Lab. No. 4

Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Bacilli

AerobicAerobic, , non-fermentativenon-fermentative, , motilemotile, , oxidase-positiveoxidase-positive gram-negative gram-negative bacilli.bacilli.

Most Important SpeciesMost Important Species P.aeruginosa P.aeruginosa opportunistic pathogen opportunistic pathogen causes UTI, wound infections and otitis causes UTI, wound infections and otitis

mediamedia

A) Gram’s Stain:A) Gram’s Stain:Gram –ve Gram –ve

Non-sporeforming bacilli , Non-sporeforming bacilli ,

having single arrangementhaving single arrangement..

B) Examination B) Examination of Motility:of Motility:

Using the Using the “Hanging “Hanging Drop technique”Drop technique”

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

It grows on It grows on simple media.simple media.

It usually produces It usually produces exopigments.exopigments.

1) Growth on nutrient agar:1) Growth on nutrient agar: Its growth on nutrient agar Its growth on nutrient agar

showing showing greenish discolourationgreenish discolouration

due to due to exopigmentexopigment production. production.

Cetrimide agar is a Cetrimide agar is a highly selective highly selective mediummedium for pseudomonas species due for pseudomonas species due to presences of to presences of cetrimide cetrimide which which inhibits the growth of other bacteria.inhibits the growth of other bacteria.

It contains also It contains also MgClMgCl22 & & KK22SoSo44 to to facilitate production of the facilitate production of the charactaristic charactaristic green pigment green pigment of of pseudomonas. pseudomonas.

2) Growth on Cetrimide Agar: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.

Procedure:

1. Inoculate Cetrimide agar plate with the test organism by streaking.

2. Incubate the plate at 35oC for 24 hrs.

Flam & Cool

Flam & Cool

Flam & Cool

MacConkey’s agar is a selective and differential medium

selective medium for selective medium for gram –ve bacteriagram –ve bacteria ( (bile saltbile salt & & crystal violetcrystal violet inhibit the growth of gram +ve bacteria). inhibit the growth of gram +ve bacteria).

Test sugar:Test sugar: lactose.lactose. pH indicator:pH indicator: neutral redneutral red ( yellow in alkaline, pink in ( yellow in alkaline, pink in

acidic pH).acidic pH).

3) Growth on MacConkey’s agar:3) Growth on MacConkey’s agar:

Principle:

Gram –ve bacteriaGram –ve bacteria are classified into:are classified into:

Lactose fermenter (Pink colonies)

Lactose non-fermenter (pale colonies)

Procedure:

1. Inoculate MacConkey’s agar plate with the test organism by streaking.

2. Incubate the plate at 35oC for 24 hrs.

Flam & Cool

Flam & Cool

Flam & Cool

Results:

Pink colonies

Lactose fermenter

Pale coloniesLactose nonfermenter

1)Oxidase test: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.

purple colour

+ve test

No colour

-ve test

Results:

+ve Test: Appearance of purple colour within 1-2 min.

purple colour

+ve test

No colour

-ve test

2) Nitrate Test:2) 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

Procedure:

Nitrate broth

test m.o

Nit.A

Nit. B

Red colour

No red colour

Add zinc dust

Incubate at 35oC for 24

hrs

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

3) 3) 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 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:

O F

1 ml liquid paraffin

Positive Test:

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

Results:

Fermentative OxidativeNon Saccharolytic

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:1. No Fermentation:

Butt: alkaline (red) Slant: alkaline (red)

2. Dextrose Fermentation:2. Dextrose Fermentation:

a) Initial reaction:

Dextrose

acid

(after 10 – 12 hrs)

Butt: acidic (yellow) Slant: acidic (yellow)

2. Dextrose Fermentation:2. Dextrose Fermentation:

b) Delayed reaction:(after 24 hrs)

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: alkaline (red)

Small amount of acid

3. Lactose Fermentation:3. Lactose Fermentation:

Lactose

Large amount of acid

Peptone O

2

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:

Butt:

Slant:

H2S Production:

acidic (yellow)

acidic (yellow)

-ve

acidic (yellow)

alkaline (red)

-ve

acidic (yellow)

alkaline (red)

+ve

Practicle WorkPracticle Work

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