Lab. No. 6

I. Pseudomonas Most Important Strains: Most Important Strains:  Ps. aeruginosa which is opportunistic microorganism, causes UTI, wound infections and otitis media

I. Pseudomonas Gram’s Stain: Gram’s Stain: Gram’s –ve rods, having single arrangement.

I. Pseudomonas Examination of Motility: Examination of Motility: Using the “Hanging Drop Preparation” Pseudomonas is highly motile by means of polar flagella.

Macroscopical Examination: Macroscopical Examination: Its growth on nutrient agar showing greenish colouration due to exopigment production. I. Pseudomonas

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

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

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

I. Pseudomonas Oxidation Fermentation (O/F) Test: Oxidation Fermentation (O/F) Test: Principle: O/F medium ( Hugh and Leifson Medium) is a specifically formulated medium (sensitive O/F medium) to detect weak acids produced from saccharolytic Gram’s –ve bacteria. i.e: Its composition deffers from ordinary fermentation medium to be more sensitive to detect the small amount of weak acids produced by Gram’s –ve bacteria.

I. Pseudomonas Oxidation Fermentation (O/F) Test: Oxidation Fermentation (O/F) Test: Principle: 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.

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

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

I. Pseudomonas Nitrate Test: Nitrate Test: Principle: Nitrate Nitrate reductase nitrite α -naphthyl amine (nit. A) Sulphanilic acid (nit. B) Red diazonium salt If no red colour! Further reduction Nirtogen (N 2 ) Add zinc dust (reducing agent) zinc dust

I. Pseudomonas Nitrate Test: Nitrate Test: Procedure: Nitrate broth test m.o Nit.A Nit. B Red colour No red colour Add zinc dust Incubate at 35 o C for 24 hrs

I. Pseudomonas Nitrate Test: Nitrate Test: Results: Red colour after addition of nit.A & nit.B Reduction of Nitrate to nitrite Red colour after addition of zinc dust -ve reduction No red colour after addition of zinc dust Further reduction to Nitrogen

 Cetrimide agar is a highly selective medium for Pseudomonas species due to presences of cetrimide which inhibits the growth of other bacteria.  It contains also MgCl 2 & K 2 So 4 to facilitate production of the charactaristic green pigmentation of Pseudomonas. I. Pseudomonas Growth on Cetrimide Agar: Growth on Cetrimide Agar: Principle:

I. Pseudomonas Growth on Cetrimide Agar: Growth on Cetrimide Agar: Procedure: 1. Inoculate Cetrimide agar plate with the test organism by streaking. 2. Incubate the plate at 35 o C for 24 hrs. Flame & Cool

I. Pseudomonas Growth on Cetrimide Agar: Growth on Cetrimide Agar: Results: Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.

Practicle Work Gram’s stain (spot) Gram’s stain (spot) Oxidase test. Oxidase test. O/F test. O/F test. Nitrate test. Nitrate test. Growth on Cetrimide agar Growth on Cetrimide agar

With my Best Wishes,,, Manal Abu El-Khair