1. Lab. No. 4

3. 1. Gram’s Stain: Gram’s +ve Bacilli Spore forming Non spore forming AerobicAnaerobic Bacillus Clostridium Corynebacterium

4. Identification of Clostridium Species Most Important Strains: Most Important Strains:  Cl. tetani causes tetanus.  Cl. perfringens causes gas gangrene.  Cl. botulinum causes food poisoning.

5. Identification of Clostridium Species Gram’s Stain: Gram’s Stain: Gram’s +ve spore forming bacilli Drum-Stick appearance of Cl.tetani

6. Identification of Clostridium Species Anaerobic growth condition: Anaerobic growth condition: 1. A naerobic jar. 2. A naerobic media.

7. Identification of Clostridium Species Anaerobic growth condition: Anaerobic growth condition: 1. Anaerobic jar:

8. Identification of Clostridium Species Anaerobic growth condition: Anaerobic growth condition: 2. Anaerobic media:   Fluid thioglycolate.  Cooked meat medium.  Litmus milk medium.

9. It is anaerobic medium due to presence of:  Sodium thioglycolate and cystaine which act as reducing agents.  Small amount of agar to increase viscosity of the medium thus decreasing convection current.  Redox indicator (Methylene Blue) which change colour of the medium when 30% of the medium become oxidized. Fluid thioglycolate: Fluid thioglycolate: Anaerobic media

10. Fluid thioglycolate: Fluid thioglycolate: Anaerobic media It should be prepared in long tube It should be prepared in long tube O2O2 aerobic bacteria anaerobic bacteria

11. It is anaerobic medium due to presence of:  Meat particles (prepared from heart muscles) which act as reducing agents. Cooked meat medium: Cooked meat medium: Anaerobic media

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

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

14. Reactions on Litmus milk medium: Reactions on Litmus milk medium: Anaerobic media 1. Acidic reaction: Lactose (milk sugar) Fermentation acid Litmus indicator pink colour

15. Reactions on Litmus milk medium: Reactions on Litmus milk medium: Anaerobic media 2. Alkaline reaction: Casine (milk protein) Digestion Alkaline amines Litmus indicator Blue colour

16. Reactions on Litmus milk medium: Reactions on Litmus milk medium: Anaerobic media 3. Stormy Clot Formation: Fermentation (Lactose) milk sugar acid + gas (Casine) milk protein Coagulation clot Stormy clot

17. Reactions on Litmus milk medium: Reactions on Litmus milk medium: Anaerobic media 4. Litmus Reduction: Litmus act as redox indicator White tube