Question

6. What is the function of the enzyme urease? 7. What is the purpose of including phenol red (PR) in the urea broth? What compound is responsible for the rise in pH? edients . فمفد 8. What happens to the gelatin inoculated with a gelatinase-positive organism? 9. What reagent is used to test for the presence of indole? What happens to the reagent layer if the indole test is positive? 10. What does SIM medium test for? m SECTION 5 Differential Tests 353

Answer 1

6. Urease catalyses the hydrolysis of urea forming ammonia and carbon dioxide.

(NH2)2CO2 + H2O → CO2 + 2NH3

Hydrolysisof occurs in two stages. At first ammonia and carbamate are produced. Then the carbamate is hydrolyses into ammonia and carbonic acid.

7. Urea broth is used to test the ability of an organism to produce an exoenzyme called urease that hydrolyses urea to produce ammonia and carbon dioxide. Phenol red works as a pH indicator. If the pH is acidic it will turn into yellow colour and if the pH is alkaline it will turn into fuchsia/pink colour.

If the urea is hydrolysed then ammonia is produced, that will ultimately lead to rise in pH level by creating an alkaline environment. Consequently the media will turn into pink colour.

8. Gelatin is a protein derived from the animal protein collagen. Whereas gelatinase is an extracellular proteolytic enzyme that hydrolyses gelatin. So, if we inoculate gelatinase-positive organism to gelatin media then the gelatinase produced from that bacteria will degrade or hydrolyse the gelatin and produce peptides in first step. Then the polypeptides are hydrolysed again to finally produce amino acids.

Gelatin → Polypeptides → Amino acids

9. Kovac's reagent is used to test for the presence of indole. This basically contains para-dimethylaminobenzaldehyde, isoamyl alcohol and concentrated hydrochloric acid.

If the indole test is positive then the surface alcohol layer of the broth will turn into red or red violet colour. The red colour occurs through a series of reactions. At first the para-dimethylaminobenzaldehyde reacts with the indole present in the medium and forms a red coloured rosindole dye. The isoamyl alcohol reacts with the rosindole dye and causes it to precipitate. The remaining isoamyl alcohol then rise to the surface of the medium.

10. SIM medium or Sulphide Indole Motility medium is a bacterial growth medium. The application of SIM medium lies in its name. It is basically used to test for the ability of the bacteria to reduce sulfate, the ability to produce indoles and motility of the bacteria.

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