Principle

The citrate test detects the ability of an organism to use citrate

Citrate

A citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid. An example of the former, a salt is trisodium citrate; an ester is triethyl citrate.-Other citric acid ions:...

 as the sole source of carbon and energy. Bacteria are inoculated on a medium containing sodium citrate

Sodium citrate

Trisodium citrate has the chemical formula of Na3C6H5O7. It is sometimes referred to simply as sodium citrate, though sodium citrate can refer to any of the three sodium salts of citric acid. It possesses a saline, mildly tart flavor. For this reason, citrates of certain alkaline and alkaline earth...

 and a pH indicator

PH indicator

A pH indicator is a halochromic chemical compound that is added in small amounts to a solution so that the pH of the solution can be determined visually. Hence a pH indicator is a chemical detector for hydronium ions or hydrogen ions in the Arrhenius model. Normally, the indicator causes the...

 such as bromothymol blue

Bromothymol blue

Bromothymol blue is a chemical indicator for weak acids and bases. The chemical is also used for observing photosynthetic activities or respiratory indicators .Bromothymol blue acts as a weak acid in solution...

. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate

Acetate

An acetate is a derivative of acetic acid. This term includes salts and esters, as well as the anion found in solution. Most of the approximately 5 billion kilograms of acetic acid produced annually in industry are used in the production of acetates, which usually take the form of polymers. In...

. Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO₂). Production of sodium bicarbonate

Sodium bicarbonate

Sodium bicarbonate or sodium hydrogen carbonate is the chemical compound with the formula Na HCO3. Sodium bicarbonate is a white solid that is crystalline but often appears as a fine powder. It has a slightly salty, alkaline taste resembling that of washing soda . The natural mineral form is...

 (NaHCO₃) as well as ammonia

Ammonia

Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...

 (NH₃) from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a change of the medium’s color from green to blue.

Bacterial colonies are picked up from a straight wire and inoculated into slope of Simmons citrate agar

Agar

Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...

 and incubated overnight at 37 °C. If the organism has the ability to use citrate, the medium changes its color from green to blue.

Examples:

• Escherichia coli
  Escherichia coli
  Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
  
  : Negative
• Klebsiella pneumoniae
  Klebsiella pneumoniae
  Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines....
  
  : Positive
• Frateuria aurantia
  Frateuria aurantia
  Frateuria aurantia is a species of Proteobacteria. It is named after the Belgian microbiologist Joseph Frateur. The cells are mostly straight rods. Frateuria aurantia was isolated from the plant Lilium auratum and from the fruit of the raspberry Rubus parvifolius....
  
  : Positive

Method

Inoculate Simmons citrate agar (do not inoculate heavily) by using straight wire from an 18 to 24 hour old colony. Inoculating from a broth culture is not recommended because the inoculum would be too heavy.
Incubate at 35°C for up to seven days.

Results

Positive : Growth on the medium even without colour change will be considered as positive.
A colour change in the medium would be observed if the test organism produces acid or alkali during its growth. The usual colour change observed is from green (neutral) to blue (alkaline).

Negative : No growth observed.