GENERAL STAINING METHODS.
Much information may be obtained from unstained sections, and in most
cases one section should be examined unstained, but the specimens
mounted in this way are so transparent that it is difficult to study
the details of the tissue. They are therefore usually prepared by
treating them with some staining reagent, not merely to render them
less transparent, but also to “differentiate” the elements of the
section, by staining one part more deeply than another, or of a
different colour. Thus hæmatoxyline stains the nuclei and rapidly
growing parts of the tissue, leaving the formed material, as a rule,
much more lightly tinted. Methyl violet again stains healthy tissues
blue, and parts affected with waxy degeneration a red-violet colour.
By combining stains also much differentiation of the tissue elements
may be obtained. Sections should be stained with several reagents, as
their effect on individual specimens varies a good deal.
The following are the most useful stains for general purposes:--
*Logwood.*--This or its purified principle hæmatoxyline is the most
useful general stain. The hæmatoxyline itself is preferable, giving
more constant results, and less diffuse staining.
For general staining purposes the following formula will be found to
give excellent results:--*Hæmatoxyline. Schuchardt’s formula.*--
(_a_) Hæmatoxyline 3 grms. 30 grs.
Absolute alcohol 16 c.c. 2-1/2 drms.
(_b_) Pure alum 3 grms. 30 grs.
Distilled water 100 c.c. 2 ozs.
Add (_a_) to (_b_) _drop by drop and with constant agitation_. Keep
for some days exposed to diffuse daylight until its colour is so deep
that it will not transmit the light. It should then be filtered, and a
crystal of thymol added. It will not give very satisfactory staining
reactions at first, and should be allowed to ripen at least a month or
six weeks before using. It improves as a dye with every month that it
is kept. Whenever hæmatoxyline has been made up with alum as in the
above formula, an abundant reddish-brown precipitate forms after some
time. This in no way interferes with the activity of the solution, but
it must always be filtered before being used.
*Barrett’s formula.*--Introduced by Dr. W. H. Barrett, of Belfast. It
gives almost as good results as the above. It is made from ordinary
English extract of logwood, and is considerably cheaper.
The extract should be dried, and finely powdered, and then extracted
with absolute alcohol for several days.
Powdered extract of logwood 2 grms. 1-1/2 drms.
Absolute alcohol 10 c.c. 1 oz.
Filter and add slowly to
Benzoate of sodium 1 grm. 36 grs.
Alum 1 grm. 36 grs.
Distilled water 100 c.c. 10 ozs.
The strength of the solution will vary with different samples of
logwood and must be estimated by trial. This solution is comparatively
cheap and is useful for class purposes.
*Ehrlich’s hæmatoxyline.*--This very useful nuclear stain is made as
follows:--
(_a_) Hæmatoxyline 2 grms. 9 grs.
Absolute alcohol 100 c.c. 2 ozs.
(_b_) Glycerine 100 c.c. 2 ozs.
Distilled water 100 c.c. 2 ozs.
Alum 120 grms. 2-1/2 ozs.
Glacial acetic acid 5 c.c. 24 mins.
Add (_a_) slowly to (_b_) with constant agitation.
Allow to ripen in sunlight for two months before using. It may be
employed as a rapid stain undiluted but far better results are
obtained by using a weak solution, a few drops to a watch-glass
full of distilled water, and staining slowly for from half an hour
to two hours. The solution improves by keeping. If after a time the
staining becomes diffuse it is an indication that the acetic acid has
evaporated, and a few drops more should be added.
*Kleinenberg’s hæmatoxyline.*--This formula differs from the previous
one in being an alcoholic solution. The calcium chloride is added
because it “sets up diffusion currents between the alcohol in the
material to be stained and the alcoholic staining solution, so
enabling the latter to penetrate more rapidly” (Squire). It is much
used in staining embryonic specimens in bulk before embedding in
paraffin, and was strongly recommended for that purpose by Foster and
Maitland Balfour.
Various formulæ have been given from time to time. That advised by
Squire (_Methods and Formulæ_, p. 25) can be accurately made up without
much difficulty.
(_a_) _Crystallised_ calcium chloride 20 grs. 1/2 oz.
Distilled water 10 c.c. 2 drms.
(_b_) Alum 3 grms. 32 grs.
Distilled water 16 c.c. 170 mins.
Mix and add
Rectified spirit 240 c.c. 8 ozs.
Allow it to stand and any excess of calcium sulphate, &c., to separate.
