Citrate utilization test-Dr C R Meera

Aim

To demonstrate the ability of microorganisms to utilize citrate as the sole source of carbon.

Principle

In the absence of fermentable glucose or lactose, some microorganisms are capable of using citrate as a carbon source for their energy.  This ability depends on the presence of a citrate permease that facilitates the transport of citrate in the cell.  Citrate is the first major intermediate in the Kreb’s cycle and is produced by the condensation of active acetyl with oxaloacetic acid.  Citrate is acted on by the enzyme citrase, which produces oxaloacetic acid and acetate.  These products are then enzymatically converted to pyruvic acid and carbon dioxide.  During this reaction the medium becomes alkaline- the carbon dioxide that is generated combines with sodium and water to form sodium carbonate, an alkaline product.  The presence of sodium carbonate changes the Bromothymol blue indicator incorporated into the medium from green to deep Prussian blue.  Bromothymol blue is green when acidic (pH 6.8 and below) and blue when alkaline (pH 7.6 and higher). 

Following incubation, citrate positive cultures are identified by the presence of growth on the surface of the slant, which is accompanied by blue coloration.  Citrate negative cultures will show no growth and the medium will remain green.

Requirements

24 hr nutrient broth cultures of species Escherichia coli, Proteus, Pseudomonas, Streptococcus, Staphylococcus and Bacillus.

Simmons citrate agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

1.  Aseptically inoculate each organism into appropriately labeled tubes by means of streak       inoculation.  One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

 

Observations

Escherichia coli, Streptococcus, and Staphylococcus sp.  showed no color change (medium remained green), indicating citrate negative. Proteus, Pseudomonas and Bacillus sp. showed growth with an intense Prussian blue color, indicating citrate positive.    

Result

Among the given cultures, Proteus, Pseudomonas and Bacillus sp. are Citrate positive whereas and E.coli, Streptococcus, and Staphylococcus sp. are Citrate negative.

§        Simmons citrate agar

Ammonium dihydrogen phosphate    1.0 g

Dipotassium phosphate                       1.0 g

Sodium chloride                                  5.0 g

Sodium citrate                                     2.0 g

Magnesium sulphate                           0.2 g

Agar                                                    15.0 g

Bromothymol blue                              0.08 g             

Distilled water                                    1 litre

pH                                                       6.9