Acetolysis and Light Microscopy

Acetolysis is an indispensable method for illustrating pollen grains with the LM. Untreated or stained pollen grains will hide much of the important information for the description of a pollen grain.

Acetolysis is a combination of chlorination and acetylation:

For chlorination, the samples are transferred to a test tube and covered with a layer (1.5 cm) of glacial acetic acid and a layer (approx. 3 cm) of a freshly prepared solution of saturated sodium chlorate. After adding 3 or 4 drops of concentrated HCl, the mixture is stirred with a glass rod, heated in a bath of boiling water for 3 min, centrifuged, and the liquid fraction decanted. The residue is carefully washed to eliminate any remaining chemicals and then finally washed in concentrated acetic acid or acetic anhydride to remove the water.

For the acetylation step, the samples are put into a mixture of 9 parts acetic anhydride and 1 part concentrated sulfuric acid and heated to 100° C for approximately 4 min. After the mixture has been cen-trifuged and the liquid fraction decanted, the residue is washed in acetic acid and water. Glycerine is then added to the sample to form a suspension.

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