1. Clostridium - Microscopic appearance of different species. - Differentiation between species according to biochemical reactions.

2. Clostridium perfringens

4. On Blood Agar C. perfringens produces large beta-haemolytic colonies are produced. Some strains produce a double zone of haemolysis.

5. 1- Prepare a plate of lactose egg yolk milk agar 2- Turn the plate over, and using a wax pencil, draw a line across the centre of the plate 3- Using a sterile swab, cover one half of the medium with C. perfringens antitoxin. Allow to dry

6. 5- Inoculate also a non-toxin producing control organism that will grow anaerobically. 6- Incubate the plate anaerobically at 35–37 ºC overnight. 7- Look for an opacity around the inoculum in the half of the plate containing no antitoxin and no opacity in the half containing the antitoxin. 4- Inoculate the test organism at right angles to the centre line (inoculum passes from the antitoxin-free half of the plate to the antitoxin-covered half.

7. A heavy inoculum of the test organism is incubated for up to 4 hours in a tube containing litmus milk. Reduction of the litmus milk is indicated by a change in colour of the medium from mauve to white or pale yellow

8. C.perfringens can reduce nitrate to nitrite which detected by addition of sulfanilic acid which react with nitrite to form diazonium salt which react with added alpha-naphthylamine to form red colour.

9. Lecithinase C activity: Seen as an opacity in the medium due to the breakdown of lecithin in the egg yolk. Lipase hydrolysis: Seen as (fatty) layer covering colonies and sometimes extending into the medium. Lactose fermentation: There is a reddening in the medium. The colonies become red on exposure to air. Proteinase activity (proteolysis): Shown by an area of clearing around the colonies due to the breakdown of casein in the milk by the enzyme proteinase.

10. On lactose egg yolk medium, C. perfringens: ● Produces lecithinase C (alpha toxin) ● Ferments lactose

11. C.perfringens produce proteolytic enzyme (gelatinase) that liquefy gelatin.

12. On Robertson’s cooked meat medium C. perfringens is saccharolytic and slightly proteolytic.

13. Clostridium botulinum

16. On Blood Agar C. botulinum produces large semi-transparent colonies with a wavy outline. Most strains are beta-haemolytic

17. On Robertson’s cooked meat medium C. botulinum is proteolytic.

18. Clostridium tetani

21. On Blood Agar C. tetani produces a fine film of growth. Use a hand lens to examine the plate. On fresh blood agar C. tetani is haemolytic (alpha first followed by beta haemolysis).

22. When C.tetani is cultured in a medium which contains tryptophan. Indole production is detected by Kovac’s reagent which contains 4 (p)- dimethylaminobenzaldehyde which reacts with the indole to produce a red coloured compound.

23. Clostridium difficle

25. On Blood Agar C. difficile produces large non-haemolytic colonies.

26. C. difficile can grow in bile esculin agar and turns the indicator ferric ammonium citrate to a dark brown color results from combination of esculetin end product of esculin hydrolysis with ferric ions to form a phenolic iron complex.