Citrate Utilization test- Aim, Principle, Procedure, Results and Interpretation

 

Aim

This test is used to differentiate of Enterobacteriaceae based on their ability to use citrate as the sole carbon source.

Principle

Sodium citrate is a salt of citric acid, a simple organic compound found as one of the metabolites in the tricarboxylic acid cycle (Krebs cycle). Some bacteria can obtain energy in a manner other than by the fermentation of carbohydrates by using citrate as the sole source of carbon. The measurement of this characteristic is important in the identification of many members of the Enterobacteriaceae. Any medium used to detect citrate utilization by test bacteria must be devoid of protein and carbohydrates as sources of carbon. The utilization of citrate by a test bacterium is detected in citrate medium by the production of alkaline by-products. The medium includes sodium citrate, an anion, as the sole source of carbon, and ammonium phosphate as the sole source of nitrogen. Bacteria that can use citrate can also extract nitrogen from the ammonium salts in the medium, producing sodium bicarbonate (NaHCO3) and ammonia (NH3), which results in an alkaline pH. Bromthymol blue—yellow below pH 6.0 and blue above pH 7.6—is theindicator.

Image source: Koneman’s Color Atlas and Text book of Diagnistic Microbiology

Media and Reagents

The citrate medium most commonly used is the formula of Simmons. The medium is poured into a tube on a slant. The formula of Simmons citrate medium is shown below:

Composition
Ammonium dihydrogen phosphate	1 g
Dipotassium phosphate	1 g
Sodium chloride	5 g
Sodium citrate	2 g
Magnesium sulfate	0.20 g
Agar	15 g
Bromthymol blue	0.08 g
Distilled water	1 L

Final pH - 6.9

Quality Control

Each new batch of medium should be tested with a positive-and a negative-reacting organism. The following species are suggested controls:

Positive control: Klebsiella pneumoniae

Negative control: Escherichia coli

Test Procedure

Streak on the slant surface of the citrate agar tube with a well isolated colony is picked from the surface of a primarily isolation medium.

The tube is incubated at 37°C for 24–48 hours.

Results and Interpretation

Positive Reaction: The development of a deep blue color within 24–48 hours.

Negative Reaction: No growth and no color change appear in slant; Slant remains green

                                                     Citrate Utilization test

A positive test is represented development of a deep blue color, indicating that the test organism has been able to utilize the citrate contained in the medium, with the production of alkaline products. A positive test may also be read without a blue color if there is visible colony growth along the inoculation streak line. This is possible because, for growth to be visible, the organism must enter the log phase of growth, possible only if carbon and nitrogen have been assimilated. A positive interpretation from reading the streak line can be confirmed by incubating the tube for an additional 24 hours, when a blue color usually develops.

Limitations

Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.

Reference:

1. BBL Manual of Products and Laboratory Procedures. Ed. 5. Cockeysville, MD: BioQuest, 1968:115, 138.
2. Blazevic DJ, Ederer GM. Principles of Biochemical Tests in Diagnostic Microbiology. New York, NY: Wiley,1975:15–18.
3. Koser SA. Utilization of the salts of organic acids by the colon-aerogenes groups. J Bacteriol 1923;8:493–520.
4. MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria. 2nd Ed. Baltimore, MD: Williams& Wilkins, 1980:59–63.
5. Simmons JS. A culture medium for differentiating organisms of typhoid-colon aerogenes groups and forisolation of certain fungi. J Infect Dis 1926;39:209–214.
6. Koneman’s Color Atlas and Text book of Diagnistic Microbiology. 
7. Bailey and Scott’s Diagnostic Microbiology.
8. Mackie and McCartney Practical Medical Microbiology.