HARDENING PROCESSES.
For the satisfactory examination of tissues it is necessary that they
should be “hardened” in certain fluids. The object of this is to give
the specimens greater consistence, so that thin sections may be more
readily obtained and more safely manipulated, and also to “fix” the
tissue element as far as possible in the same relative position as in
the living body. The hardening process also acts on the protoplasm of
the cells, and prevents their swelling up when placed in water, and in
the various staining fluids.
The fluid used must be one which will not itself injure the specimen,
and which can be thoroughly removed by washing, so that it may not
interfere with staining operations. The specimens should be kept while
hardening in wide mouthed bottles, on the bottom of which a little
cotton wool or tow has been laid. This allows the hardening fluid to
come freely in contact with the under surface of the pieces of tissue,
and prevents their being flattened against the hard glass bottom.
The hardening fluid requires changing occasionally. This should always
be done at the end of twenty-four hours, in order to get rid of any
deposit of blood, &c., that may have accumulated. Besides this, the
tissue when placed in the fluid contained a good deal of water which
will have diluted it and consequently an early change is desirable.
Afterwards the fluid requires to be changed only as often as it becomes
turbid, or any deposit occurs, usually about once a week.
While hardening, specimens should be kept in a cool place, as warmth
favours changes in the cells, &c.
In manipulating the portions of organs, forceps should always be used
and these with great gentleness. The specimens should never be impaled
with needles, or unsightly holes, which may even be mistaken for
pathological appearances, will appear when a section is examined under
the microscope.
It requires some practice to know when the tissue is sufficiently
hardened. The object aimed at is to make them not really hard but
tough. It is almost unnecessary to add that in testing this with the
fingers the utmost gentleness must be observed, or serious damage may
be done to the tissue.
When the tissue is sufficiently hardened the hardening fluid must be
thoroughly dissolved out. This is most quickly effected by placing the
specimen in a basin into which cold water from a tap is constantly
running. The tissue may then be removed (forceps always being used and
never the needle) and placed in an imbedding medium as subsequently
directed; or, if it is not to be cut at once, into equal parts of
methylated spirit and water, in which it may be kept indefinitely, the
fluid being changed if it becomes at all cloudy.
It is unnecessary for ordinary work to have more than the following
hardening fluids:--*Müller’s fluid*:--
Potassium Bichromate 2-1/4 grms. 3-1/2 drachms.
Sodium Sulphate 1 grm. 1-1/2 drachms.
Water to 100 c.c. 1 pint.
Two drachms of carbolic acid are sometimes added to each pint of the
fluid but as a rule it is not necessary.
Müller’s fluid is the most generally useful of the various fluids
employed, for the following reasons:--
1. It causes very little shrinking of the elements of the tissue, and
hence may be employed for most delicate objects, _e.g._, the retina and
embryos.
2. In consequence of its not making the tissues shrink, it does not
squeeze the blood out of the vessels and where the organ has been
congested before death, we may, by using Müller’s fluid, preserve a
natural injection of the capillaries.
3. There is comparatively little danger of over-hardening the tissue
and rendering it brittle.
4. Sections of organs hardened in Müller’s fluid are usually firm
and easy to manipulate. They do not tend to curl up or adhere to one
another as much as those hardened in spirit.
5. It readily permeates the tissues, and hence large portions of
organs, or even the entire organ may be satisfactorily hardened in it.
6. It is very cheap. A gallon can be made up for about eightpence.
The fluid has however certain slight drawbacks:--
1. The hardening process is a slow one occupying four to eight weeks.
2. The fluid gives a permanent dingy colour to the tissue. This does
not cause any inconvenience for microscopic purposes, but it is a
disadvantage when it is intended to preserve the rest of the specimen,
as a naked eye preparation. In such cases the organ should be hardened
in spirit, carbolic acid, or formal.
Müller’s fluid can be used for almost any tissue. It is especially
useful for those which contain a large quantity of fluid, or of blood,
and is essential for nerve tissues which it is intended to stain by
Pal’s method (p. 89).
