Citrate Utilization Test

Theoretical Background

• The purpose of the citrate test is to determine the ability of the bacteria to use sodium citrate as the only source of carbon and inorganic ammonium hydrogen phosphate (NH4H2PO4) as a source of nitrogen. The citrate utilization test, a part of the IMViC test series, is possible only if the organisms are capable of fermenting citrate. The process that takes place via the enzymes is called citrase.

Citrate Test using Citrase Enzymes:

The citrate agar test is conducted in the medium of simmons citrate agar and the mixture is prepared using the following ingredients:

• Ingredients Simmon citrate agar (g/L)
• Ammonium hydrogen phosphate (NH4H2PO4) 1g
• Dipotassium phosphate (HK2O4P) 1g
• Sodium chloride (NaCl) 5g
• Sodium citrate (C₆H₅Na₃O₇) 2g
• Magnesium sulfate (MgSO4) 0.20g
• Bromothymol blue 0.08g
• Agar 15g
• Distilled water 1L

Final pH 6.9

Citrate Test Principle of Work

• Sodium citrate is considered both a carbon source and an energy source.
• The Nitrogen source is NH4+.
• The presence of enzymes such as citrate permease (citrase) facilitates the citrate into the bacterium.
• Bromothymol blue is considered a pH indicator.
• For the citrate utilization test oxygen is required and the process is done on the slants.
• The citrate is oxidized from bacteria and is extracted with the release of CO2 from the medium.
• Sodium from sodium citrate combines with CO2 and water, to form an alkaline product (sodium carbonate).
• The change in the pH of the solution gives details about the presence and absence of the test.
• The Color change of the solution to blue indicates the positive citrate test.
• If there is no color shift, that means there is no growth in the medium, which indicates the absence of the citrate test.

To use citrate, proteins such as permeases, need to be transported by citrate. Upon cell absorption of citrate, it is cleaved into oxaloacetate and acetate by the citrate lyase. Oxaloacetate is further metabolized to carbon dioxide and pyruvate.

Citrate = acetate + oxaloacetate

Oxaloacetate = pyruvate + CO2

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After the formation of pyruvic acid metabolic degradation depends on the pH of the solution.

1. Under basic conditions, acetate and formate are formed during the metabolism of pyruvate.

2. Under acidic conditions, (pH is below 7) lactate is formed.

The citrate test plays a crucial  role in Enterobacteriaceae differentiation, distinguishing among genera based on their ability to utilize sodium 

citrate as a sole carbon source.