BOT/MICRO/ZOOL 5364
DEHYDRATION
Dehydration is necessary when embedding specimens in water-insoluble media.
Most dehydrating agents are strong organic solvents which bring about some shrinkage and extraction of cell components. To minimize these effects, dehydrating agents are used in a graded series for short periods of time. Water is gradually replaced so that violent osmotic changes do not produce distortions. Concentrations of the agent should begin high enough to insolubilize unstabilized proteins.
Dehydrating Agents
I. Ethanol
Advantages:
1. Appears to cause less extraction of cellular components in general than other agents.
2. Inexpensive and easily obtained.
Disadvantages:
1. Extracts more lipids than acetone.
2. May cause more shrinkage of specimen.
3. May react with an unreduced 0s04 remaining in specimen.
4. Only slightly miscible with most resins.
Methanol may extract even fewer cellular components than ethanol and may be substituted for ethanol.
II. Acetone
Advantages:
1. May cause less shrinkage of specimen than ethanol.
2. Not reactive with 0s04 remaining in specimen.
3. Miscible with most embedding resins.
Disadvantages:
1. Absolute acetone easily contaminated with water. May result in incomplete dehydration.
2. Uranyl acetate and phosphotungstic acid only soluble in dilute solutions of acetone.
III. Propylene oxide
Advantages:
1. Completely miscible with embedding resins.
2. Low viscosity. Infiltrates tissue readily and reduces viscosity of embedding resin mixtures.
Disadvantages:
1. Very reactive even at low temperatures. May combine with reactive groups in cells and effect certain cytochemical and staining reactions.
2. May extract even fixed lipids. Can be minimized by low temperature and short duration of dehydration.
3. Traces may be retained in polymerized resin. May react with epoxy groups and partially inhibit polymerization which adversely affects hardness and cutting properties of blocks.