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Citrate Test

Citrate testing is used 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 is possible only if the organisms are capable of fermenting citrate. The process takes place via the enzymes is called citrase.


Citrate Test using Citrase Enzymes 1

Citrate Test using Citrase Enzymes 2

Medium for the Citrate Utilization Test

Citrate test is conducted in the medium of simmon 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

Principle of the Citrate Utilization Test

  • Sodium citrate is considered as both a carbon source and an energy source.
  • Nitrogen source is NH4+.
  • The presence of enzymes such as citrate permease (citrase) facilitates the citrate into the bacterium.
  • Bromothymol blue is considered as 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.
  • Colour change of the solution to blue indicates the positive citrate test.
  • If there is no colour 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

Citrate test principle

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.

Procedure to Follow the Citrate Utilization Test

  • Consider a slant, inject Simmons citrate agar gently on the slant by slightly touching the tip of the needle to the colony, which is 18 – 24 hours old.
  • In the citrate medium, organisms require more time to grow. Therefore, the solution must be incubated at 35°C to 37°C for 18 – 24 hours. Some organisms may also require seven days of incubation.
  • Observe the colour change in the solution, the development of blue colour indicates the alkylation process.

Interpretation of the Citrate Utilization Test

Positive result:

The reagent used – Bromthymol blue

Growth on the medium indicates the change in the colour of the reagent from green to blue.

Negative result:

Very less or no growth in the medium and colour of the solution remains the same.

List of Bacteria Gives Positive Citrate Test

1. Providencia

2. Apart from Typhi and Paratyphi A, Salmonella

3. Serratia marcescens

4. Proteus mirabilis

5. Enterobacter species

List of Bacteria Gives Negative Citrate Test

1. Yersinia enterocolitica

2. Salmonella Typhi

3. Escherichia coli

4. Salmonella Paratyphi A

5. Yersinia enterocolitica

List of Bacteria Gives Variable Citrate Test

These bacteria give different results in different strains.

1. Vibrio cholerae

2. Vibrio parahaemolyticus

3. Proteus vulgaris

Bacteria Citrate Test
Citrobacter + (Blue)
Klebsiella + (Blue)
Providencia + (Blue)
Serratia + (Blue)
Enterobacter + (Blue)
Proteus +/-
Salmonella +/-
Shigella – (Green)
Morganella – (Green)
Escherichia – (Green)

Uses of the Citrate Test

  • Used to identify the gram-negative pathogens of environmental isolates and the Enterobacteriaceae family.
  • Test kits, including API- 20E and many others contain citrate tests as one of the diagnostic criteria.
Test Your Knowledge on Citrate Test


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