Biochemical tests

Biochemical Tests

1-Catalase test
Most aerobic microorganism contain catalase enzyme. The function of catalase is to remove toxic hydrogen peroxide that forms during the oxidation-reduction reactions. Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen, and can be differentiate between Staphylococci (+) and Streptococci (-) 2 H2o2 Catalase 2 H2o + o2

PROCEDURE 1-Transfer 2-3 drops of 3% H2o2 to the surface of clean slide. 2-Transfer bacterial colonies and mixed with drops of H2o2. 3- Observe the result positive reaction: bubbles forming negative reaction : no bubbles forming.

Note : A false positive reaction may be obtained if the culture medium contains catalase ( e.g.blood agar) which found in RBCs.

2-Oxidase test

Oxidase enzymes play an important role in the operation of the electron transport system during aerobic respiration. This test used to determine the transport of electron between electron donor ( bacteria ) to electron acceptor ( dye ), The dye most commonly used : Tetra methyl _P_phenylene_ diamine dihydrochloride.

Many bacteria that live in the presence of oxygen produce cytochrome oxidase, but some do not. In this test differentiates these microbial groups. This test is used to differentiate between Pseudomonas Spp. (+ve ) from Entrobateriaceae (-)

PROCEDURE: 1- filter paper will saturate with the dye of : (tetra methyl _ P_ phenylene_ diamine_ dihydrochloride). 2-then transfer bacterial colonies to the filter paper and observe the change of color within 10 sec. 3- positive result :Dye is oxidize to indophenol blue (deep purple or violate) color negative result :colorless

NOTE: false positive result traces of ions will catalyze the reaction and give false positive result so we transfer bacteria by toothpick.


3- Coagulase test
This test is used to detect the ability of microorganisms to coagulate plasma by action of this enzyme ( coagulase ), this enzyme has prothrombin like effect, that convert fibrinogen to fibrin. produce by Staph. aureus which use this enzyme to forming a fibrin clot around themselves and avoid attack by the hosts defenses. This test used to differentiate between species of staphylococci: Staph. aureus (+) Staph . epidermidis (-) Staph saprophyticus (-)

PROCEDURE: Tube test : By adding 0.5ml of bacterial broth culture to 0.5ml of rabbit plasma in sterile test tube. Rotated, then incubate at 37C for 1-4 hrs. Examine the clot formation. Result (+) : clotting Result (-) : no clotting N0TE Can take sample from blood used Citrate and EDTA as blood anticoagulants, to prevent false positive results.

4-Urea hydrolysis Test

Urea is a common metabolic product that is toxic to most living organisms Some bacteria are able to produce an enzyme called urease. The enzyme urease catalyzes the urea in to ammonia and carbon dioxide as shown here

The urease test is used to distinguish member of Proteus Spp.(+) from other non lactose fermenting enteric bacteria(Salmonella-Shigella).(-)


Urease activity is detected by growing bacteria in a medium containing urea and using pH indecator( phenol red ). When urea is hydrolyzed,ammonia accumulate in the medium and make it alkaline. Increase of PH ,and the color will be change.

PROCEDURE: 1- Christensens urea media which are used in this test inoculated with examine bacteria. 2- Incubate at 37 C, and examine after 24hrs. The change in PH can be detected by phenol red in this media {indicator} this indicator is deep pink in PH 7.5 and yellow in PH 6.9. 3- observe the color of medium Result (+) : deep pink Result (-) : yellow(Orange)

positive

negative

5-Gelatin hydrolysis test

Gelatinase is an enzyme which removes gelatin that solidifying a medium(Liquefaction). Gelatinase production is use to identify species of bacteria. Gelatin dissolves in warm water and get solid when cool, if it was hydrolyzed, it does not gel but remains liquid when cooled. Gelatin suspensions are in the gel state ( semisolid ) below 25Co and ( liquid ) above that temperature.


Note heat liquefied gelatin. Remember that after incubation at 37Cothe liquid state of a warm gelatin culture can be confused with the result of bacterial liquefaction. Therefore always chill your gelatin cultures before reporting gelatin liquefaction.

Procedure: Label 2 tubes containing nutrient gelatin(15% of gelatin to nutrient broth ). Inculate each gelatin tube with bacteria then incubate the culture at least 48 hours, At 37o Place your gelatin culture tubes upright in a refrigerator of at least 30 minutes. Examine each tube for evidence of liquefaction. Result : (+ve) liquefied of gelatin. (-ve) no liquefied of gelatin.


Staph. aureus, Pseud. aeruginosa Proteus vulgaris E.coli (-ve)

(+ ve).