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PHT 381 Lab# 6 1- Catalase Test Principle: Catalase enzyme H 2 O2 Procedure: 3H2O2 1 2 H 2o + O2 Air bubbles Results: Positive test: rapid appearance of gas bubbles. Catalase +ve Catalase –ve 2-. Coagulase Test: Principle: Fibrinogen Plasma Coagulase enzyme Fibrin Visible Clot Procedure: 2 1 1 ml rabbit plasma 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 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 Insoluble In acid DNase enzyme Nucleotides 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 Cloudiness in all the plate S.epidermidis Clear zone around the growth while the rest of the plate appears cloudy S.aureus 1. Sugar fermentation: Sugar fermentation can be indicated by change of color of the medium from red to yellow. Glucose +ve Maltose +ve Sucrose +ve Glucose +ve Maltose +ve Sucrose - ve 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) 2.Glucose Fermentation: a) Initial reaction: (after 10 – 12 hrs) Dextrose acid Butt: acidic (yellow) Slant: acidic (yellow) 2. glucose Fermentation: b) Delayed reaction: (after 24 hrs) Small amount of acid Butt: acidic (yellow) Slant: alkaline (red) 3. Lactose/ sucrose Fermentation: Lactose Large amount of acid 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: Butt: acidic (yellow) acidic (yellow) acidic (yellow) Slant: acidic (yellow) alkaline (red) alkaline (red) H2S Production: -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 Results: Positive Test: O-/FNon Saccharolytic O+/F+ Fermentative O+/FOxidative O-/F+ 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: Results: Positive Test: O-/FNon Saccharolytic O+/F+ Fermentative O+/FOxidative O-/F+ 4- MR-VP Test: Principle: Dextrose Acidic pathway Mixed acids pH less than 4.4 MR indicator Red colour MR +ve OR Neutral pathway Acetyl methyl carbinol α-naphthol KOH Brick red colour VP +ve 4- Indole test Principle: Tryptophane Tryptophanase enzyme Indole Kovac’s reagent Purple red color in the upper organic layer Results: +ve -ve