What is Citrate Utilization Test?

Many biochemical tests are studied and experimented in the laboratories. These tests help us to find the nature of the micro-organisms and their characteristics against certain enzymes and indicators used. One such test is citrate Utilization test.

Citrate tests are usually performed to determine the ability of the bacteria to utilize sodium citrate as the source for carbon and inorganic ammonium hydrogen phosphate as the source for nitrogen.

This citrate utilization test is possible only for the organisms which are capable of fermenting the citrate. This processes usually takes place with the help of an enzyme citrase.

Characteristic of Citrate Utilization Test

Citrate utilization test is often performed along with IMViC test. IMViC test is an abbreviated form of Indole, Methyl Red, Vogues-Proskauer and citrate test.

This test helps in differentiating the organisms on the basis of their ability to use citrate as a source of energy. This citrate is test mostly performed with other IMViC tests which helps us to differentiate Gram-negative bacilli of the Enterobacteriaceae family from other species.

It is considered as one of the important tests as it has a capability to detect the species-level identification in the family of Enterobacteriaceae.

However, it also has exception with the few species of the Enterobacteriaceae family. Some species such as Salmonella, Edwardsiella, Serratia Providencia, Citrobacter, Enterobacter, etc. gives a positive reaction in a citrate utilization test, and other species like Escherichia, Morganella, Shigella and Yersinia gives a negative reaction during this test. Proteus is considered as citrate variable.

Citrate utilization test is most commonly called as Simmon’s citrate test; as it helps in utilizing Simmon’s citrate agar that contains a major source of energy in the form of carbon.

This medium also comprises of ammonium hydrogen phosphate as the sole source of nitrogen.

The organisms which gives positive result during this test has the capability to ferment citrate during the presence of an enzyme citrate.

Citrate Utilization Test Principle

Citrate agar is used to test the ability of an organism whether the particular has citrate as the source of energy.

The agar medium contains citrate as the source of carbon and the other inorganic ammonium salts as the source of nitrogen.

Thus, the growth of organism in the medium is usually indicates by the amount of citrate that is utilized, as citrate is one of the intermediate compounds in the process of Krebs’s cycle.

The enzyme citrase breaks the citrate into oxoacetate and acetate.

Oxoacetate is again broken down into pyruvate and carbon dioxide. Hence the carbon dioxide released, induces the metabolism of ammonium salts, and results in the formation of ammonia or sodium carbonate, which increases the alkalinity in the medium.

On adding an indicator bromothymol blue, there will be a shift in pH and the color of the medium changes from green to blue, when the pH is above 7.6.

Thus, the growth of the organism is detected by change in colour of the medium, as a result Citrate metabolism determines a positive citrate test.

Citrate Utilization Test Requirements

Media: For citrate utilization test, simmon’s citrate is generally used as the medium to test the ability of the organism to utilize citrate as a sole source of energy.

Simmon’s citrate agar is now a days sold commercially in the form of dehydrated powder by commercial vendors.

At the same time, it can also be prepared in the laboratory using the necessary ingredients.

The ingredients and their composition are listed below;

Ingredients	Gram/liter
Ammonium dihydrogen phosphate	1.0
Magnesium sulphate	0.2
Dipotassium phosphate	1.0
Sodium citrate	2.0
Sodium chloride	5.0
Bromothymol blue	0.08
Bacteriological agar	15.00

Supplies:

 Inoculating loops or needles

 Incubator

Citrate Utilization Test Procedure

About 24.28 grams of dehydrated powder or the media prepared in laboratory are added in a beaker containing 1000 milliliters of pure distilled water.

Then the solution is heated until it boils, so that the medium dissolves completely.

Further the dissolved medium is shifted into the tubes and the tubes are sterilized in an autoclaved at a temperature of 121ºC for about 15 minutes.

After completion of the autoclaving process, the tubes are taken out and they are cooled at temperature of about 40 to 45ºC in a slanted position and this position is maintained till obtaining the butts in a depth of about 1.5 to 2.0cm

Utilization Test

An isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating loop.

Then the citrate agar tubes are inoculated by streaking the surface of the slant, in the back and froth direction using the inoculating sticks.

The caps of the tubes are usually left loosened in order to ensure adequate aeration and they are incubated aerobically at a temperature of 35 to 37ºC for about 4 days.

This test is examined each day.

It must be kept for a minimum to 4 days to finalize the results.

The change in color is observed if present.

Citrate Utilization Test Quality Control

Aseptic simmon citrate agar is maintained in a forest green color which is slightly opaque at the gel slants.

The agar is inspected for the evidence of freezing, any contamination, dehydration, cracks or formation of bubbles prior to use.

If any tubes are seen as blue perform usage then it must be discarded.

Positive control of the tests includes Klebsiella pneumoniae, and the negative control include Escherichia coli.

Citrate Utilization Test Results

Positive Result: Positive test is usually determined by growth in the medium along with the color change. Here the color changes from green to deep blue along the direction of the slant.

Negative Results: The test is concluded as negative if there is no growth or change in color in the medium, such that the color remains the same, green.