Filter and add
Hæmatoxyline 2-1/2 grms. 25 grs.
A little thymol should be added as a preservative.
In making up these solutions care must be taken that _only distilled
water_ is used, and that all the vessels employed have been previously
rinsed out with it, otherwise precipitation of the hæmatoxyline will
occur.
Should sections be overstained in hæmatoxyline, this may be remedied
by washing it in a half per cent. solution of acetic acid, until
sufficient of the stain is discharged, but the staining is more diffuse
than if the happy mean had been hit in the first instance.
Hæmatoxyline stains the nuclei of the cells a beautiful violet colour,
and also tints, more or less lightly, the cell protoplasm and the
fibrous elements. It also stains the axis cylinders of nerves, and is
much used in special staining of the nerve centres as will be described
later, (pp. 88–91).
The stain is permanent. Sections may be mounted either in Farrant’s
solution, or in Canada balsam, the latter being preferable.
*Eosine.*--Much more satisfactory results are obtained from the
commercial eosine (an amorphous orange powder used in dyeing and in the
manufacture of red ink), than from the pure crystalline form.
It may be used as an aqueous solution (1/30 per cent.) or as a
solution in absolute alcohol (1/15 per cent.). Sections stained in the
former should be rapidly passed through a one per cent. solution of
acetic acid in order to “fix” the stain, and then washed in distilled
water.
It is a very transparent stain, and the most delicate details of a
section stained with it are perfectly visible.
It stains the nucleus but slightly, while it stains the cell protoplasm
and fibrous tissues and especially muscular tissues a beautiful rose
colour.
It will be seen, therefore, that it stains those parts which are left
unstained by hæmatoxyline, and _vice versâ_. This complementary action
is applied in the following method.
*Double staining with eosine and hæmatoxyline.*--Sections having been
stained in hæmatoxyline in the ordinary way, are washed in distilled
water, and dehydrated in a solution (about 1 in 1500) of eosine in
absolute alcohol. They should remain in this for about two minutes, and
then be passed through oil of cloves and mounted in Canada balsam in
the ordinary way.
This method gives extremely useful and beautiful results with almost
all tissues, and is superior to picrocarmine for differentiating
the tissue elements. Thus, the nuclei are stained violet, the cell
protoplasm a much paler and warmer violet, the fibrous tissues pink,
and red blood corpuscles orange or brick red.
The alcoholic solution of eosine is also used as a contrast stain after
staining for micro-organisms with blue or violet dyes.
*Carmine.*--It is made as follows:--
Carmine (best) 2 1 drachm.
Strong ammonia 2 1 drachm.
Distilled water 100 6 ounces.
Rub the carmine with a little water in a mortar, add the ammonia,
when the liquid will turn black. Gradually add the rest of the water,
rubbing it up all the time. It should be bottled, allowed to stand for
a few days, and then filtered, and a piece of camphor put in the bottle.
*Lithium carmine* resembles closely ammonia carmine in its staining
effects. It is usually a matter of individual preference which is
employed.
Carmine 2-1/2 grms. 10-1/2 grs.
Saturated aqueous solution
of lithium carbonate 100 c.c. 2 ozs.
Dissolve and filter.
Sections may be sufficiently stained in either of these fluids in from
three to five minutes, but more satisfactory results are to be obtained
by diluting with twenty times the bulk of distilled water, and leaving
sections to stain for twenty-four hours.
After staining in carmine the sections must be passed through a half
per cent. solution of acetic acid, in order to fix the carmine in the
tissues, as otherwise the water will dissolve the stain out.
*Borax carmine*--
(_a_) Borax 4 grms. 3 drachms.
Carmine 2 grms. 1-1/2 drachms.
Distilled water 100 c.c. 5 ounces.
Dissolve with the aid of heat and add slowly to (_b_).
(_b_) Alcohol 70 c.c. 3-1/2 ounces.
Distilled water 30 c.c. 1-1/2 ounce.
Allow to stand for a fortnight. Filter, and add a lump of camphor.
To use it, place sections, or the tissue in bulk, in it for from four
to twenty-four hours, according to size, and then transfer to alcohol
(seventy per cent.) containing a drop to the ounce of hydrochloric
acid, for twenty-four hours, and then wash thoroughly in water. The
tissue may then be placed in gum if it is to be frozen, dehydrated in
alcohol if paraffin or celloidin is to be employed.
