Four techniques are listed as follows:
For each pollen sample, prepare a matching SEM stub by covering the stub's upper surface with double stick tape and labeling the lower surface. Remove anthers from an herbarium specimen and place on the sticky surface of the stub. To separate the pollen, squash the anthers gently with the edge of a sterile probe that was rinsed first in acetone and then in 100% ETOH. Next, to reduce charging, paint carbon paint around the periphery of the stub, over the double stick tape. Place stubs in a desiccator for at least 24 hours to dry the carbon paint. Once dry, the stubs are ready for coating and examination.
For each pollen sample, remove 15-25 dried flowers that had been gathered and dried from a vouchered pollen packet. Extract anthers with sterile forceps and placed into a 15 ml glass centrifuge tube.
Add about 3 ml of 5% potassium hydroxide (KOH) to each centrifuge tube. To soften the anthers, stir the KOH-anther mixture with a sterile glass rod for about 30 seconds and then allow to sit for another 30 seconds. Dilute the mixture of distilled water to prevent possible pollen destruction. Strain the KOH-anther-water mixture through a stainless steel wire screen. A 200 µm mesh screen can be used for small grains, and a 450 µm mesh screen for grains. Gently mash the anthers and press them into the screen with a clean spatula. The screen and mashed anthers are now with five ml of distilled water; collect the rinse water in a beaker.
To remove pollen from the rinse water, pour each pollen-solution back into its original centrifuge tube and centrifuge at 4,000 r.p.m. for three minutes. Decant the supernatant, and mix the pollen residue using a vortex stirrer. Any water-pollen solution remaining in the beaker is added to the centrifuge tube and processed accordingly. To remove pollen from the sides of the beaker, rinse the beaker with a fine jet of distilled water and then with 95% ETOH. The rinse liquid poured into the centrifuge tube. Centrifuge, decant, and vortex the samples.
To remove traces of ETOH and KOH, rinse the samples twice with distilled water, each time centrifuging, decanting, and vortexing. Next, rinse the samples with glacial acetic acid, centrifuge, decant, and vortex. Recently dried and fresh samples can be acetylated (Erdtman 1960, 1963; Lieux 1980a) for 12 minutes in a hot block at 100o C. Samples from herbarium specimens can be acetylated for 10 minutes; acetylate fragile pollen grains for five minutes.
The acetylated samples are now rinsed once with glacial acetic acid and twice with distilled water; centrifuging, decanting, and vortexing each time. Samples to be air dried are rinsed with 95% ETOH, centrifuged, and decanted. After the last wash and centrifugation process, the pollen samples in the centrifuged tubes were ready for SEM preparation.
Prepare SEM stubs by rinsing them in acetone and allow them to air dry. Label each stub to match a sample and paint the unlabeled surface with Mikrostikª. Vortex each pollen sample for 15 seconds, and a small amount of pollen residue with a glass pipette. Hold the pipette about three inches above the stub, and drop one drop of pollen residue onto the appropriately labeled stub. The three inch fall allows the residue to spread on the stub, separating the pollen grains. Air dry specimens in a desiccator for at least 24 hours. After drying, coat the specimens with gold-palladium and examine.
The third technique is used to prepare polleniferous material that collapses during air drying. The initial procedure is the same as Technique 2; except the steps subsequent to acetylation. After acetylation, the samples are rinsed once with glacial acetic acid and twice with distilled water, centrifuging for 3 minutes, decanting , and vortexing. Next, the samples are rinsed five times in a graded dehydration series of 10, 20, 50, 75, 90% ETOH, followed by three rinses in 100% ETOH. Centrifuging, decanting, and vortexing after each rinse.
After the final ETOH rinse, each pollen residue is placed into a 0.8 µm porosity, filter paper with an appropriate label and folded so the pollen can not be dislodged. The folded filter paper, with its pollen, is placed into small critical point baskets and submerged in 100% ETOH until several samples are ready for critical point drying. Samples are now critical point dried.
After drying, pollen is dusted onto the surface of SEM stubs that had been covered with double-stick tape, Mikrostikª or Tempfixª. The stubs are placed into a desiccator until ready for coating. Any remaining pollen can remain in its respective filter paper and placed in a desiccator.
This procedure is used for pollen sensitive to acetylation, such as Lauraceae grains. Polleniferous material is hydrated in distilled water and washed in an ETOH series of 25, 50, 95, and 100%. If the grains can be air dried, they are strewn onto cover slips that are attached to aluminum stubs with silver paint (Lieux 1980a). If the samples could not be air dried, they are critical point dried and mounted onto SEM stubs as previously described.