Eijkman Test Principle and Procedure

 The IMViC test has two drawbacks. The first, It has many controversial procedures and second is test results do not give satisfactory differentiation between fecal and non-fecal coliforms. In 1904 Eijkman proposed another test to differentiate fecal and non-fecal coliform.

Principle of Eijkman Test

  • Only fecal coliforms of warm blooded animals grow at 46°C and ferment lactose with Acid & Gas production
  • Most strains of fecal E.coli can ferment lactose in a special buffered broth when incubated at 45°C, where as very few or less frequently the Enterobacter aerogenes do so.
  • The test is called as Eijkman test or elevated temperature test.

Procedure of Eijkman Test

  • A buffered tryptose lactose broth in tubes with inverted Durham's tube is inoculated with a culture of coliforms.
  • It is then incubated in water jacketed incubator at 45°C for 48 hours.
  • Gas production after incubation constitutes a positive test for fecal coliforms.
  • Another method, instead of tryptone lactose broth, buffered boric acid lactose broth (BALB) medium is used.
  • The advantage is that, the medium used is selective for fecal Escherichia.
  • It selectively inhibits growth and gas production by Enterobacter and other intermediate members of coliforms.
  • Sterile medium is first warmed to 37°C and then inoculated with culture & incubated at 45°C for 48 hrs.
  • Gas production indicates positive test.
  • Eijkman test gives better result than IMViC tests.
  • Therefore, it is generally preferred in water examination.

IMViC Biochemical Tests: Principles Procedures and Results

IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. In this the first test is Indole test, the second one is Methyl Red, the third one is Voges Proskauer and the fourth one is Citrate Utilization test.

  • In the quantitative test for coliforms if completed test is positive, further testing is essential to differentiate fecal and nonfecal coliforms.
  • Escherichia coli and Enterobacter aerogens are the important contaminants of water respectively.
  • Escherichia coli is a fecal coliform as it is mainly found in human feces while Enterobacter aerogenes is considered as non fecal as it also occurs in soil & plant material.
  • They closely resemble each other in their morphological and cultural characteristics. 
  • Therefore, the biochemical tests are performed to differentiate them.
  • Tests are collectively designated as the IMViC tests.
  • The name was coined by Parr from the first letters of the four tests namely - I for Indole, M for Methyl Red, V for Voges Proskauer and C for Citrate Utilization test.
  • The letter i between V and C is added solely for euphony.

Indole Test

  • Indole Test is used to detect indole production from amino acid tryptophan.
  • E. coli has the ability to breakdown the tryptophan by enzyme tryptophanase with release of indole, pyruvic acid and ammonia.
  • Enterobacter doesn't produce enzyme tryptophanase. Therefore, they are not producing indol from an amino acid tryptophan.
  • Test is performed by inoculating the test organism in 1 % tryptone water or 2 % peptone water, incubation at 37°C for 24 hrs.
  • Indole production can be detected by adding few drops of xylene and Kovac's or Ehrlich's reagent which contains p-dimethyl aminobenzaldehyde.
  • This p-dimethyl aminobenzaldehyde reacts with the indole and produce a cherry red(pink) coloured compound. This is a reduction type of reaction.
  • Xylene extracts the indole in upper layer of the medium.

Methyl Red Test

  • Methyl Red Test is carried out to detect acid production ability of test organism from glucose.
  • It is performed by inoculating test organism in glucose phosphate broth tube and incubating at 37°C for 24 hrs
  • Methyl red indicator is then added to detect acid production which gives red colour in positive reaction and yellow in negative.
  • Escherichia coli rapidly ferments glucose with production of acids and reduce the pH to about 5.0
  • This pH / acidity prevents further growth of E.coli in glucose phosphate broth tube.
  • Enterobacter aerogens initially produce acids but later on it is converted to non acid products such as ethanol, acetyl-methyl-carbinol (acetoin) and 2, 3- butane-di-ol ( reduction product of acetoin).
  • Due to this, E. aerogens continues to grow without producing its limiting pH.
  • Thus, Escherichia coli gives positive methyl red test while Enterobacter aerogeys gives negative test.
  • Methyl red is a pH indicator which is red at pH 4.4 while yellow at pH 6.2

Voges Proskauer

  • The test used for the detection of acetyl methyl carbinol (acetoin) production from glucose, by the test organisms.
  • It is also performed by inoculating the test organisms in glucose phosphate broth medium and incubating at 37°C for 24 hrs.
  • This is followed by addition of 40 % potassium hydroxide and 5 % a-naphthol solution with the shaking of the tube.
  • For the Detection of acetoin requires its further oxidation to diacetyl, In presence of catalyst α-naphthol, alkali(KOH) and air, acetoin is further oxidised to diacetyl.
  • Diacetyl in presence of peptone, gives a red colour.
  • The constituent of peptone responsible for red colour is guanidine nucleus of the amino acid arginine.
  • Thus, a positive test is indicated by development of red colour.
  • Enterobacter aerogenes produces acetoin from pyruvic acid(Positive test) while Escherichia coli doesn't produce it(Negative test).

IMViC tests Results

Citrate Utilization Test

  • Citrate Utilization test is carried out to detect the ability of organism to utilize citrate as a sole source of carbon and energy.
  • The utilization of citrate depends upon the enzyme citrate permease that facilitates citrate transport into the cell.
  • E. aerogenes produce citrate permease and are able to utilize citrate as the sole source of carbon while E.coli do not produce
  • The test is performed by inoculating the test organisms in Koser's citrate medium which sodium citrate as the sole source of carbon; and incubating at 37°C for 24 hrs.
  • Ability to use citrate is indicated by the development of turbidity in medium.
  • Citrate Utilization test can also be performed by inoculating the test organisms on the Simmon's citrate agar slant and incubating at 37°C for 24 hrs.
  • The Simmon's citrate agar is the modified processed agar media which contains bromothymol blue as a pH indicator
  • Enterobacter converts citrate to oxaloacetate and acetic acid by enzyme citrase.
  • These products are further converted to pyruvic acid and CO2.
  • The CO2 reacts with sodium and water to form sodium carbonate
  • Sodium carbonate is an alkaline product and it raises the pH of medium Bromothymol blue (pH indicator) is blue in alkaline and green in acidic condition.
  • The change in colour of slant from green to blue indicates positive test.

 Results :


The IMViC test has two drawbacks as- 
  1. It has many controversial procedures 
  2. Test results do not give satisfactory differentiation between fecal and non-fecal coliforms.