1. Lab. No. 6

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

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

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

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

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

8. 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

9. 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

10. 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.

11. 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.

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

13. 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

14. 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

15. 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

16. 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

17.  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:

18. 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

19. 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.

20. 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

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