426
Appendix
cytoplasm and the nucleus. Osmium tetroxide, an oxidizing agent, is also used as a f
xative For electron
microscopy.
Alcohols
, either methyl or ethyl alcohol, are also used as f
xatives. Because they cause the tissue to
become quite hard and, thereFore, brittle, they are usually used in special applications such as smears oF
tissue or blood. Alcohol in various dilutions is also commonly used in tissue processing.
Mercuric chloride
is used in combination with other substances in a variety oF f
xatives. Though
the method oF action at the cellular level is not well understood and f
xatives containing this substance
may penetrate the tissue slowly, they do provide excellent cellular detail.
Acetic acid
, though rarely used as a f xative on its own, is used in combination with other agents
in a variety oF f xatives such as Bouin, Carnoy, and Clarke solutions. Although acetic acid may be used
in concentrated Form in certain applications, it is primarily used in combinations with other f
xatives
(and is thereby diluted); it is also commonly used by itselF in dilutions oF 1% to 5%.
Methods of Fixation
±ixation is usually accomplished in one oF two ways. The f
rst is by
immersion ± xation
. The f
xative
is prepared and a small piece oF tissue removed and immersed in the f
xative. It is common to suspend
the tissue sample in the f
xative; one method is to drape a delicate piece oF cheesecloth in the f
xative
and place the sample in the sling thus created. This is advantageous because the f xative can penetrate
From all sides oF the tissue block. This method is useFul when the tissue sample is small and the f xative
penetrates rapidly.
The second is
perfusion ± xation
. In this method the f
xative is perFused through the intact vascular
system oF the organism. The f xative is preceded by a wash oF physiological saline containing heparin
and a substance that will paralyze the vessel walls, such as procaine hydrochloride, to ensure that the
vascular bed will not contract in response to the f
xative solution. This wash is Followed immediately
by the f xative. AFter perFusion, the tissue samples are removed and placed in more oF the same f
xative
used in the perFusion. This method provides superior f
xation oF large pieces oF tissue and is commonly
used in many research applications.
FREEZING
is also used as a method oF f
xation, especially in the clinical setting when a rapid diagno-
sis is needed during a medical procedure. A Fresh tissue sample is retrieved From the patient or organ-
ism and immersed in liquid carbon dioxide or in a substance (such as isopentane) cooled extremely
rapidly by dry ice. The best results are obtained with small tissue samples that are rapidly cooled to
very low temperatures (–40° to –60° C). In general, rapid cooling causes very small ice crystals and
minimal tissue disruption; slow cooling (like in your Freezer) causes large ice crystals and maximum
tissue disruption.
Processing of Fixed Tissue
Once the tissue sample is satisFactorily f
xed, it must be taken through a series oF steps that result in a
thin slice oF tissue mounted on a glass slide (For light microscopy) or an even thinner slice mounted on a
copper grid (For electron microscopy). In general, this process requires three basic steps: (1) The water
in the tissue needs to be removed, (2) the tissue must be passed through a solution that is miscible with
water and with the substance in which the tissue will be embedded, and (3) the tissue must be passed
through the embedding medium. Because the specif
c details oF these steps may relate to the type oF
tissue being processed, only general comments are made here.
Dehydration:
The goal oF dehydration is to remove the water From the tissue. The most common
method is to start with alcohol in a concentration oF 75% to 80%. Recall that alcohol will mix with
water; thereFore, it can be used to remove water From the tissue. The tissue sample is passed, stepwise,
through progressively higher concentrations (From 75%–80% up to 100%) oF alcohol; usually more
than one step is used at 95% and 100%. Through this process, the water is completely removed From
the tissue and replaced with alcohol.
Clearing:
The clearing reagent is a substance that will mix both with alcohol and with the embed-
ding medium. The most commonly used are xylene, toluene, and chloroForm. The tissue is passed From
100% alcohol through changes oF the clearing reagent. This stepwise process progressively removes
the alcohol From the tissue and replaces it with the clearing reagent. This reagent is miscible with the
embedding medium.
Impregnation and embedding:
Because the clearing reagent will mix with the embedding medium,
the tissue sample is taken From the last step in this reagent and placed in melted embedding medium
(such as paraplast or bioloid—both are types oF wax). The sample is then progressively passed through
several changes oF the embedding material. This stepwise process progressively removes the clearing
reagent and replaces it with the embedding medium that will harden when cooled. Embedding is gen-
erally done in two steps. ±irst, the tissue is removed From the last impregnation step and immediately
placed in melted medium in a vacuum oven. The last traces oF the clearing reagent and any minute
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