PHT 381 Lab# 6 Biochemical reactions

Enzyme production

1- Catalase Test Principle: Procedure: 3 1 2 Catalase enzyme H2O2 H2o + O2 Air bubbles Procedure: 3 H2O2 1 2

Results: Positive test: rapid appearance of gas bubbles. Catalase +ve

2-. Coagulase Test: Principle: Procedure: 2 1 3 Fibrinogen Plasma Coagulase enzyme Fibrin Visible Clot Procedure: 2 1 ml rabbit plasma 1 3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

Results: Positive test: formation of visible clot. Coagulase -ve S.epidermidis Coagulase +ve S.aureus

Tetramethyl p-phenylene diamine 3- Oxidase Test: Principle: Tetramethyl p-phenylene diamine (oxidase reagent) colourless Cytochrome oxidase enzyme Indophenol (Purple colour)

Oxidase test: Results: +ve Test: Appearance of purple colour within 1-2 min. No colour purple colour -ve test +ve test

Oxidase test: Results: +ve Test: Appearance of purple colour within 1-2 min. No colour purple colour -ve test +ve test

4- Urease Test: Principle: Urease enzyme Urea NH3 ( alkaline) Phenol red indicator Pink colour

Results: +ve -ve

5- Deoxyribonuclease (DNase) Test: Principle: DNA DNase enzyme Nucleotides Insoluble In acid soluble In acid Procedure: 1. Inoculate DNase agar plate with the test organism. 2. Incubate the plate at 35oC for 24 hrs. 3. Flood the plate with 1M HCl.

Results: DNase activity is indicated by a clear zone around the growth after addition of Hcl Clear zone around the growth while the rest of the plate appears cloudy Cloudiness in all the plate S.aureus S.epidermidis

Sugar Fermentation

1. Sugar fermentation: Sugar fermentation can be indicated by change of color of the medium from red to yellow. Glucose Maltose Sucrose Glucose Maltose Sucrose +ve +ve +ve +ve +ve - ve

(glucose 0.1%, lactose 1%, sucrose 1%). 2- Triple Sugar Iron (TSI) agar: Principle: (glucose 0.1%, lactose 1%, sucrose 1%). butt slant

1. No Fermentation: Butt: alkaline (red) Slant: alkaline (red)

a) Initial reaction: 2.Glucose Fermentation: (after 10 – 12 hrs) Dextrose acid Butt: acidic (yellow) Slant: acidic (yellow)

b) Delayed reaction: 2. glucose Fermentation: (after 24 hrs) O2 Peptone O2 Small amount of acid Alkaline amines Butt: acidic (yellow) Slant: alkaline (red)

3. Lactose/ sucrose Fermentation: Peptone O2 Large amount of acid Alkaline amines Butt: acidic (yellow) Slant: acidic (yellow)

This medium contains also ferrous sulfate as an indicator for H2S production. H2S + FeSo4 FeS Black ppt. of ferrous sulfid

Results: acidic (yellow) acidic (yellow) acidic (yellow) Butt: Slant: H2S Production: acidic (yellow) alkaline (red) alkaline (red) -ve +ve -ve

3-O/F Test (Oxidation Fermentation Test) Principle: Saccharolytic bacteria attack carbohydrates either: fermentatively ( in absence of oxygen) to yield relatively strong acids, or oxidatively ( in presence of oxygen) to yield weak acids. Oxidation process is much more easier than Fermentation process.

Procedure: 1 ml liquid paraffin O F

O-/F+ O-/F- O+/F+ O+/F- Results: Positive Test: Non Saccharolytic Fermentative Oxidative

sensitive O/F Test (Oxidation Fermentation Test) Principle: sensitive O/F medium is a specifically formulated medium to detect weak acids produced from saccharolytic Gram’s –ve bacteria.

To be more sensitive this medium contains: Higher conc. Of sugar to increase amount of acid produced. Lower amount of peptone to reduce formation of alkaline amines which neutralize weak acids formed. Lower conc. Of agar making the medium semisold to facilitate diffusion of acid throughout the medium.

Oxidation Fermentation (O/F) Test: Procedure: 1 ml liquid paraffin O F

Oxidation Fermentation (O/F) Test: Positive Test: Results: O-/F+ O-/F- O+/F+ O+/F- Non Saccharolytic Fermentative Oxidative

4- MR-VP Test: Principle: Dextrose Acidic pathway Neutral pathway OR Neutral pathway Mixed acids  pH less than 4.4 Acetyl methyl carbinol α-naphthol KOH MR indicator Brick red colour Red colour MR +ve VP +ve

Amino acid Metabolism

Purple red color in the upper organic layer 4- Indole test Principle: Tryptophanase enzyme Tryptophane Indole Kovac’s reagent Purple red color in the upper organic layer

Results: +ve -ve

Thank you