Citrate Utilization Test

Objective:
-To determine the ability of bacteria utilize citrate as sole carbon source.
T-o use this fact to differentiate enteric organisms.

Principle:

           

            Some organisms are utilizing citrate as a carbon source for their energy.  The enzyme citrate permease facilitates the transport of citrate into the cell.  Then citrate is acted upon by enzyme citrase which produces oxaloacetic acid and acetate. That are further converted to pyruvic acid and CO_2. This CO₂ combines with medium (Simmon’s Citrate) and water to form sodium carbonate (NaHCO₃) and ammonia (NH₃). This products change the pH of the medium into alkaline. Bromothymol blue (indicator) turns to deep prussian blue in alkaine pH. At neutral pH , bromothymol is green.

Materials required:
 Bacterial cultures (Klebsiella sp., E.coli)

 Simmon’s Citarte Agar

Procedure :
1.Inoculate the bacterial cultures on simmon’s citare agar tubes (slant) by stab and streaking.
2.The tubes are incubating at 37° C for 24 hrs.
3. To observe and record the result based on color change.

Observation:
-  Prussian blue /deep blue color change in  Klebsiella sp tube 
- Green color / No color change in tube with E.coli

Result : -
 - Klebsiella sp  : Citrate  Positive 
- E. coli : Citrate Negative

 Interpretation:

           

            Klebsiella sp., using citrate as sole carbon source with help of citrate permease and releases sodium carbonate and ammonia and this products change the pH of the medium into alkaline. In this pH condition the indicator bromothymol blue change the color from green to deep blue/Prussian blue and give positive result.  E.coli doesn’t produce the enzymes like citrate permease and citrase, which indicate the negative result and no change in medium color. 

 Citrate Positive Organisms:

Klebsiella sp., Enterobacter sp., Proteus mirabilis, 

                                                                   Citrate Positive

                                                                        Citrate Negative

Composition of Simmon's Citaret agar :

Ingredients Per liter of distilled water

Sodium Citrate (dehydrate)    -   2.0 g

Ammonium dihydrogen
 phosphate -   1.0 g

Dipotassium phosphate -   1.0 g 

Magnesium sulfate  (heptahydrate) -   0.2 g

Sodium chloride (NaCl) -   5.0 g

Bromothymol Blue (indicator) -   0.08 g

Agar  -   15.0 g

pH : 6.9