1. PHT382
Lab. No. 4

2. Classification of Bacteria

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

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

5. 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

7. (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.

8. 1. Anaerobic jar:

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

10. 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.

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

12. 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.

13. 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.

15. (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).

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

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

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

19. Thank you