Citrate Test Different Bacteria Showing Citrate Utilization Test

The Citrate Test is part of a group of tests called IMViC (Indole, Methyl-Red, Voges-Proskauer, and Citrate) used to tell the difference between different types of Gram-Negative bacilli in the Enterobacteriaceae family.

The Citrate Test can only be done if the bacteria can ferment citrate. In these tests, the most common citrate medium used is the Simmons formula.

Uses of Citrate Test

The Citrate Test is used to see if organisms can use citrate as an energy source. It’s also used to see if organisms can use citrate as a carbon source. Organisms that can use citrate as their only carbon source may also use ammonium salts as their only nitrogen source. It’s also used to tell the difference between faecal coli and members of the aerogenes group based on how they use citrate.

Principle of the Citrate Test

The basic idea of this test is to see if organisms can use citrate as their only carbon source for their own metabolism, and then make the environment more alkaline. There’s another test medium for citrate that doesn’t need organisms to use citrate as their only carbon source. This is a specific way to see if organisms can use citrate as their only carbon source.

• It’s known that wild-type E. coli can’t use citrate as a carbon source when there’s air because it doesn’t have a way to move citrate. But it can use citrate when there’s no air by using a special transporter called CitT.
• The citrate test is used to see if bacteria can use sodium citrate as their only carbon source and inorganic ammonium dihydrogen phosphate (NH4H2PO4) as their only nitrogen source.
• The test organism is grown on a medium that has sodium citrate and a special dye called bromothymol blue. If the organism can use citrate, the medium changes from green to blue.

Alkaline Carbonates and Bicarbonates

Alkaline carbonates and bicarbonates formed as by-products of citrate catabolism raise the pH of the medium are above 7.6, causing the bromothymol blue indicator to change colour from its original green to blue.

When an organic acid such as citrate (think of the Krebs cycle) is used as a source of carbon and energy, alkaline carbonates and bicarbonates are formed, which raise the pH of the medium, resulting in a colour change.

When bacteria metabolize citrate, the inorganic ammonium salt is broken down into ammonia, which increases alkalinity and changes the blue bromothymol indicator from green to blue. Sodium citrate combines with CO₂ and water to form an alkaline product (sodium carbonate).

Bacteria Produced By Citrate Permease

Bacteria capable of growing on this medium produce citrate permease, which converts citric acid to pyruvate.

Similarly, citrate-positive organisms first grow on glucose and then switch to citrate metabolism to form cherry (alkaline) colonies through the same reaction pathway as described for Simmons citrate agar. A positive result was obtained (the right tube was positive for citrate).

• The solution colour turns blue to indicate a positive citrate test. If Simmons citrate agar does not turn blue after further incubation, repeat the citrate test with a smaller inoculum.
• Two bacteria (both Enterobacteriaceae) on citrate agar showed the results of the citrate test and results. Christensen Citrate Agar (Sigma) is a selective differential medium for diagnosing the use of citrate by gut bacteria.
• Citrate utilization was detected using differential media and quantified by Mass Spectrometry (MS) of media filtrates at different growth stages.
• For comparative analysis of the transcriptome and growth proteome of glucose, citrate, and mixed carbon sources, cells were cultured in CGXII minimal medium supplemented with 0.03 g L-1 protocatechuic acid and 12 g L-1 MgSO₄ .

To assess the overall gene expression response of C. glutamic to citrate, avoiding comparison with glucose or other carbon sources in minimal medium, use complex LB medium for growth.

Glutamic

Recent studies have shown that glutamic growth in the presence of citrate as the sole carbon source if the medium contains high concentrations of Mg²⁺ ions, possibly due to the co-transport of Mg²⁺ and citrate.

Recent studies have shown that glutamic is able to grow on citrate as the sole source of carbon and energy and induces two citrate uptake systems in the presence of citrate, CitM and TctABC.

Only bacteria capable of using citrate as their sole source of carbon and energy can grow on Simmons’ citrate medium, so citrate-negative test cultures are nearly indistinguishable from uninoculated slants. Some citrate-negative microorganisms may grow weakly on sloped surfaces without causing discolouration.

Incubation Period

Some organisms may require incubation times of up to 7 days due to limited growth rates on citrate medium. As shown, bacterial growth correlated with citrate consumption in the medium.

Large colonies formed after 2-3 days of incubation on longitudinal redundancy check (LRC) agar were also tested on Simmons and Christensen citrate agar. Large blue colonies on Simmons citrate agar from M9C medium were not clonal due to the deposition of predominantly elongated pre-dividing cells.

• Several genes encoding enzymes for central carbon and energy metabolism have shown elevated levels of mRNA in cells grown with citrate.
• Here, when cells were grown on citrate, the malic enzyme encoding malic acid showed an approximately two-fold increase in mRNA levels, indicating differential regulation of gluconeogenic carbon sources.
• Candidates showing higher gene expression and protein levels on citrate than on glucose are indicated by enzyme names in addition to reactions. The use of citrate is due to the enzyme citrus, which breaks down citrate into oxaloacetate and acetate.
• In a minimal environment containing ammonium sulfate as a nitrogen source, cells can use many different compounds as sole carbon sources, including d-glucose, d-fructose, d-ribose, L-malate, L-aspartate, citrate, N-acetylglucosamine, gluconate and mannitol.
• To exploit the collection of E. coli K-12 Keio mutants that provide kanamycin (Kn) tagged mutations in every non-essential gene, systematic analysis of citrate utilization with this strain were performed.
• Other available test methods for citrate use are IMViC, test kit method, and API test kit method.
• As you know, Escherichia coli tests are negative for citrate use; although rare, natural variants of E. coli positive for citrate have been isolated.

Bacteria Showing Citrate Utilization Test FAQs

Which organisms are positive for citrate utilization?

Citrate positive organisms are Klebsiella pneumonia and Proteus mirabilis. Escherichia coli and Shigella dysenteries are citrate negative.

Which enzyme is detected by the citrate utilization test?

Citrate enzyme is detected by the citrate utilization test. The basic principle is to detect the ability of an organism that can utilize citrate as a sole source of carbon for their metabolism with resulting alkalinity. The enzyme hydrolyses the citrate to form oxaloacetic acid and acetic acid.

Why is the indole production test important?

The given test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to indole, which accumulates in the medium. The Indole production test is important in the identification of Enterobacteriaceae.