For steps #1-#9 Protozoa are spun with clinical centrifuge(750 RPM for 30 secs spin time: our lab centrifuge setting is 4; timer set to 2'( since zero time is actually 1' and also need time to get up to speed)
MAKE SURE CELLS ARE IN POLYPROPYLENE TUBES, NOT POLYSTYRENE
1. Protozoa are fixed overnight at 4oC in 1.5 % GTA in 0.1M NaCac pH 7.4
2. Protozoa are spun with clinical centrifuge(750 RPM for 30 secs; setting 4; timer set to 2' ( is actually 1' and time to get up to speed) and rinsed in 0.15 M Na Cacodylate, Ph 7.4.
3. Protozoa are fixed again at RT in 1 % GTA in 0.1M NaCac pH 7.3, with 1% Tannic acid (LMW(@1700g, from Mallinckrodt) for 1 hour. Make solution is fresh and filter(#1 Whatman) just before use.
4. Protozoa are rinsed in 0.1 M Na Cacodylate at Ph 7.4.
5. Protozoa are post-fixed in 1% OsO4 in 0.1 M Na Cacodylate at Ph 7.4 at 4oC for 1 hr.
6. Protozoa are rinsed in 0.1 M Na Cacodylate at Ph 7.4.
7. Dehydrate thru ETOH series: 30%, 50%, 70%, 85%, 95%, 5 mins. ea.
7. Three 5 minute changes in 100% ETOH.
8. Two 10 min. changed in Propylene oxide(PO).
9. Protozoa are immersed in LX112:PO - 1.5:1 (10.2 ml:6.8 ml). Cap vials for 2 hrs. Remove caps and place in vacuum desiccator overnight.
10. Transfer Protozoa to BEEM capsules and fill with fresh LX112. Place in vacuum desiccator overnight.
Spin protozoa down: 800-1000 RPM for 15 mins. BEEM capsules will fit into cotton cushioned 15 ml polypropylene centrifuge tubes. Put two layers of tape onto covered top of BEEM capsule. Remove BEEM capsules, using tweezers to grasp the tape.
11. Polymerize at 60 C for 48 hrs.
Remove blocks from heat to cool.
Last Updated: 10/2/08