The following procedure is to be performed in a hood with a low speed centrifuge.
To change each solution centrifuge at a low speed for three minutes, decant the solution and re-suspend specimens with air bubbled into the next solution.
1A. If the amount of aspirate is known beforehand, add an equal amount of 8% buffered glutaraldehyde fixative at 4º C. in a 10 ml glass test tube.
1B. If the amount of aspirate is unknown beforehand place it in a graduated 10 ml glass test tube or other suitable container and add 2 to 3 times the amount of 4% buffered glutaraldehyde fixative at 4º C.
1C. Decant the nasal scraping fluid into a graduated 10 ml glass test tube , centrifuge and suspend the resulting pellet in 0.1M sodium cacodylate buffer at 4º C. for at least 15 min. up to a week. Proceed to step 3.
2. Place either preparation in the refrigerator for 90 min. and centrifuge, decant the fixative-sera portion and resuspend in 0.1M sodium cacodylate buffer at 4º C. for at least 15 min. up to a week.
3. Make three changes of 0.1M sodium cacodylate buffer for at least 15 min. each at 4º C.
4. Resuspend in a covered test tube 2% osmium tetroxide with 0.1M sodium cacodylate buffer for one hour at 4º C.
The remainder of this procedure may be done in an uncovered test tube at room temperature.
5. Place in 0.1M sodium cacodylate buffer for 5 minutes.
6. Place in in 50% ethanol/distilled water for 5 minutes.
7. Place in in 70% ethanol/distilled water for 10 minutes.
8. Place in in 90% ethanol/ distilled water for 10 minutes.
9. Place in three changes,20 min. each 100% reagent grade ethanol.
10. Place in 50% L. R. White/100% reagent grade ethanol for 15 minutes.
11. Place in 75% L. R. White/100% reagent grade ethanol for 15 minutes.
All changes now done at high speed in the high speed centrifuge.
12. Two changes,30 min. each in 100% L. R. White.Centrifuge,after the first change decant the L.R. White mixture mixture and pipette the remainder into size 00 conical tip Beem capsules. Fill the remainder with 100% L. R. White and leave for thirty minutes.
13. Centrifuge and then decant the L. R. White from the top of the capsules Fill with 100% L. R. White. Place the caps on the Beem capsules and fill #000 gelatin capsules with L. R. White almost to the top,place the Beem Capsule into the gelatin capsule leaving only liquid under the Beem Capsule and cap the gelatin capsule.
14. Place in a 60º C oven for 22 to 26 hours.