Its advantage is that it is very diffusible, and so can be used
to stain tissues in bulk. It takes a considerable time to stain
sufficiently deeply, but there is little fear of overstaining.
It stains nerve-cells and axis cylinders brightly, and also the
connective tissue, bringing a sclerosed patch out very prominently.
*Alum carmine*:--
Alum five per cent. solution
in distilled water 100 c.c. 1 oz.
Pure carmine 1 grm. 4-1/2 grs.
Boil for twenty minutes. Filter. Add a few drops of carbolic acid.
In using this reagent it should be filtered into a watch glass, and
the sections placed in it for at least an hour. There is no fear of
overstaining, and they may be left all night. After they have been
stained they must be _thoroughly_ washed in water to remove the alum,
otherwise numerous crystals of it will be seen in the field when the
section is mounted. Sections may be mounted in Farrant’s solution or in
Canada balsam. The staining effect improves very much after the section
has been kept a few days.
If desired its staining action may be complemented by dehydrating it in
an alcoholic solution, either of eosine (1 in 1500) or of picric acid,
and then clearing up in oil of cloves, and mounting in Canada balsam.
By itself it gives a stain very like that of hæmatoxyline, only warmer.
It picks out the nuclei and axis cylinders of nerves, stains cell
protoplasm slightly, and the fibrous elements scarcely at all.
It may be used for the same purposes as hæmatoxyline. The colour is
less attractive, and not so deep as that of the latter, but as it does
not overstain sections, even when left in it for a week, it is a very
convenient stain for general purposes.
It is particularly useful as a contrast stain for sections of brain
and spinal cord, after the Weigert-Pal hæmatoxylin process (p. 88).
*Ammonia-picrocarmine* was formerly very largely used as a staining
reagent. Its place has now to a large extent been taken by
lithio-picrocarmine.
In its preparation the best carmine must be used.
It is made as follows:--
Carmine 1 part.
Liq. ammon. fort. 3 parts.
Distilled water 3 parts.
Dissolve with gentle heat, and add
Cold saturated aqueous solution
of picric acid 200 parts.
Bring the mixture to the boiling point, and then place in a shallow
vessel, covered with a glass plate, and leave it in full sunlight for
a month or more. Filter, bottle, and add six drops of carbolic acid to
each ounce of the mixture. It will keep indefinitely and improves with
age. It requires filtering from time to time, as a gelatinous crimson
mud tends to deposit from the solution.
*Lithio-picrocarmine.*--Prepared as follows:--
Carmine 2·5 grms. 10 grs.
Saturated solution lithium carbonate 100 c.c. 1 oz.
Dissolve, and add
Saturated solution picric acid 250 c.c. 2-1/2 oz.
Add a few drops of carbolic acid to each ounce.
It should stand a day or two in sunlight and then be filtered. It
improves by keeping.
It should be kept in a stoppered bottle with a glass rod fused into the
stopper.
When sections are to be stained they are to be floated out on a clean
glass slide as described on page 55. The slide should then be tilted
to allow the water to drain off, and superfluous moisture round the
section removed by a soft rag, or blotting paper. A drop or two of the
stain should then be transferred to the slide, which should be left
lying quite flat for about ten minutes. Unless the room is very warm it
is advisable to heat the slide very gently over a spirit lamp, as this
causes the tissues to stain more brightly and more rapidly.
The excess of the picrocarmine should be allowed to run off the slide,
and the latter wiped. Some of the stain should, however, be left on the
section, as its effects go on increasing, and are often not fully seen
until a few weeks have elapsed. They should be mounted in Farrant’s
medium. As a rule those mounted in Canada balsam do not give such good
results. Should there be special reasons for using this medium, as in
mounting spinal cord sections, &c., they should be dehydrated after
staining in picrocarmine in an alcoholic solution of picric acid (one
part of a saturated alcoholic solution to five of alcohol), before
clarifying in oil of cloves, as otherwise the alcohol will dissolve out
the picric acid, and much of the differential staining effect will be
lost. The nuclei should be stained a bright crimson, the protoplasm of
the cells yellow, or a dull pink, the fibrous elements a bright pink,
red corpuscles green, and all dead material, _e.g._, caseous matter,
bright yellow. It also stains nerve-cells, and the axis cylinders of
nerve fibres very brightly. It is, however, a rather uncertain dye.
The results are most brilliant in the case of fresh sections.
*Osmic acid* is invaluable for staining fatty particles in the cells.
