Procedures for seroty

Procedures for Serotyping of Streptococcus pneumoniae by co-agglutination assay (modified) - Dr Rewa Kanungo (05.11.04)

Reagents:
1. 7.2 Phostpahte buffered saline (PBS)- 1000 ml with 0.05% Na Azide (for washing)
2. 1.5% formaldehyde- 10 ml
3. water bath 800C
4. 10 ml pipettes
5. centrifuge tube- x20
6. smaller glass tubes- x20
7. biochemical tubes –x20
8. micropipette- x20 (100 μl)
9. PBS (Ph 7.2)- 200 ml with 0.1% Na Azide
10. PBS (Ph 7.2)- 100 ml without Na Azide


Preparation of Staphylococcus aureus
1. In the morning, inoculate freshly cultured Cowan type 1 strain of Staphylococcus aureus into MHBA (6 plates)
2. Incubate at 370C overnight in CO2 jar.
3. Next day, wash plates with PBS (Ph 7.2) with 0.05% Na Azide)
4. Scrape gently with sterile glass rod.
5. Collect into a centrifuge tube
6. Centrifuge at 3000 x g for 20 min then discard supernatant
7. Combine pellets from all 30 tubes in 1 or 2 tubes
8. Depending on the volume of the pellet, add 2 times the quality of PBS (Ph 7.2) with 0.05% Na Azide to tube containing pellet.
9. Mix thoroughly
10. Centrifuge at 3000 g for 15 min then discard the supernatant
11. Add 10 ml fresh PBS (Ph 7.2) with 0.05% Na Azide to the tube containing the pellet
12. Repeat steps 10 & 11 three times
13. Add 1.5% formaldehyde 10 times the volume of the pellet to stabilize staph (e.g. to 1 ml pellet add 10 ml 1.5% formaldehyde)
14. Mix thoroughly and stand at room temperature for 90 min
15. Centrifuge at 3000 g for 15 min then discard the supernatant
16. Wash 3 times as before then discard supernatant
17. Add 10 ml of PBS (Ph 7.2) (wash buffer)
18. Put tube in 800C water bath for 5 minutes (to kill staph)
19. Wash 2 times
20. Make 10% suspension of pellet (to 1 ml pellet add 10 ml PBS Ph7.2 with 0.1% Na Azide
21. Transfer 1 ml from pellet suspension to 2 tubes , keep 1 aside as control
22. 1st tube: Make 2.5% suspension as control (not sensitized) and keep aside
23. 2nd tube to be used for sensitization


Sensitization Steps:
1. Add 0.1 ml of antiserum to a fresh, dry, sterile tube
2. Add 1 ml of 10 % pellet suspension (2nd tube) to the antiserum (drop wise) while shaking the tube.
3. Stand at room temperature for 30 min with intermittent shaking
4. Centrifuge at 3000 g for 15 minutes then discard supernatant
5. Add 5 ml PBS (Ph7.2) with 0.1% Na Azide (this will make a 2% solution) and distribute in 1 ml
6. Label the coagglutination antisera solutions appropriately (A-F,H, and P-T)
7. Store at 2-80C (i.e. refrigerate)


Test Procedure
1. Carry out a clean glass slide
2. Make 25 μl of the culture suspension in PBS (Ph 7.2) ( without Na Azide)
3. Add each of the pool coagglutination antisera solutions (25 μl) as per the chart provided with the Pneumostat kit ( A-F, H, and P-T)
4. Rock the slide by hand for 2 minutes
5. Observe the results
6. Test is positive if cell clumping is observed along with clearing of suspension within 2 minutes and control slide shows no clumping

Microbiology Antigen/Antisera Shigella ()
ANTISERA, TOXOIDS, VACCINES AND TUBERCULINS IN PROPHYLAXIS AND TREATMENT.