PHT313 Lab. No. 4

Classification of Bacteria

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

Identification of pseudomonas species (I) 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.

(II) Cultural characteristics: 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.

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.

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

Procedure: Inoculate Cetrimide agar plate with the test organism by streaking. Flam & Cool 2. Incubate the plate at 35oC for 24 hrs.

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

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

Inoculate MacConkey’s agar plate with the test organism by streaking. Procedure: Inoculate MacConkey’s agar plate with the test organism by streaking. Flam & Cool 2. Incubate the plate at 35oC for 24 hrs.

Results: Pink colonies Lactose fermenter Pale colonies Lactose nonfermenter

Tetramethyl p-phenylene diamine (III) Biochemical reactions: 1)Oxidase test: Principle: Tetramethyl p-phenylene diamine (oxidase reagent) colourless Cytochrome oxidase enzyme Indophenol (Purple colour)

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

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

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

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

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

3) 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 differs from ordinary fermentation medium to be more sensitive to detect the small amount of weak acids produced by Gram’s –ve bacteria.

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.

Procedure: 1 ml liquid paraffin O F

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

4) Growth on Triple Sugar Iron (TSI) agar: Principle: This medium contains three types of sugars (lactose, sucrose and dextrose). The conc. of lactose and sucrose is 10 times that of dextrose. It also contains phenol red indicator.

Principle: butt slant

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

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

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

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

Principle: This medium contains also ferrous sulfate as an indicator for H2S production. H2S + FeSo4 FeS Black ppt. of ferrous sulfid

Procedure:

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

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