CITRATE UTILIZATION TEST

CITRATE UTILIZATION TEST

AIM

         To detect the ability of the bacteria to utilize Sodium citrate as sole carbon source and Ammonium salt as sole source of nitrogen.

PRINCIPLE   

        Citrate utilization test is used to determine the ability of bacteria to utilize Sodium citrate as its only Carbon source and Inorganic ammonium salts as its only Nitrogen source. Citrate utilization test can be used to differentiate the coliforms such as Klebsiella (formerly Enterobacter) aerogenes (Positive) which occur naturally in the Soil and Aquatic environments from the Fecal coliforms such as Escherichia coli (Negative) whose presence would be indicative of fecal contamination. Simmon’s Citrate agar is used for the Citrate utilization test. The medium contains Citrate as the sole Carbon source and inorganic Ammonium salts (NH₄H₂PO₄) as the sole source of Nitrogen. Bacteria that can grow on this medium produce an enzyme, Citrate permease which is capable of converting Citrate to Pyruvate. The Pyruvate can then enter the microorganism metabolic cycle for the production of energy. Growth is indicative of utilization of citrate, an intermediate metabolite in the Krebs cycle. When the bacteria metabolize Citrate, the Ammonium salts are broken down to Ammonia, which increases alkalinity (above pH 7.6). The shift in pH turns the Bromthymol blue indicator in the medium from Green to Blue.

Citrate utilization positive bacteria – Salmonella sp., Edwardsiella sp., Citrobacter sp., Klebsiella sp., Enterobacter sp., Serratia sp., Providencia sp. and Vibrio vulnificus.

Citrate utilization negative bacteria – Escherichia coli, Shigella sp., Morganella sp. & Yersinia sp.

MATERIALS REQUIRED

1. Sterile Test tubes
2. Simmon’s Citrate Agar
3. Inoculation loop
4. Incubator

PROCEDURE

a)     Inoculate the test microorganism into the Simmon’s citrate agar slant.

b)   Incubate the inoculated Simmon’s citrate agar slant at 37 °C for overnight or 24 hours (Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium).

c)     Observe the development of Blue colour denoting Alkalinization.

OBSERVATION AND RESULTS

·       Positive Citrate utilization test – Colour change of slant from Green to Blue.

·       Negative Citrate utilization test – No colour change of slant. Slant remain Green colour.

 

                                                                          A                             B

(A – Citrate utilization positive; B – Citrate utilization negative)

Figure – 1: Citrate utilization test