To harden a specimen in it at least twenty times the bulk of fluid must
be employed.
The fluid must be changed on the third day, and afterwards about every
week as may be required.
*Methylated spirit* is a very useful hardening agent. It hardens in one
to three weeks according to the size of the tissue and the quantity of
spirit used. Its disadvantages are:--
1. It is more apt to overharden than Müller’s fluid.
2. It causes a great deal of shrinking of the tissue and thus squeezes
much of the blood out of the vessels.
It is most useful in hardening tissues containing much epithelium,
_e.g._, kidney, epithelioma, &c.
Spirit is also frequently employed to complete the hardening by
Müller’s fluid and to preserve tissues after they have been hardened.
About ten or fifteen times the bulk of spirit should be used for one
of the tissues. The fluid should be changed on the third day and
afterwards as required.
*Müller’s fluid and spirit.*--This is a useful combination for many
purposes. It is made thus:--Müller’s fluid, three parts; methylated
spirit, one part.
The fluid must be allowed to cool after mixing before being used, and
if necessary filtered. It will harden specimens satisfactorily in three
weeks.
*Müller’s fluid and formal.*--Is an extremely useful mixture made by
adding one part of formal to nine of Müller’s fluid. It hardens in a
much shorter time than Müller’s fluid and causes very little shrinkage.
*Absolute alcohol.*--Used as a hardening agent where the tissues are
to be examined for micro-organisms, and for specimens to be stained
by Nissl’s method (p. 101). A cheaper and equally effective hardening
medium is made by dehydrating methylated spirit by adding one ounce of
fused carbonate of potassium to each pint of methylated spirit, and
decanting.
Small pieces must be used. The depths of the block should not exceed
3/8 inch. The fluid should be changed on the third day. Hardening will
be completed in about ten days or even earlier.
*Osmic acid.*--For rapidity of action, and for rapid fixing of all the
tissue elements in their natural position osmic acid is one of the best
hardening reagents we possess.
Its disadvantages as a hardening agent are:--
1. Its expense.
2. Its irritating and corrosive vapour.
3. The fact that only small pieces of tissue can be hardened in it,
since the external surface is very rapidly hardened and thus the fluid
is prevented from penetrating into the centre of the lump.
It is most frequently used as a hardening agent for very delicate
structures, such as the retina, or embryos, or for fresh sections of
brain (p. 95).
The acid itself may be procured in sealed tubes, each containing one
gramme. These should be broken in a bottle under sufficient distilled
water to make a one per cent. solution. The bottle containing it
should be covered with brown paper to exclude the light. For hardening
purposes small pieces of the tissue, not much larger than a pea, should
be placed in the acid, the one per cent. solution being diluted with
five to ten volumes of distilled water. The tissues may be left in this
for from three to five days. They must then be *thoroughly* washed in
distilled water and may afterwards be preserved in methylated spirit.
Both the hardening and the subsequent washing must be carried on *in
the dark*.
Osmic acid is also a most valuable staining reagent (see p. 81).
*Carbolic acid* (5 per cent.).--May be used to harden almost any
tissue, but is particularly useful for hardening nervous tissues such
as brain or spinal cord which are afterwards to be preserved as museum
specimens. It does not discharge the colour of a specimen so rapidly as
spirit.
Three or four times the bulk of fluid should be used. It requires
changing at the end of twenty-four hours, and again at the end of the
first week.
Saturated aqueous solution of *corrosive sublimate* is one of the
most convenient hardening reagents for small pieces of delicate
tissue, _e.g._, embryos. It hardens them in a few days. When they are
sufficiently hardened the mercurial salt should be removed by washing
first in methylated spirit for a few hours and then in running water.
