PHT382 Lab. No. 4

Classification of Bacteria

Classification of Gram's +ve Bacilli Gram +ve Bacilli Spore forming Non spore forming Anaerobic Aerobic Corynebacterium Clostridium Bacillus

Most Important species: Clostridium Most Important species: Cl. tetani → causes tetanus. Cl. perfringens → causes gas gangrene. Cl. botulinum → causes food poisoning (botulism).

Identification of Clostridium Species Gram’s +ve, spore forming bacilli (the position of the spores helps in species identification). (I) Microscopical examination (morphology): Drum-Stick appearance of Cl.tetani

(II) Cultural characteristics: These organisms require anaerobic cultivation which can be achieved through: 1- Removal of O2& replacing it with an inert gas→ Blood agar plates in Anaerobic Jar. 2- Special anaerobic media containing a reducing agent.

1. Anaerobic jar:

2. Anaerobic media: Thioglycollate broth. Cooked meat medium.

1-Thioglycollate broth: It is a nutrient broth which serves as anaerobic medium due to presence of 1- Sodium thioglycollate which acts as a reducing agent. 2- Small amount of agar to increase viscosity of the medium thus decreasing convection current. The medium contains Redox “oxidation-reduction potential” indicator (Methylene Blue) which changes color of the medium when 30% of the medium becomes oxidized.

Thioglycolate broth: It should be prepared in long tube O2 aerobic bacteria anaerobic bacteria

2- Cooked meat medium: It is anaerobic medium due to presence of: Meat particles (prepared from heart muscles) which contain hematin and glutathione that act as reducing agents.

Reactions on Cooked meat medium: 1.Saccharolytic reaction: Causes fermentation of the muscle glycogen with production of acid and gas. The meat particles remain intact. e.g. Cl.perfringes. 2. Proteolytic reaction: Causes digestion of the meat particles leading to formation of black, foul smelling sulphur compounds.

(III) Biochemical reactions: Litmus milk 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 medium: 1. Acidic reaction: Lactose (milk sugar) Fermentation acid Litmus indicator pink colour

2. Alkaline reaction: Casine (milk protein) Digestion Alkaline amines Litmus indicator Blue colour

3. Stormy Clot Formation: Fermentation (Lactose) milk sugar acid + gas Stormy clot (Casine) milk protein Coagulation clot

Thank you