Chlamydomonas culture medium + 2% glutaraldehyde (5 ml medium + 0.9 ml 25% glutaraldehyde
100 mM sodium cacodylate, pH 7.2
1% glutaraldehyde in 100 mM sodium cacodylate, pH 7.2
1% OsO4 in 100 mM sodium cacodylate
Suspend cells in 1/3 plastic:2/3 acetone and leave the tube uncovered overnight in the hood. If possible, put the tubes on a rotary shaker. The acetone will gradually evaporate and increase the concentration of plastic.
The next day gently pellet the cells, remove the acetone:plastic mix and add fresh 100% plastic. I generally underlay the cells with plastic, let them sit for ~30 min, and place the tubes in a vacuum chamber - pumped by house vacuum. Cells will settle to the bottom of the tube within 1-2 hrs. Pellet the cells in the clinical centrifuge and remove the plastic above the cells.
Put BEEM capsules in the bottom of plastic or glass centrifuge tubes, fill about half full with fresh plastic, and overlay with cells from the above pellets. Spin at top speed for ~ 10 min. Put a label in the top of the capsule and put the tubes in the oven.
Polymerize at 40 degrees C overnight and raise to 65-80 degrees C for a second day or just put at 65 degrees C for 1-2 days.