SPECIAL STAINING METHODS.--SPECIAL METHODS FOR STAINING THE NERVE
CENTRES.
1. For staining *nerve fibres*.
Three methods (two of which are modifications of the first) are
employed far more often than any others. By these methods the myelin
coating is stained. Tissues must have been hardened previously for many
weeks in Müller’s fluid, or some other bichromate solution. They are
then overstained in a solution of hæmatoxyline, and the section treated
with a suitable bleaching reagent, when the colour is discharged from
all the tissue elements except the nerve fibres. This method displays
not merely the nerve fibres in the white matter, but also the fine
network in the grey matter of the brain and spinal cord. Degenerated
fibres are left unstained and so degenerated tracts shew up as
unstained spots on a dark background. The sections may be subsequently
stained with alum carmine or eosine to shew the cells and neuroglia.
*Weigert’s method.*--The piece of cord to be cut after prolonged
hardening in Müller’s fluid is transferred without washing to absolute
alcohol and dehydrated preparatory to embedding in celloidin (p. 30).
When sections are cut they are transferred at once to Weigert’s
hæmatoxyline solution:--
Hæmatoxyline 1 4 grains.
Alcohol 10 45 minims.
Distilled water 100 1 ounce.
They are stained in this for twenty-four hours or longer, until they
are quite black.
The staining will take place much more rapidly if the fluid be kept
at 100° F. in the incubator. After staining they are transferred to
Weigert’s differentiating solution:--
Borax 2 160 grs.
Potassium ferricyanide 2·5 200 grs.
Distilled water 100 1 pint.
They are left in this solution for several hours, until the ground work
becomes nearly decolourised.
Sections will sometimes stain more satisfactorily if they are treated,
according to Weigert’s original directions, for a few hours with a half
saturated solution of acetate of copper. If the hardening in Müller’s
fluid has been sufficiently prolonged, this step is usually superfluous.
*Pal’s modification* of Weigert’s method.--By this method quicker and
more complete decolouration of the neuroglia, nerve cells, &c., is
obtained. Sections are prepared in exactly the same way as in Weigert’s
method and then transferred to Weigert’s hæmatoxyline. Pal recommends
that this solution be diluted to half the strength and a few drops of
a saturated solution of lithium carbonate added. The writer finds the
results equally good if the ordinary Weigert solution be employed.
When the sections have been thoroughly stained they are washed in
distilled water and placed in a three-quarter per cent. solution of
permanganate of potassium. The time required in this solution depends
on the time the specimen has been in Müller’s fluid. It should not be
less than half a minute, and in very thoroughly hardened specimens,
five minutes may be allowed with advantage. In this solution the
sections will become of an opaque brown colour. They are washed in
distilled water, and transferred to:--
*Pal’s differentiating solution.*
Potassium sulphite 1 grm. 40 gr.
Oxalic acid 1 grm. 40 gr.
Distilled water 200 cc. 1 pint.
They are kept in this for one to five minutes, according to the depth
of staining, until the white and grey matter are clearly defined and
the brown colour is completely discharged. If the brown stain does not
readily clear up, the section should be returned to the permanganate
solution for about half a minute, and again treated with “Pal’s
solution.” This manœuvre may be repeated several times. As soon as the
sections are thoroughly differentiated they are transferred one by one
to a large vessel of water and thoroughly washed. The blue stain of the
hæmatoxyline becomes brighter during the washing process. The sections
may be mounted at once, but more beautiful results will be obtained
if they are stained in alum carmine for 24 hours. They should then
be washed, dehydrated in alcohol, clarified in oil of bergamot, and
mounted in Canada balsam.
The very prolonged hardening in Müller’s fluid which is a necessary
preliminary for this method led to the introduction of:--
*Schäfer’s modification* of Pal’s method.--In this method hardening in
Müller’s fluid for three or four weeks is sufficient. The sections are
made exactly as in the previous method, and transferred to Marchi’s
fluid (p. 24) for six hours. They are washed and stained all night in
the following:--
Hæmatoxylin 1 4 grs.
Alcohol 10 45 min.
Acetic acid (2 per cent. aqueous solution) 100 1 oz.
The subsequent processes of differentiation, bleaching, &c., are
exactly the same as in Pal’s method.
*Osmic acid.*--Employed with fresh and also with hardened specimens to
demonstrate the medullary sheath. Much the best results are obtained
with the former. The nerves, or small pieces of the central nervous
system are placed in half to one per cent. solution of osmic acid as
soon as possible after death and kept in the dark for about a week.
The tissue must be very thoroughly washed in running water to remove
all traces of osmic acid, and then stained for a couple of days in
borax carmine to demonstrate the nuclei and axis cylinders. Sections
may be made in gum, or the tissue may be teased with needles and then
mounted in Farrant. Embedding in celloidin, and mounting in balsam are
inadvisable, because the ether tends to dissolve out myelin, and the
clarifying oil to render it too transparent.
