Bio chemical tests

	· ·	Catalase Test Put the one drop 3 % H2O2(hydrogen peroxide) Visible bubble split formation
EX:
	1.	Stephylococci
	2.	Bacillus subtitils
	3.	Pseudomonas aeuroginosa
	4.	Candida Albicans
	5.	Aspergillus brasillensis
	6.	Micrococci
	7.	E.coli
	8.	Protious vlugartus 9. Salmonella enterica

 ·         Catalase is the Enzyme that breaks Hydrogen peroxide (H2O2) in to H2 , O2.

 ·    2H2O2----------------------------- 2H2 O + O2

·         3% hydrogen peroxide is topical disinfectant in wound. And the bubbling that is seen is due to the evolution of O2 gas.

·         H-O-O-H

Oxidase test:

·         Bacterium produced the Cytochrome oxidase enzyme production

·         This enzyme is indicative of aerobic respiration.

·         Oxidase disk/oxidase reagents.

Positive Test:

·         Purpule (dark purpule) due to free of oxigen. Negative Test:

·         Light pink (no colour change)

EX:

1.       Psedomonas auroginosa

2.       Vibrio cholerae

      ·         Organism is a aerobe or aerobic respiration.

·         Electron transport chain that the final phase normally oxygen is final electron transfer but

·         In this Oxidase test a artificial final electron acceptor (N,N,N1,N1 –tetra methyl 1-4 phenlenedimine di chloride) is used in the place of oxygen.

·         This process Artificial chemical that change colour to a dark blue/purple colour.

·         When it takes the electron from last element (Cytochorome Oxidase) in the electron transport chain.

                                          Coagulation Test

·         Blood clotting test.

·         0.5 ml Mammalian (rabbit or horse) plasma + 1ml culture

·         Incubate 30-35⁰C 1 day incubation after gel formation.

          Ex : Staphylococcus aureus.

Indole test

·         Indole test used to determine the ability of an organism to spilt amino acid tryptophan to from the Compound Indole.

·    Tryptophan/amino acids ---------------------covert ------------------------ Indole.

·         Indole test differentiate the Entero a teria’s or other genera.

  

Method:

·         Inoculate the tryptophan broth add 1 ml of young culture.

·         Incubate at 37°C for 24-28 hours in ambient air.

·         Add 0.5 ml or 5 drops of Kova ’s reagent or Ehrli h’s reagent to the broth culture

Expected results:

Positive: Pink or Red –Violet colored ring or layer after addition of appropriate reagent

Negative: No color change even after the addition of appropriate reagent. e.g. Klebsiella Pneumoniae

Indole positive organisms: Most strains of E.coli, P. vulgaris are indole positive.

Kovac’s reagent or Ehrlich’s  reagent

Isomyl Alcohol, Para- Dimethyl amino benzaldehyde, concentrated hydrochloric Acid.

Motility test

1. Hanging Drop motility test:

·         Place a drop of bacterial culture(young broth culture) in middle of a cover slip.

·         Take a slide.

·         Slide around the petrolium jelly or paraffin wax applied.

·         The slide is inverted (gently press the cover slip)

·         The bacterial drop hangs the cover slip.

·         Observe under microscope.

·         Use immersion oil in under 100X objective.

·         Use a lower power lens to find the bacteria.

2. Biochemical test:

·         Take the TTC motility agar tubes.

·         Inoculate the culture (young culture) with the help of the needle, all the way to the bottom.

·         Incubate the tube 30-35⁰C.

·         Observed the tube

·         Motile bacteria spread (red colored) the throughout tube.

·         Red colour dye –Tetrazolin which turn to red as a result of the bacterium metabolizing.

Expected Results /Response Motility

Eschericha coli -------------------Growth Positive

Salmonella ------------------------Growth Positive

Staphylococcus aureus ---------Growth Negative (non-motile)