*Formal.*--An aqueous solution containing about 35 per cent. of
formaldehyde. It is a rapid hardening agent, causes very little
shrinkage of the tissues, and does not discharge the colour of the
specimens as much as alcohol. For hardening formal should be used as
a two to five per cent. solution in distilled water. It may also be
used as a ten per cent. solution for mounting museum preparations, but
there is some tendency for a cloudy deposit to form on the glass after
a time. It is the most suitable hardening agent at our disposal for
eyes. It rapidly fixes the tissue elements, but does not cause much
contraction. It may also be used for hardening the brain and spinal
cord. Large quantities of fluid must be used for the latter purpose
and it must be frequently changed. As soon as they are sufficiently
hardened they should be transferred to methylated spirit.
*Marchi’s fluid.*--This consists of:--
Müller’s fluid 2 parts.
Osmic acid solution (one per cent.) 1 part.
It is used for hardening specimens as a preliminary to Golgi’s method
for staining nerve cells (p. 97), and also to complete the hardening of
sections of spinal cord, &c., before employing Schäfer’s modification
of the Weigert Pal hæmatoxyline method (p. 91).
It is also used as a stain for recently degenerated nerve tracts and
fibres, especially after experimental lesions.
The fluid has little penetrating power, and therefore tissues must be
cut into small pieces, about 3/8 inch cube. It is not necessary to
place them in this fluid at once on removal from the body, but the
preliminary hardening must be in Müller’s fluid and not in alcohol, &c.
SPECIAL HARDENING REAGENTS FOR RAPID FIXATION IN ORDER TO STUDY CELL
STRUCTURE.
1. *Alcohol.*
2. *Flemming’s solution* (modified by Friedmann):--
Osmic acid (one per cent.) 3 c.c. (♏xxx.).
Glacial acetic acid 2 c.c. (♏xx.).
Chromic acid (one per cent.) 42 c.c. (℥j.)
Small pieces should be hardened in this fluid for twelve to twenty-four
hours, and then washed and transferred to alcohol for some days before
staining.
3. *Nitric acid.*--A ten per cent. solution in distilled water. It
hardens the tissue in three to four hours, and should be followed by
70 per cent. alcohol, the hardening being completed in absolute alcohol.
In using any of these methods it is necessary that the tissue be
removed from the body during life or immediately after death. They are
employed for revealing the changes in the cells and their nuclei in
rapidly growing or inflamed tissue, for studying karyokinesis in cancer
cells, and investigating the appearance of nerve cells and gland cells
at rest, when actively employed and when fatigued; and they are also
most useful in preparing specimens of the “parasitic bodies” which have
been described in many cancer cells.
DECALCIFYING FLUIDS.
Used in the preparation of bone, tooth, osseous tumours, &c. The two
best fluids for general use are:--
*Chromic and nitric fluid.*--This is made as follows:--
Chromic acid 1 gramme 45 grains.
Nitric acid 2 grammes 1-1/2 drachms.
Water 200 c.c. 1 pint.
If the bone is not very compact the fluid may be used diluted with an
equal quantity of water. A large quantity of fluid should be used, and
like all decalcifying fluids, it should be frequently changed.
As soon as the specimen is sufficiently flexible, it should be
thoroughly washed in running water for some hours, and then transferred
to spirit until it is convenient to cut sections.
*Von Ebner’s solution*:--
Hydrochloric acid 1 gramme 1-1/2 drachms.
Common salt 10 grammes 2 ounces.
Water to 100 c.c. 1 pint.
It is a very useful decalcifying agent, but causes the fibrous elements
to swell up rather more than chromic and nitric fluid. A large quantity
of the fluid must be used, and it should be changed daily. It must be
very thoroughly washed out in running water when the decalcification is
completed.
*Bleaching solution* (eau de Javelle).
(1) “Chloride of lime” (bleaching powder) 20 1/2 oz.
Water 100 2-1/2 oz.
Shake up well.
(2) Carbonate of potassium 20 1/2 oz.
Water 100 2-1/2 oz.
Mix the two solutions. Allow them to stand for an hour and filter.
It is used chiefly for clearing vegetable sections but may also be used
for sections containing a large quantity of pigment. It is particularly
useful in decolourizing sections of “madura foot” due to the presence
of a black fungus.