2. *Intra-muscular ramifications of nerves*:--
*Sihler’s chloral hæmatoxyline method.*--This method reveals the
intra-muscular nerve-endings, and also brings into prominence the
curious “muscle spindles” which Sherrington has shown to be connected
with the posterior nerve roots, and which are believed by some to be
the end organs subserving muscular sense.
A piece of muscle is taken as soon as possible after death, or from an
amputated limb, and slices cut about one-tenth of an inch thick with
the freezing microtome. Transfer for twenty-four hours to the following
solution:--
Acetic acid 1 part.
Glycerine 1 "
One per cent. aqueous solution of chloral hydrate 6 parts.
The tissues swell up in this fluid and become translucent and
gelatinous in appearance. They are now placed in pure glycerine until
saturated as shown by their sinking to the bottom of the dish. This
usually takes several days. They may now be stained in the following
solution:--
Ehrlich’s hæmatoxyline (p. 70) 1 part.
Glycerine 1 "
One per cent. aqueous solution of chloral hydrate 6 parts.
They may be left in this from three days to a week with little fear of
overstaining. Portions may then be teased with needles, and mounted in
glycerine, or the stained tissue may be pressed out into a sufficiently
thin layer by squeezing it forcibly between two glass slides.
*Motor and sensory nerve endings.*--These are best stained by the
chloride of gold method (p. 82).
Specimens must be taken from the body immediately after death. The
method is therefore useless for the post-mortem room, but may be used
for tissues removed by operation. Small pieces of tissue must be
employed and must be stained in bulk, sections being made subsequently.
For motor nerve endings the muscle of a frog or human muscle from a
limb just amputated may be taken. Specimens should be prepared after
staining by teasing in preference to making sections. Mount in Farrant.
Sensory nerve endings may be conveniently studied in the cornea of a
recently killed frog or rabbit, or in a freshly extirpated human eye.
Tactile end organs may be studied in the lip or finger tips, taste buds
in the papilla foliata of the rabbit’s tongue, and Pacini’s corpuscles
are well seen in the mesentery of a thin cat.
3. *Staining nerve cells.*
*Bevan Lewis’s aniline blue-black method*:--This method is the best for
demonstrating the wealth of nerve cells in the fresh cerebral cortex.
The solution of aniline blue-black should be of the strength of 1 in
400, about a grain to the ounce. A piece of the cerebral cortex with
pia mater attached, should be removed as soon as possible after death
by parallel cuts about 1/8 inch apart, and perpendicular to the surface
of the convolution, placed on the plate of the freezing microtome and
just frozen--not too hard or the tissue will be brittle and will also
injure the edge of the razor. As soon as a good section is obtained
the razor should be plunged into a large bowl of cold water to detach
the section, which is at once floated on a glass slide, and osmic acid
solution, 1/4 per cent. allowed to flow over it from a pipette. This
will fix the tissue elements in about two minutes. The section is again
floated off into the bowl of water and thoroughly washed to free it
from the osmic acid. It is then stained either on the slide, or in a
watch-glass, with the aniline blue-black solution for an hour in the
cold, or half-an-hour if the solution is slightly warmed. The dye is
thoroughly washed away with distilled water, excess of moisture wiped
off the slide with blotting paper, and the section allowed to dry
under a glass bell jar. It is not practicable to dehydrate by means of
alcohol as it would cause sudden shrinking of the tissues. When the
section is dry a drop of Canada balsam is applied and it is covered
with alcohol in the usual way. The nerve cells and their processes are
stained a deep slate colour, as are the nuclei of the connective tissue
cells, while the ground work of the neuroglia is faintly stained and
of a neutral grey tint. This method gives beautiful results both with
normal and morbid specimens.
Various other aniline dyes, indulin, methylene blue, gentian violet,
have been employed in the same way, but none of them give such good or
uniform results as aniline blue-black.
Hardened specimens may also be stained with aniline blue-black, but the
results are not to be compared with those obtained by the fresh method.
The stain is usually diffuse, but this can be improved by placing the
sections for 1/2 - 2 minutes in a 2 per cent. solution of chloral
hydrate in distilled water after staining.
*Golgi’s metallic stains* for nerve cells.
Golgi introduced the methods of producing a metallic deposit of mercury
or silver in the nerve cell, revealing both the cell and its processes.
This method has been very fruitful in discoveries, especially in the
hands of Ramon y Cajal, Köllicher, Van Gehucten and others. It gives
best results with embryonic tissues. To ensure good results it is
important that the tissue be removed immediately after death. Sections
of brains removed some hours after death usually give disappointing
results.
There are several methods now in vogue, all slight modifications of
Golgi’s original methods.
