Citrate utilization Test

IMViC Test Full Form

IMViC Test stands for Indole, Methyl red, Voges-Proskauer, Citrate Test. This as a set of four biochemical tests used to differentiate between Escherichia coli, Klebsiella Enterobacter, and Enterobacteriaceae family organisms.

Citrate utilization Test

Citrate Utilization Test is chiefly used in the differentiation of Gram positive rods, by checking their ability to utilize the carbon of sodium citrate as the sole source of carbon.

Some bacteria can convert salts of organic acids to alkaline carbonates. This was first demonstrated in milk where an alkaline reaction was further confirmed in a medium where nitrogen was supplied containing sodium ammonium phosphate as nitrogen source (peptone free nitrogen base).

It was also found that the growth of certain organisms was enhanced by sodium citrate, whereas the growth of other was completely inhibited.  

In 1923, Koser devised a liquid basal broth medium in which ammonium phosphate supplied as the source of nitrogen, to which he added each organic acid in a concentration of 0.2%. He studied 18 organic acids and evaluated the ability of various organisms to utilize these acids as carbon sources.

The most striking result of this work was the recognition that the aerogenes group of organisms could utilize citrate for growth and the coli group of organisms was completely unable to grow in this medium. This was the introduction of carbon utilization as a diagnostic aid.          

Since determining growth on solid medium is easier than interpreting turbidity in broth, Simon’s added agar to Koser’s medium and incorporated an indicator, bromothymol blue, which was green at a pH of 6.8 and blue at pH greater than 7.6.

The alkaline reaction involved in citrate utilization is not due to release of ammonia. The alkaline reaction mostly occurs because an excess of carbon dioxide is generated as citrate is cleaved to oxaloacetate, which is then decarboxylated to pyruvate and carbon dioxide.

The excess of carbon dioxide may combine with sodium and water to form sodium carbonate, which is sufficiently alkaline to change the indicator from green to deep blue.

It is also possible that the sodium ion may remain in excess in this reaction and may attract the hydroxyl group from the available water in the medium and form sodium hydroxide.  

The enzyme that catalyzedthe cleavage of citrate has been given a number of names: citrate lyase, citratase, citrase, citrate aldolase, citridesmolase.

The primary products of cleavage are oxaloacetate and acetate which are subsequently converted to pyruvate and carbon dioxide by an oxaloacetate decarboxylase.

E. coli has been shown to possess citrate lyase although it cannot utilize citrate in Koser’s or Simmon’s media. It apparently lacks the transport system or permease that would permit the citrate to enter the cell, to be used as a carbon source. The permease for citrate utilization is inducible.

When performing this test, it is important to use a light inoculum and minimize carryover of any nutritive constituents of the culture medium from which the inoculums is taken.

Requirements of citrate utilization Test

  1. Test culture suspension. 
  2. Sterile Koser’s citrate medium (1.0 ml in each tube) / Simon’s citrate medium (agar slant) 
  3. Koser’s citrate medium: 
Sodium chloride0.5g
Tri-sodium citrate0.5g
Ammonium dihydrogen phosphate1.0g
Magnesium sulfate0.02g
Potassium dihydrogen phosphate0.1g
Distilled water100ml

Medium is autoclave at 110oC for 10 minutes.

Citrate Utilization Test Procedure

  1. Inoculate the medium (Koser’s broth or Simon’s agar) with the culture suspension. 
  2. Incubate at 37oC for 24 hours. 
  3. Check for turbidity (indicating positive test) in Koser’s medium; growth and change in colour of indicator to blue on Simon’s citrate agar (positive test).