VMC 221: Veterinary Immunology and Serology (1+1)

PROCEDURE 

Preparation of Staphylococci

1. Cultivate Staph.aureus ( Cowan-1 strain ) in tryptic soy broth overnight.
2. Add 5 ml of broth containing the bacteria to each 1 litre Roux flask containing 125 ml of tryptic soy agar.
3. Incubate at 37^◦ C for 24 hours.
4. Harvest the growth with PBS.
5. Wash the growth for three times in PBS.
6. Prepare 10% suspension of cells and add 0.5% formalin.
7. Allow to stand at room temperature for three hours with occasional agitation.
8. Wash the cells for three times in PBS.
9. Make 10% cell suspension in PBS and store at 4^◦ C until used.
10. Add 1 ml of 10% cell suspension to 0.1 ml of 1:5 dilution of antiserum (anti choleragen).
11. Incubate for 1 hour at room temperature with occasional agitation.
12. Add 4 ml of PBS and centrifuge the mixture.
13. Dilute the sensitized cells to a final working suspension of 2% in PBS and store at 4^◦ C .