*Silver nitrate method.*--Small pieces not more than a quarter of an
inch cube, are transferred straight from the body to a large quantity
of Marchi’s fluid (p. 24) and kept in it for about a week, or longer
in the case of adult specimens. On removal from Marchi’s solution the
tissue should be washed for a few seconds in distilled water, and then
placed in a large quantity of a 3/4 per cent. solution of nitrate of
silver solution in distilled water for at least a week. The lump of
tissue becomes of a brick red colour owing to a coating of silver
chromate. On removal from the silver solution the tissue should be
washed in methylated spirit for a few minutes and the incrustation of
silver chromate brushed off. Sections may be cut in gum and celloidin;
or they may be fixed on a cork with celloidin or spirit varnish and cut
without embedding: very thin sections are not required. Dehydrate in
alcohol, clear in xylol, and mount in balsam. Goodall advises a mixture
of pyridine and xylol for clearing, and mounts in strong xylol-dammar
solution, without a cover-glass.
Very careful attention to details and much practice are required before
uniformly good results can be obtained. The results are extremely
beautiful and well repay the labour expended on them. The cells and
their processes appear black on a yellowish ground.
A method has been employed for deepening the colour of the stain,
but the writer has no experience of it. Kallus (_Zeitsch. f. Wiss.
Mikr._, 1893, 477) dilutes an ordinary hydrokinone developing solution
(prepared as for developing an ordinary photographic plate) with about
ten times its volume of distilled water. Just before using a third
part of absolute alcohol is added. Sections which have been through the
silver process when placed in it become grey or black in a few minutes,
and, after washing in methylated spirit, are transferred to a 20 per
cent. aqueous solution of hyposulphite of soda for a couple of minutes
and then washed very thoroughly in distilled water for twenty-four
hours. They are then dehydrated and mounted in balsam.
*Buckley’s modification of the silver method.*--Described in _Brain_,
Winter number, 1895.
The method is applicable to specimens that have been hardened in
Müller’s fluid. Thin slices are cut in the usual way, and then immersed
in
Bichromate of potassium, 3 per cent. solution 5 parts
Osmic acid, 1 per cent. solution 1 part
for three to five days. Excess of bichromate is removed from the
sections by blotting paper, and they are transferred to the _freshly
prepared_ staining mixture:--
Phospho-molybdic acid (10 per cent.) 1 minim 2 drops.
Nitrate of silver (1 per cent.) 1 ounce 60 c.c.
which must not be filtered. Stain for several days.
The sections should be cut at once after removal from the staining
solution. It is claimed that the minute details of structure of the
cell processes are better shewn by this method.
*Corrosive sublimate* method.--This method is similar in its mode of
action to the last, mercury being deposited in the cell instead of
silver. It is rather less certain and requires more practice. It seldom
stains uniformly. One cell will be found exquisitely stained while
those in its vicinity are unaffected.
Small pieces of cortex are hardened for several weeks in Müller’s
fluid, or other bichromate solution, and are then transferred direct
to a one-half per cent. aqueous solution of corrosive sublimate, in
which they should be left from three to six weeks. Shorter periods will
only give disappointing and inconstant results. Sections should be
cut, if possible, in gum. They may be mounted in Farrant, or dehydrated
and mounted in balsam. Tal has proposed to render the effect sharper
by transforming the deposit of mercury into mercuric sulphide, by
treating the sections with a solution of sulphide of sodium, which
he prepares by saturating a ten per cent. solution of caustic soda
with sulphuretted hydrogen and then adding an equal quantity of fresh
soda solution. They are stained in this for a few minutes and then
thoroughly washed.
By this method the pyramidal cells and their delicate processes appear
as black opaque objects on a light ground. The neuroglia cells with
their fine delicate processes are often also beautifully stained.
*Nissl’s aniline method.*--This method is complementary to Golgi’s
method. The latter impregnates the cell rendering it opaque and shewing
its form with great definiteness.
Nissl’s method stains the protoplasm without greatly reducing its
transparency and allows us to study details of cell structure. Small
portions of tissue, _removed as soon as possible after death_, are
hardened in alcohol. Sections are then cut, preferably in gum, as
celloidin is inconvenient owing to its staining so deeply with aniline
dyes.
Sections are transferred from alcohol to a one-half per cent. aqueous
solution of methylene blue, which is heated in a watch glass till
it steams freely, but short of the boiling point. Stain for about
a quarter of an hour and allow to cool. Transfer the sections to
a mixture containing one part of aniline oil and ten of absolute
alcohol, and move them about till no more colour comes away. Transfer
the section to a slide with a section lifter, drain, and dry well by
pressing folded filter paper carefully on the section. Allow some
origanum oil to flow over the section and remove excess of this by
pressure with blotting paper. Moisten with benzine,[1] and add a
drop of colophonium resin dissolved in benzine. The slide is warmed
cautiously till the benzine is driven off and the colophonium liquefied
by heat alone, and then the cover-glass is applied.
[1] The student will bear in mind the danger of working with benzine
near a naked light.
Magenta and other aniline dyes may also be employed in a similar
manner.