Biochemical Tests to differentiate microorganisms
Colony forming characteristics of diff. organisms in selective media.

1] Amylase test or starch hydrolysis: Starch is a complex carbohydrate classed with polysaccharides. Some bacteria elaborate an extra cellular enzyme called ‘amylase’
(diastase) which has the power to hydrolyze colloidal starch molecule to simple
sugar i.e. dextrin, maltose, glucose. Maltose is diffusible and can enter the bacterial cell when it is attacked by the intracellular or respiratory enzymes
Inoculate starch agar slant containing 1% starch with B. subtilis and incubate for 48hrs. Observe the clarification of starch and test it with iodine. Absence of blue colour indicates complete hydrolysis of starch.

2] Nitrate reduction test : The term nitrate reduction includes all processes in which nitrate disappears under the influence of bacterial action and appears in some less oxidized state. Most bacterial species do not carry out the reduction beyond the stage of nitrite. However insome instances, the reduction may proceed to NH3 stage an even to molecular nitrogen. The reduction of nitrate proceeds in the presence of anaerobic environment. Inoculate the nitrate broth tube’s with E. coli and incubate for 24-96 hrs.
After the growth has taken place transfer 1ml of broth into test tube add 2 drops of sulfanilicacid reagent, 2 drops of a- hapthalamine. A appearance of pink colour indicate nitrate formation.

3] Protease test or Hydrolysis of Gelatin: – Gelatin is convenient as a substrate to test for proteolyses enzymes in micro-organisms. In the concentrations. used water solution of gelatin will solidity at cool temperateness. It the gelatin will solidify at cool temperatures. If the gelatin has been hydrolyzed by the action of the micro-organisms being tested, the medium will remain liquid.

Inoculate 4 tubes of nutrient gelatin with E – coli B. subtilis ; Streptococcus faecalis and Proteus Vulgarism.Inccubute at 370c , Test for 2 days and up to 7 days to obtain a positive reaction. To examine for hydrolysis, chill the tube in ice water. The control tubes and the tubes with negative reaction will solidify. Hydrolyzed gelatin will remain fluid. Protein breakdown is very slow as compared to cryohydrates; hence longer period of inabution is essential for visual detection.
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4] Urease test or Hydrolysis of Area : Hydrolysis of area is brought about by an enzyme urease produced by certain baste rid, This results in the production of ammonia.
Decompusition of area is particularly criteria for differentiation of certain closely related bacteria eg. Proteus from salmonla Escherichia.
Urea agar slant or area broth certaining 2% area and phenol red is in osculated with protons Vulgarism and incubated 370c for 48 hrs. The production of ammonia from hydrolysis of area increases the PH of the medium which changes the colour of the medium from yellow to pink or red.

5] Sugar fermentation: Organisms vary considerably in their ability to attack and ferment various car Bo hydrates. Some organisms are able to attack a carbohydrate and produce acid and gas. Others are able to produce acid but no gas, still others fail to ferment the comp.
Inoculate the tubes of lactose or glucose broth with yeast (s.cervisiae) and incubate for 48hrs. The formation of gas bubbles in the Durham’s tubes indicate sugar fermentation. Add production may be tested may be tested with bromothymol blue indicator which turns yellow.

6] Patronization and fermentation of milk : Milk is used as a differential medium to demonstrate the ability of an organism to produce 1) a fermentuneous fermentation and patronization. A rapid utilization of lactose results in a terminative reaction. Under these conditions casein is curdled and accompanied by separation of clear whey. The PH is strongly acidic sufficient usually to prevent further bacterial attack. The presence of a rennin card indicates that the organism either does not attack the lactose or ferment it very slowly. In the former case, a slow action on the milk sugar results in a simultaneous fermentation and protein decomposition.
Inoculate tubes of litmus milk with lactobacillus buglers and incubate for 48 hrs.
Colony forming characteristics of diff. organisms in selective media.
1] S.aureus –> Vogel Johnson agar —> Black colonies by yellow zones confirmed by coagulase test and gram staining

2] Pseudomos aero. —-> Cetrimide agar —-> Bluish green colonies with green fluorescence confirmed by oxidase test and gram staining

3] E coli —-> Mc. Conkey’s agar —-> Brick pink colonies confirmed by gram staining
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