Question

Explain how you would inoculate your bacteria to do the citrate test.

a.If the test turned blue after incubation what does that say about your bacteria?

Answer 1

Citrate test or Citrate utilisation test is used to detect the ability of an organism to use citrate as the sole source of carbon and energy. This test is among the suit of IMViC Tests (Indole, Methyl-red,Vogues-Proskauer/VP, Citrate) that are used to differentiate among Gram-negative bacilli in the family Enterobacteriaceae based on their metaboloc by-products.Citrate utilization can be used to distinguish between coliforms such as Klebsiella (formerly Enterobacter) aerogenes (+ve) which occur naturally in the soil and in aquatic environments and fecal coliforms such as Escherichia coli (-ve) whose presence would be indicative of fecal contamination.

Principle of Citrate Utilization Test

Citrate agar is used to test the ability of an organism to utilize citrate as the sole source of energy.The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen.

When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced ultimately. In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source.

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..

When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.

Media used in Citrate Test :

Simmon's Citrate Agar.

Composition :

Sodium Chloride : 5.0 gm

Sodium Citrate : 2.0 gm

Ammonium Dihydrogem Phosphate : 1.0 gm

Dipotassium Phosphate : 1.0 gm

Magnesium Sulphate : 0.2 gm

Bromothymol Blue : 0.08 gm

Agar : 15.0 gm

Deionized water = 1,000 ml
Final pH 6.9 +/- 0.2 at 25 degrees C.

Preparation :

1. Dissolve above salts in deionized water.
2. Adjust pH to 6.9.
3. Add agar and Bromothymol blue.
4. Gently heat, with mixing, to boiling until agar is dissolved.
5. Dispense 4.0 to 5.0 ml into 16-mm tubes.
6. Autoclave at 121 degree C under 15 psi pressure for 15 minutes.
7. Cool in slanted position (long slant, shallow butt).
8. Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
9. The uninoculated medium will be a deep forest green due to the pH of the sample and the bromothymol blue.

Procedure :

1. Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old.
2. Incubate 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 rate of growth on citrate medium.
3. Observe the colour change from green to blue along the slant: denoting alkalization.

a) If the test turned blue after incubation :

• Denotes the test is 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

Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia, etc.