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313 PHT Lab. No. 8

313 PHT Lab. No. 8. Classification of Bacteria. Pseudomonas. Aerobic , non-fermentative , motile , oxidase-positive gram-negative bacilli. Most Important Species P.aeruginosa opportunistic pathogen causes UTI, wound infections and otitis media. Microscopical examination:

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313 PHT Lab. No. 8

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  1. 313 PHT Lab. No. 8

  2. Classification of Bacteria

  3. Pseudomonas • Aerobic, non-fermentative, motile, oxidase-positive gram-negative bacilli. • Most Important Species P.aeruginosa • opportunistic pathogen • causes UTI, wound infections and otitis media

  4. Microscopical examination: (morphology) A) Gram’s Stain: Gram –ve Non-sporeforming bacilli , having single arrangement.

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

  6. Cultural characteristic: It grows on simple media. It usually produces exopigments. 1) Growth on nutrient agar: Its growth on nutrient agar showing greenish discolouration due to exopigment production.

  7. 2) Growth on Cetrimide Agar: Principle: • 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 MgCl2 & K2So4 to facilitate production of the charactaristic green pigment of pseudomonas.

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

  9. 3) Growth on MacConkey’s agar: Principle: • MacConkey’s agar is a selective and differential medium • selective medium for enteric gram –ve bacteria (bile salt inhibit the growth of non enteric bacteria). • Test sugar:lactose. • pH indicator:neutral red ( yellow in alkaline, pink in acidic pH).

  10. Gram –ve bacteriaare classified into: Lactose fermenter (Pink colonies) Lactose non-fermenter (pale colonies)

  11. Results: Pink colonies Lactose fermenter Pale colonies Lactose nonfermenter

  12. Biochemical reaction: 1)Oxidase test. 2) Nitrate Test. 3) Oxidation Fermentation (O/F) Test. 4) Growth on Triple Sugar Iron (TSI) agar.

  13. 1)Oxidase test: Results: +ve Test: Appearance of purple colour within few seconds. No colour purple colour -ve test Enterobacteriaceae +ve test Pseudomonas

  14. 2) Nitrate Test: Principle: Further reduction Nitrate reductase nitrite Nitrate Nirtogen (N2) α-naphthylamine (nit. A) Sulphanilic acid (nit. B) Red diazonium salt Enterobacteriaceae If no red colour! Add zinc dust (reducing agent)

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

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

  17. 3) Oxidation Fermentation (O/F) Test: Sensitive O/F test

  18. Positive Test: Results: O-/F- O+/F- O+/F+ O-/F+ Non Saccharolytic Fermentative Enterobacteriaceae Oxidative Pseudomonas

  19. 4) Growth on Triple Sugar Iron (TSI) agar: Principle: butt slant

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

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

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

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

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

  25. Pseudomonas aeruginosa I'm very resistant to most antibiotics, so it's very hard to get rid me.

  26. Thank you

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