CENTRIFUGE = 5 minutes at 500 RPM
ALLOW 1 hour 1. Pick out/seperate pollen masses or anthers from other plant material. Engrave glass tubes with sample #s.
ALLOW 1/2 hr. 2. Hydrate, if dry. You may need to use a wetting agent (a drop of Photoflo or detergent). Wash with water. Centrifuge and decant.
3. Wash with glacial acetic acid. Centrifuge and decant.
ALLOW 1/2 hr. 4. Place about 1-2 ml of acetolysis fluid into the test tube with the pollen/anthers. Stir and then place in a boiling water bath for 10 minutes.
Acetolysis fluid is made by mixing 9 parts acetic anhydride with 1 part concentrated sulfuric acid. Do this in small volumes (less than 25 ml at a time). Also, be sure to add the acid to the anhydride ONE drop at a time. If you squirt all the acid in at once, there could be a violent release of heat and a spill.
5. Centrifuge the pollen and decant the spent acetolysis fluid. The tube and the liquid in it will be hot. Handle with care.
ALLOW 1 hour 6. Wash in glacial acetic acid. Centrifuge and decant.
7. Rehydrate with several washes of water. Check under dissecting scope. If it is necessary to remove any anthers or other debris, filter through 183 mesh Nitex filter. The acetolyzed pollen will be a light to dark brown, depending on the type of pollen. If the pollen is very dark, you may destroy the details you need to study.
Drying and Mounting options for SEM:
ALLOW 12 hr. 1. Concentrate pollen in MPF dH2O; allow to dry onto a poly-l-lysine coated cover slip. (Sigma #p8920, with preservative added, for adhering tissue to glass slides). Mount onto stub. Coat with AuPd. Coat with carbon, if necessary.
ALLOW 12 hr. 2. Concentrate pollen in MPF dH2O. Allow to dry. Mount pollen onto stubs(use both mounting media for each sample) with sticky tabs and warm VERY THIN layer of apiezon wax.
3. If air-drying collapses pollen:
1 hr. to 70% ETOH a. Concentrate pollen in MPF dH2O; allow grains to settle onto a
2 hr. to CPD poly-l-lysine coated cover slip for 30 minutes. dehydrate through 100% ETOH and CPD; Mount cover slip onto stubs with sticky tabs. Coat with AuPd. Coat with carbon, if necessary.
1 hr. to 70% ETOH b. Concentrate pollen in MPF dH2O; dehydrate through 100% ETOH; ALLOW 12 hr. 1 change of Tetramethysilane(TMS):100% ETOH; 1 change of straight TMS; then incubate 10 min.. in straight TMS; Allow to dry. Mount pollen onto stubs(use both mounting media for each sample) with sticky tabs and warm VERY THIN layer of Apiezon W-100 wax.
NOTE on WAX: Heat wax, skim off to very thin layer(so pollen won't sink into wax); remove from heat (you want wax soft but not melted, as melted hot wax will wick up sides of pollen grains) and immediately sprinkle pollen onto stub. Wax must be hot enough for pollen to stick (no charging), but not so hot as to wick up the pollen sides.
If mounting in glycerin jelly, add a drop of concentrated pollen to a piece of glycerin jelly on a slide. Warm slowly and stir to mix. Seal with a cover glass with nail polish around the edges. Store the slide flat to avoid loss of pollen.
SEM Notes: BOTH air-drying and drying with TMS; gave good results. The best mounting options for air-drying and TMS were to .1) mount pollen onto stubs, using with sticky tabs and 2) mount pollen onto stubs using a warm, VERY THIN layer of apiezon wax.
Last Updated: 10/2/08