BOT/MICRO/ZOO 5364

A Sample Protocol for the Periodic Acid-Thiocarbohydrazide-Silver Proteinate Reaction

Reference: Thiéry, J-P. 1967. Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J. Microscopie 6:987-1018.

This stain is used on the grid and is an analog to the PAS (periodic acid-Schiff's) reaction of light microscopy. Material is prepared by fixation in glutaraldehyde and post-fixation in osmium tetroxide, dehydration in ethanol and embedded in araldite (other plastics work also). Due to secondary labeling of osmium, material that is prepared without osmium may be preferable. Some of the steps are light sensitive. Material should be picked up on NICKEL or GOLD grids only.

  1. 1% periodic acid in water, 20 to 30 minutes.

  2. Wash in water (several changes)

  3. 0.2% thiocarbohydrazide in 20% acetic acid (or 1% thiosemicarbozide in 10% acetic acid.) Stain at RT° for 30-40 minutes, 24 hr, 48 hr, or 72 hr, depending on desired sensitivity. (I used 6 hr in the dark.) TCH may be stored for several days at 4°C.

  4. Wash in graded acetic acid series to water. Suggested sequence: 10% acetic acid - 2 rapid washes, followed by 3 washes at 20 minutes each. This is followed by 20 minute changes in 5% acetic acid, 2% acetic acid, and then 2 rapid changes in water and 3 changes of double distilled water at 20 minutes apiece.

  5. Stain for 30 minutes in 1% silver proteinate (Roques) in double distilled water. Mix by swirling under safelight or equivalent. Stored at 4°C in the dark, this lasts several weeks.

  6. Wash in 2 rapid changes of double distilled water in the dark followed by 3 changes of double distilled water 20 minutes apiece in the light.