Lab. No. 6

Bacteria Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Rods

I. PseudomonasI. Pseudomonas

Most Important Strains:Most Important Strains:

Ps. aeruginosa Ps. aeruginosa which is which is opportunistic microorganism, opportunistic microorganism, causes UTI, wound infections and causes UTI, wound infections and otitis mediaotitis media

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

arrangement.arrangement.

I. PseudomonasI. Pseudomonas

Examination of Motility:Examination of Motility:Using the “Hanging Drop Preparation” Using the “Hanging Drop Preparation”

Pseudomonas is highly motile by means of Pseudomonas is highly motile by means of polar flagella.polar flagella.

Macroscopical Examination:Macroscopical Examination:Its growth on nutrient agar showing greenish Its growth on nutrient agar showing greenish

colouration due to exopigment production.colouration due to exopigment production.

I. PseudomonasI. Pseudomonas

I. PseudomonasI. Pseudomonas Oxidase test:Oxidase test:

Principle:Tetramethyl p-phenylene diamine

(oxidase reagent)colourless

Cytochrome oxidase enzyme

Indophenol(Purple colour)

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

Procedure:

O F

1 ml liquid paraffin

Positive Test:

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

O-/F- O+/F+ O+/F- O-/F+

Results:

Fermentative OxidativeNon Saccharolytic

I. PseudomonasI. Pseudomonas Nitrate Test:Nitrate Test:

Principle:

NitrateNitrate reductase

nitrite

α-naphthyl amine (nit. A)

Sulphanilic acid (nit. B)

Red diazonium salt

If no red colour!

Further reductionNirtogen (N2)

Add zinc dust (reducing agent)

zinc dust

I. PseudomonasI. 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 35oC for 24

hrs

I. PseudomonasI. 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 Cetrimide agar is a highly selective medium for Pseudomonas species medium for Pseudomonas species due to presences of cetrimide which due to presences of cetrimide which inhibits the growth of other bacteria.inhibits the growth of other bacteria.

It contains also MgClIt contains also MgCl22 & K & K22SoSo44 to to facilitate production of the facilitate production of the charactaristic green pigmentation of charactaristic green pigmentation of Pseudomonas. Pseudomonas.

I. PseudomonasI. Pseudomonas Growth on Cetrimide Agar:Growth on Cetrimide Agar:

Principle:

I. PseudomonasI. 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 35oC for 24 hrs.

Flame & Cool

Flame & Cool

Flame & Cool

I. PseudomonasI. 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 WorkPracticle 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