich’s reagent and tubes are observed for formation of the pink colour ring.


Indole is an aromatic heterocyclic organic compound. Indole can be produced by some members of Enterobacteriaceae family by hydrolysis of tryptophan. Indole production is a protein utilization test. Here we are going to use a protein rich that is tryptophan-rich broth. This media contains tryptophan as a source of protein. The bacterial cells that are able to produce tryptophanase enzyme hydrolyse and deaminate tryptophan into Indole, pyruvate and ammonia. Further, these degraded products are treated with Kovac’s reagent or Ehrlich’s reagent in presence of heat. If the degraded products in the tube contain Indole then this Indole reacts with Kovac’s reagent or Ehrlich’s reagent and form pink colour rose Indole complex. In this way, Indole production is detected.

[Note – Indole is soluble in the organic compound so it is recommended to add xylene/chloroform/ether by adding this substance Indole get extracted from the whole medium and forms a separate layer on the surface of the medium. So that Kovac’s reagent or Ehrlich’s reagent can easily react with the indole and form a pink colour ring.

Result – If there is the formation of the pink colour ring then the Indole production test is positive and if there is no ring formation then the test is negative.

Methyl Red Test {MR test}


To carry out Methyl-red test to determine fermentation capacity of given bacterial culture.


  • Glucose phosphate broth
  • Methyl-red indicator
  • Test culture.

Composition of media

Glucose phosphate broth

  1. Glucose  – 0.5 gms
  2. K2HPO4 –  0.5 gms
  3. Peptone – 0.5 gms
  4. Distil water – 100 ml
  5. pH – 7


  1. Glucose phosphate broth is prepared and distributed in test tubes these test tubes are further sterilized by autoclaving.
  2. The sterile test tubes are inoculated with test culture and incubated at 37 ° C for 24 hours.
  3. After incubation, the five drops of a methyl-red indicator is added to the medium and the tubes are observed for development of red colour.


As we know we carry out IMViC test for determination of members of Enterobacteriaceae family. The micro-organisms from Enterobacteriaceae family carry out fermentation pathway they metabolise glucose to pyruvic acid to formic acid and this formic acid formation is called formic acid fermentation process.

In general, there are two types of formic acid fermentation pathway and they are:-

  1. Mixed acid fermentation- This type of fermentation is carried out in E.coli, Salmonella and Proteus. In this fermentation ethanol and the mixture of various acid like succinic acid, formic acid, acetic acid and lactic acid are formed.
  2. 2. Butanediol fermentation – This fermentation is carried out in Bacillus, Enterobacteriaceae, Serratia. Here pyruvate is converted to acetyl methyl carbonyl and further from this Butanediol is formed. This fermentation mainly produces neutral products. 

Now when we compare this two fermentations process, mixed acid fermentation produces more acidic products and it acidifies the incubation media. Here only mixed acid fermentation produces a sufficient amount of acid which can be detected by methyl red indicator.

As here we use glucose phosphate broth as an incubation medium this medium is highly buffered and a small amount of acid is produced the media can resist change in pH. So here we use Methyl red the pH range of methyl red is at 4.4 pH it shows red colour and whereas at 6.2 pH it shows yellow colour. So after incubation, the pH indicator methyl red and the medium is observed for change in colour of medium.

If acid is produced in medium and after addition of methyl red the medium shows red colour then the Methyl red test is positive.

The Methyl red test is shown positive in case of mixed acid fermentation as the complex mixture of acid is produced.


If there a stable red colour is developed in the media after addition of Methyl red indicator then the M-R Test is positive.

Voges-Proskauer (V-P) Test


To carry out Voges-Proskauer test determine the capacity of bacterial culture to carry out fermentation.


  • Glucose phosphate broth
  • 5 % alcoholic alpha-naphthol
  • 40 % KOH solution
  • Test culture


  1. Glucose phosphate broth is prepared and distributed in test tubes these test tubes are further sterilized by autoclaving.
  2. The sterile test tubes are inoculated with test culture and incubated at 37 ° C for 24 hours.
  3. After incubation of 0.6 ml of alpha-naphthol and 0.2 ml of KOH solution per ml of culture broth media is added.
  4. Further after addition of these two reagents the culture tubes are shaken properly and kept in slanting position to increase aeration. Keep these tubes in slanting position for about one hour and the results are noted down.


As we know there are two types of fermentation carried out by members of Enterobacteriaceae family( Details are mention above in M-R test) In methyl red test we detected the mixed acid fermentation and here in Voges-Proskauer test, we are going to determine butanediol fermentation by detecting product acetoin.

This acetoin is a precursor of butanediol and this acetoin is produced during butanediol fermentation. Here in V-P test after fermentation, the medium is treated with alpha-naphthol and KOH solution and due to the addition of this acetoin is oxidised to diacetyl. Now, this diacetyl reacts with the peptone present in the broth and produce the pink colour.

[ Peptone is present in a culture broth and this peptone contains guanidine nucleus of arginine and diacetyl reacts with guanidine nucleus of arginine and gives pink colour to the medium.]

Thus the formation of pink colour indicates the presence of acetoin and conforms Butanediol fermentation and indicates V-P test as positive.

Citrate Utilization Test


To carry out the citrate utilization test to determine the ability of a bacterial cell to utilize citrate.


  • Simmons’s citrate agar
  • Test culture.

The composition of Simmons’s citrate agar

  1. Sodium citrate                                    –0.2 gm
  2. Magnesium sulphate                         –0.02 gm
  3. Sodium chloride                                  –0.5 gm
  4. Ammonium dihydrogen phosphate – 0.1 gm
  5. Dipotassium phosphate                      – 0.1 gm
  6. Bromothymol blue                               – 0.008 gm
  7. Agar-agar                                               – 3 gm
  8. Distil water                                            – 100 ml
  9. pH                                                             – 7


  1. The Simmons’s agar is prepared according to the composition given above, sterilized and after sterilization, the slants of this media are prepared.
  2. Further, this slant is streaked heavily on the slant and incubated for 24 hours at about 37°C
  3. After incubation, the slant is observed for a change in colour and the results are recorded.


The members of Enterobacteriaceae family have the ability to obtain energy and carbon by utilization of citrate. This is an important characteristic to identify the members of the Enterobacteriaceae family. For carrying out the citrate utilization test the test culture should be exposed to a medium which contains citrate as a sole source of carbon and energy. If the bacterial cells are able to produce enzyme citrate permease can utilize citrate because this enzyme can facilitate transport of citrate into bacteria then a bacterial cell can utilize citrate. For carrying out this test we need citrate agar that contains, ammonium phosphate as a source of nitrogen, Sodium citrate as the source of carbon and bromothymol blue as a pH indicator. Bromothymol blue is yellow at acidic pH and blue at alkaline pH.

In citrate utilization test we use agar slant because for this test oxygen is required and after oxidation of citrate, CO2 is liberated. Now this CO2 reacts with the sodium and water present in the medium and form sodium carbonate and this sodium carbonate is alkaline product other hands the bacteria utilize ammonium citrate release nitrogen with the production of ammonia and convert it to ammonium hydro-oxide and this is the other alkaline product. Now this alkaline products increases the pH of the medium to the alkaline side and thus the colour of the medium changes to blue colour. Hence the change in colour of medium to blue indicates positive citrate utilization test.


Formation of a deep blue colour indicates positive citrate utilization test and the test culture has an ability to utilize citrate.

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