Lab. No. 4

Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Rods

Gram +ve Bacilli

Spore forming Non spore forming

AerobicAnaerobic

BacillusClostridium

Corynebacterium

Most Important species:Most Important species:

Cl. tetaniCl. tetani → → causes tetanus.causes tetanus. Cl. perfringensCl. perfringens → → causes gas gangrene.causes gas gangrene. Cl. botulinumCl. botulinum → → causes food poisoningcauses food poisoning

(botulism).(botulism).

Gram’s +veGram’s +ve, , spore forming spore forming bacillibacilli

(the position of the spores(the position of the spores

helps in species helps in species

identification).identification).

Drum-Stick appearance of Cl.tetani

These organisms require These organisms require

anaerobic cultivation which can be achieved which can be achieved through:through:

1- Removal of O21- Removal of O2& replacing it with & replacing it with an inert gasan inert gas→ Blood agar plates → Blood agar plates

in in Anaerobic Jar.Anaerobic Jar.2-2- Special anaerobic mediaSpecial anaerobic media

containing containing a reducing agent.a reducing agent.

1. Anaerobic jar:

2. Anaerobic media:

Thioglycollate broth. Cooked meat medium.

It is a nutrient broth which serves as It is a nutrient broth which serves as

anaerobic mediumanaerobic medium due to presence of due to presence of

1- 1- Sodium thioglycollateSodium thioglycollate which acts as a which acts as a reducing agent.reducing agent.

2- 2- Small amount of agarSmall amount of agar to increase to increase viscosity of the medium thus decreasing viscosity of the medium thus decreasing convection current.convection current.

The medium contains The medium contains Redox Redox “oxidation-reduction potential”“oxidation-reduction potential” indicatorindicator (Methylene Blue)(Methylene Blue) which changes which changes color of the medium when 30% of the color of the medium when 30% of the medium becomes oxidizedmedium becomes oxidized. .

1-Thioglycollate broth:1-Thioglycollate broth:

Thioglycolate broth:Thioglycolate broth: It should be prepared in long tubeIt should be prepared in long tube

O2

aerobic bacteria

anaerobic bacteria

It is anaerobic medium due to presence of:It is anaerobic medium due to presence of: Meat particles (prepared from heart muscles) which Meat particles (prepared from heart muscles) which

contain contain hematin hematin and and glutathione glutathione that act as reducing that act as reducing agents.agents.

2- Cooked meat medium:2- Cooked meat medium:

Reactions on Cooked meat medium:Reactions on Cooked meat medium:

1.Saccharolytic reaction:

2. Proteolytic reaction:

Causes fermentation of the muscle glycogen with production of acid and gas. The meat particles remain intact.e.g. Cl.perfringes.

Causes digestion of the meat particles leading to formation of black, foul smelling sulphur compounds.

Litmus milk Litmus milk medium:medium:It Contains: • Skimmed milk (without fat) i.e: contains only sugar (Lactose) and protein (casine)• Litmus indicator (acid base and redox indicator).

Reactions on Litmus milk Reactions on Litmus milk medium:medium:1. Acidic reaction:

Lactose (milk

sugar)

Fermentationacid

Litmus indicatorpink colour

2. Alkaline reaction:

Casine (milk

protein)

DigestionAlkaline amines

Litmus indicatorBlue colour

3. Stormy Clot Formation:

Fermentation(Lactose) milk sugar

acid + gas

(Casine) milk

protein

Coagulation clot

Stormy clot