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Friday, April 24, 2009

Tests Enzyme Catalase determination Catalase Test Coagulase Oxidize Test

CATALASE TEST:-
Catalase is an enzyme present in most bacteria. It catalyses the breakdown of the with the release of free or gas.
2H2O2 + Catalase ------> 2H2O2 + O2
Gas, in most of the cases can be readily seen us a white froth if you add a few drops of 3% H2O2 to a colony or a broth culture, catalase activity is inhibited to some extent in strongly acid media. Four important Catalase –ve genera are streptococcus, Leuconostoc, Lactobacillius and clostridium, catalase –ve organism tend to be anaerobic. The enzyme catalase is essential for the aerobic growth of most microorganisms.

Procedure:-
Inoculate on agar slant with E Coli. Keep one slant as control. Incubate for 24.48hrs. at 370c cover the agar slant with 3% solution of H2O2 bubbles of O2 around the colonies indicate presence of catalase.
Coagulase test :-
This test is used to determine the virulence of s.aureus. Active coagulate producing strains when added to small amounts of diluted plasma and inculcated at 370c cause formation of clot within 2-3 hrs. food poisoning strains of staphylococci are generally coagulate +ve.
Procedure:- Add 0.5 – 1ml of 24hrs broth culture to 0.5ml of a treated human blood plasma place the test tube in a water bulk at 370c and observe the tube at frequent intervals over a 3hrs. period formation of clot shows coagulase present.
Microscopic slide coagulation:- Place on a clean dried slid one loopful of citrated human plasma in the centre mix with one loopful of the culture. Also place one the same slide one loopful each of plasma and culture separately observe under microscope for coagulation.
Oxidize Test:- The ability to produce enzyme oxidize is a characteristic of only a few bacteria. This rapid test allows a convenient differentiation between pseudomonas and other gram –ve , lastose –ve, colons on isolation plates.
Procedure:-
Inoculate nutrient agar with pseudomonas species, incubate at 370c for 48hrs. After inculcation cover the colonies with oxidize reagent i.e.1% soln of P-amino dimethyl anifine monochloride or Tetramethyl one diaphonic diamine dihydrochloride. After a minute or two pour the excess reagent. Watch the derange in colour of colonies, pint turning to purple and gradually to black with 2 – 15 minutes.
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