Citrate Utilization Test: Principle, Procedure, Results

The citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red, VP, and Citrate) tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products.

Citrate utilization can be used to distinguish between coliforms such as Klebsiella aerogenes (formerly Enterobacter aerogenes) (+ve) which occur naturally in the soil, and aquatic environments from fecal coliforms such as Escherichia coli (-ve), whose presence would be indicative of fecal contamination.

A citrate utilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic ammonium dihydrogen phosphate (NH4H2PO4) as the sole nitrogen source.

Principle of Citrate Utilization Test 

When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced, increasing the medium’s pH, resulting in the color change.

Utilization of exogenous citrate requires the presence of citrate transport proteins (permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO₂.

Citrate = oxaloacetate + acetate

oxalacetate = pyruvate + CO₂

The further metabolic breakdown is dependent upon the pH of the medium.

A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.
pyruvate = acetate + formate

B. At pH 7.0 and below, lactate and acetoin are also produced.
pyruvate = acetate + lactate + CO₂
pyruvate = acetoin + CO₂
The released carbon dioxide will subsequently react with water and the sodium ion in the medium to produce sodium carbonate, an alkaline compound that will raise the pH.  In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source, which also helps to increase the medium pH.

Growth usually results in the bromothymol blue indicator turning from green to blue. The bromothymol blue pH indicator is deep forest green at neutral pH.  With an increase in medium pH to above 7.6, bromothymol blue changes to blue.

Media Composition

In the citrate utilization test, the citrate medium most commonly used is the formula of Simmons. The medium is poured into a tube on a slant. The composition of Simmons citrate agar is as follows:

Ingredient	Simmons Citrate Agar (g/L)
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
Bromothymol blue	0.08 g
Distilled water	1 L
Final pH	6.9

Quality Control

1. Perform QC on each new lot of media before using them. Inspect agar for evidence of freezing, contamination, cracks, dehydration, and bubbles before storage and before use. Discard blue tubes.
2. Organism
   1. Klebsiella pneumoniae ATCC 13883—citrate positive (growth; blue color)
   2. Escherichia coli ATCC 25922—citrate negative (no growth or trace of growth)

Procedure of Citrate Utilization Test

1. Inoculate Simmons citrate agar lightly on the slant* by touching the tip of a needle to a colony 18 to 24 hours old.
2. Place cap loosely on the tube.
3. Incubate aerobically at 35°C to 37°C for  18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited growth rate on citrate medium.
4. Observe the development of blue color along the slant, denoting alkalinization.

*Do not stab the slant since the test requires an aerobic environment.

Results

Citrate positive:  growth will be visible on the slant surface, and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.

Citrate negative:  trace or no growth will be visible. No color change will occur; the medium will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium. Thus, a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant. E. coli is citrate negative.

Repeat the tests with equivocal results.

Citrate Positive Organisms

Many members of the family Enterobacteriaceae are citrate positive.

1. Klebsiella pneumoniae
2. Enterobacter species (minority of strains gives negative result)
3. Citrobacter freundii
4. Salmonella other than Typhi and Paratyphi A
5. Serratia marcescens
6. Proteus mirabilis (a minority of strains gives negative result)
7. Providencia

Citrate Test: variable (different strains give different results)

1. Proteus vulgaris
2. Vibrio cholerae
3. Vibrio parahaemolyticus

Citrate Negative Organisms

1. Escherichia coli
2. Shigella spp
3. Salmonella Typhi
4. Salmonella Paratyphi A
5. Morganella morganii
6. Yersinia enterocolitica

Although uncommon, natural E. coli variants that are citrate positive have been isolated.  Citrate-negative strains of E. aerogenes have also been found.

Uses of Citrate Utilization Test

Citrate utilization test is often used as a part of a battery of tests (IMViC) used to identify gram-negative pathogens of the Enterobacteriaceae family. For instance, test kits such as the API-20E and Enterotube II  include citrate utilization medium as one of the diagnostic tests.

Limitations

1. Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with a smaller inoculum.
2. Do not inoculate from broth cultures due to the carryover of media.
3. To avoid false-positive reactions, use a light inoculum to prevent the carryover of substances from previous media.
4. The citrate utilization test results alone are insufficient for identification up to the species level.

References and further reading

1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition