313 PHT Lab. No. 8

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

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

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

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.

 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 pigment of pseudomonas. 2) Growth on Cetrimide Agar: Principle:

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

  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). 3) Growth on MacConkey’s agar: Principle:

Gram –ve bacteria are classified into: Gram –ve bacteria are classified into: Lactose fermenter (Pink colonies) Lactose non-fermenter (pale colonies)

Results: Pink colonies Lactose fermenter Pale colonies Lactose nonfermenter

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

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

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

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

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

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

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

Principle: buttslant 4) Growth on Triple Sugar Iron (TSI) agar:

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

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

2. Dextrose Fermentation: b) Delayed reaction: (after 24 hrs) Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: alkaline (red) Small amount of acid

3. Lactose Fermentation: Lactose Large amount of acid Peptone O2O2 Alkaline amines Butt: acidic (yellow)Slant: acidic (yellow)

Results: Butt: Slant: H 2 S : acidic (yellow) -ve acidic (yellow) alkaline (red) -ve acidic (yellow) alkaline (red) +ve alkaline (red) -ve

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

Thank you