BOT/MICRO/ZOOL 5364

GENERALIZED SCHEDULE FOR SPECIMEN PREPARATION

General Consideration

Fixation:

Fixation time and temperature depend on the nature and size of the specimen and the composition of the fixative.

Fixation time should be long enough so the stabilizing action of the fixative is complete, but not long enough to cause excessive extraction by the buffer.  Time depends on the size of the specimen and the rate of penetration of the fixative.

Fixation temperature depends on the desired results and the nature of the fixative.  In general the penetration rate of the fixative is higher at higher temperatures.  Low temperatures slow down enzymatic reactions which may cause changes in cells prior to stabilization by the fix.  Shrinkage of specimens is reduced at low temperatures.  Some labile cell components such as microtubules may be lost at lower temperatures.

Washing:

Specimens must be washed sufficiently between treatments to prevent precipitation, particularly if:

1.     components of treatments react with each other.

Glutaraldehyde and osmium

Uranyl acetate and phosphate or cacodylate buffer

Residual osmium and ethanol

2.     components of one treatment are not soluble in the next mixture.

Uranyl acetate and acetone or higher concentrations of alcohol

Time depends on size of specimen.

Lower temperatures reduce extraction of components.

Dehydration:

Time is less critical than time for fixation, but extraction does occur.  The time should be sufficient for complete dehydration and for osmotic adjustment but not long enough to cause excessive extraction of components.

If fixation and washing have been carried out at 0-4 C, dehydration may also be carried out in the cold.  Specimens should be brought to room temperature between 90-95% and the last change of absolute dehydrating agent.