84
4.
EMBEDDING
4.2. Dehydration
A graded series of ethyl alcohol dilutions is used most commonly for
dehydration purposes. As indicated in the previous chapter, though,
electron microscopists have learned to be concerned about materials
being leached out of osmium-fixed specimens, even while they are in the
fixative, and also while being dehydrated. It is, therefore, commonly
accepted that dehydration should be completed as rapidly as possible.
In some laboratories also it is the custom to dehydrate at near zero
temperatures. Naturally this would have little meaning unless the fixation also had been completed in the cold.
When tissue blocks are first removed from the fixative they probably
should be given a quick rinse with water, for excessive quantities of
osmium tetroxide react with alcohol. In this writer's laboratory de-ionized
water has been used for some time on the assumption that most proteins
would be less soluble in this than in salt solutions. However, other laboratories do advocate salt solutions or the buffer solution of the fixative
prepared without the addition of osmium tetroxide. Little more than a
surface washing of the specimen and a flushing of the container are
necessary before adding dilute alcohol.
Useful tissue blocks are so tiny that the exchange of water and alcohol
is very rapid. In this laboratory we pass blocks of the usual size through
50, 75, and 95% alcohol, allowing about 3-5 minutes in each bath. Thus,
the specimens ordinarily reach absolute alcohol within 15 minutes. As
an experiment in this laboratory we have, on occasion, gone directly
from fixative to absolute alcohol without apparently harming the tissue
in any way. If we had the courage of our convictions we perhaps would
do this routinely.
It is sometimes desirable to dissect or trim a slab of tissue during
dehydration. This can best be done in the 75 or 95% bath. Thus, advential layers can be removed to make subsequent sectioning easier, or
a thin sheet of material that was fixed in situ can be cut up into small
blocks.
There is little point in hurrying the process of final dehydration, for
the tissue will be exposed for hours to unpolymerized plastic, and the
hardeners or plasticizers that may be added, which will act as excellent
organic solvents that cannot be avoided. In our own laboratory we
usually use two short baths of absolute alcohol (about 5-10 minutes
4.
EMBEDDING
4.2. Dehydration
A graded series of ethyl alcohol dilutions is used most commonly for
dehydration purposes. As indicated in the previous chapter, though,
electron microscopists have learned to be concerned about materials
being leached out of osmium-fixed specimens, even while they are in the
fixative, and also while being dehydrated. It is, therefore, commonly
accepted that dehydration should be completed as rapidly as possible.
In some laboratories also it is the custom to dehydrate at near zero
temperatures. Naturally this would have little meaning unless the fixation also had been completed in the cold.
When tissue blocks are first removed from the fixative they probably
should be given a quick rinse with water, for excessive quantities of
osmium tetroxide react with alcohol. In this writer's laboratory de-ionized
water has been used for some time on the assumption that most proteins
would be less soluble in this than in salt solutions. However, other laboratories do advocate salt solutions or the buffer solution of the fixative
prepared without the addition of osmium tetroxide. Little more than a
surface washing of the specimen and a flushing of the container are
necessary before adding dilute alcohol.
Useful tissue blocks are so tiny that the exchange of water and alcohol
is very rapid. In this laboratory we pass blocks of the usual size through
50, 75, and 95% alcohol, allowing about 3-5 minutes in each bath. Thus,
the specimens ordinarily reach absolute alcohol within 15 minutes. As
an experiment in this laboratory we have, on occasion, gone directly
from fixative to absolute alcohol without apparently harming the tissue
in any way. If we had the courage of our convictions we perhaps would
do this routinely.
It is sometimes desirable to dissect or trim a slab of tissue during
dehydration. This can best be done in the 75 or 95% bath. Thus, advential layers can be removed to make subsequent sectioning easier, or
a thin sheet of material that was fixed in situ can be cut up into small
blocks.
There is little point in hurrying the process of final dehydration, for
the tissue will be exposed for hours to unpolymerized plastic, and the
hardeners or plasticizers that may be added, which will act as excellent
organic solvents that cannot be avoided. In our own laboratory we
usually use two short baths of absolute alcohol (about 5-10 minutes