Vp and citrate utilization test

NON ACID END PRODUCT TEST

 

AIM:

To observe the non acid end product in the given bacterial culture

PRINCIPLE:

The Voges-Proskauer test (named after Daniel Voges, German physician, and Bernhard Proskauer, German hygienist, in the early twentieth century) identifies bacteria that ferment glucose, leading to 2,3-butanediol accumulation in the medium. The addition of 40% KOH and a 5% solution of alpha-naphthol in absolute ethanol (Barritt’s reagent) will detect the presence of acetoin—a precursor in the synthesis of 2,3-butanediol. In the presence of the reagents and acetoin, a cherry-red color develops. Development of a red color in the culture medium 15 minutes following the addition of Barritt’s reagent represents a positive VP test; absence of a red color is a negative VP test

MAREIALS REQUIRED:

Test tubes, bacterial culture, Inoculation loop, Baritt’s reagent A & B

PROCEDURE:

1. Pre sterilized MR-VP broth was prepared in appropriate quantity in a conical flask and it was distributed in test tubes and autoclaved.

2. The MR-VP broth media tubes were labeled with the name of the bacterium to be inoculated, name, and date.

3. By following aseptic technique, each tube was inoculated with the appropriate bacterium by means of a loop inoculation.

4. All tubes were incubated with appropriate control at 35°C for 24 to 48 hours.

Biochemistry within bacteria

                                                      CO₂                       CO₂

Glucose + 1/2 O2       2 pyruvate       n-acetolactate         acetoin        2,3-butanediol

Biochemistry within tubes

Acetoin + _-naphthol  40% KOH

diacetyl + creatine (pink complex)

absolute

alcohol

           

CITRATE UTILIZATION TEST

AIM:

To observe the citrate as a sole carbon source for the given bacterial culture

PRINCIPLE:

The citrate utilization test determines the ability of bacteria to use citrate as a sole carbon source for their energy needs. This ability depends on the presence of a citrate permease that facilitates transport of citrate into the bacterium. Once inside the bacterium, citrate is converted to pyruvic acid and CO₂. Simmons citrate agar slants contain sodium citrate as the carbon source, NH₄⁺ as a nitrogen source, and the pH indicator bromothymol blue. This test is done on slants since O2 is necessary for citrate utilization. When bacteria oxidize citrate, they remove it from the medium and liberate CO2. CO2 combines with sodium (supplied by sodium citrate) and water to form sodium carbonate—an alkaline product. This raises the pH, turns the pH indicator to a blue color, and represents a positive citrate test; absence of a color change is a negative citrate test. Citrate-negative cultures will also show no growth in the medium.

MARTERIALS REQUIRED:

Test tubes, Simmon’s Citrate agar, Bacterial culture, Inoculation loop

PROCEDURE:

1. The Simmons citrate agar slants were labeled with the name of the bacterium to be inoculated, name and date.

2. By following aseptic technique, Simmons citrate agar slants were inoculated the bacterium by means of a stab-and-streak method.

3. The inoculated tubes were incubated for 24 to 48 hours at 35°C.

4. The slant cultures were examined for the presence or absence of growth and for any change in color from green to blue.

Biochemistry within bacteria

Sodium           citrate

                                                        

Citrate            permease  citrase    Pyruvic acid + oxaloacetic acid + CO₂

Biochemistry within tubes

 

Excess sodium from sodium citrate + CO₂ + H₂O            Na₂CO₃ (alkaline) pH