For ordinary use the one per cent. stock solution (p. 21) should be
diluted with ten times its bulk of distilled water, and sections
stained in it all night in a dark cupboard, or the watch glass
containing them may be placed inside a small box.
The sections must be washed _thoroughly_ in plenty of water. If desired
they may be stained subsequently in picrocarmine or methyl violet
if waxy degeneration also be present. Sections should be mounted in
Farrant’s solution, as Canada balsam usually gives disappointing
results.
It demonstrates the most minute fatty particles in degenerating cells,
&c., staining them black. It may be employed to demonstrate the
globules of fat blocking up the vessels in fat embolism.
It stains the myelin sheaths of nerves black, and will be again
referred to when speaking of methods of staining the spinal cord.
*Nitrate of silver* is employed for staining the intercellular cement
of epithelial cells. It stains this substance a deep black, while the
rest of the tissue takes on a brown colour. It is used as a half per
cent. solution in *distilled* water, and kept in a stoppered bottle
carefully covered up with brown paper. To use it take some epithelial
tissue, _e.g._, the omentum from a recently killed animal, or a section
of some epithelial tumour, immediately after excision. Wash thoroughly
in distilled water to remove all chlorides, and then place in a watch
glass containing the silver solution. Keep this in the dark for half an
hour and then wash thoroughly in plenty of water. The section should be
mounted in glycerine or Farrant’s medium and kept from the light or it
will become too darkly stained.
*Chloride of gold* is employed to demonstrate the peripheral
terminations of nerves. It can only be employed within the first half
hour after the tissue has been removed from the living body. The pieces
of tissue must be small and may be stained in bulk, sections being
subsequently made.
A half per cent. solution in distilled water is employed. The tissue
is transferred to this on its removal from the body, until it becomes
lemon coloured. It is then exposed in a one per cent. solution of
acetic acid to a strong light until it assumes a purplish tinge,
which takes from two hours to two days. Sections should be mounted in
Farrant’s medium. It stains the cells of the tissue, and nerve cells
reddish purple, and nerve fibrils, especially the terminal ones, rather
more violet. This is very well seen in the cornea.
It is useful sometimes for clinical purposes to excise a portion
of muscular tissue and examine the nerve endings by this method.
Unfortunately the stain is somewhat uncertain in its action. Better
results are obtained by Sihler’s chloral hæmatoxyline method (p. 92).
*Methyl violet.*--A very satisfactory solution may be obtained ready
made in the “telegraphen tinte,” prepared by Leonhardi, of Dresden,
as recommended by Woodhead. It may also be used as a one per cent.
solution in distilled water, a few drops of carbolic acid being added
to prevent the growth of fungi.
It is a very useful selective stain. It gives two reactions, red
violet, and blue violet. Thus it stains the matrix of hyaline
cartilage blue violet, but the cells red violet. It has also a most
important pathological application, as it picks out any parts which
have undergone “waxy” or “lardaceous” degeneration, staining them red
violet, but the rest of the section blue violet.
About ten drops of a one per cent. solution should be filtered into
a watchglassful of water, and the sections stained for about five
minutes. They must then be passed through a half per cent. solution of
acetic acid and washed *thoroughly* for some time in a large quantity
of water till no more colour comes away.
If these steps are not taken with care, the dye will diffuse out after
the section has been mounted, blurring all details and spoiling the
appearance of the section.
Sections may be mounted in Farrant’s solution (to which a spot of
formic acid may be added): if mounted in Canada balsam the sections
must be overstained as both the alcohol and oil of cloves rapidly
dissolve out the dye.
*Safranine.*--Employed as a freshly made saturated solution in aniline
oil water warmed to 60° C. (140° F.). Filter into a watch glass. Stain
for not more than a minute. Dehydrate in alcohol which will remove much
of the stain, clarify in oil of cloves or origanum oil. Mount in balsam.
Another method is to stain for about ten minutes, and then leave for
a minute in Gram’s iodide solution. The sections are then washed in
alcohol, dehydrated, clarified in oil of cloves, and mounted in balsam.
By these methods the stain is withdrawn except from certain elements,
_e.g._, those undergoing colloid or calcareous degeneration.
A quarter per cent. watery solution is sometimes employed as it stains
nucleoli and actively dividing nuclei very brightly, while the rest of
the cell is stained faintly. It may be employed to study karyokinesis
in the cells of a rapidly growing cancer.
*Ehrlich-Biondi stain.*--This stain has been much employed for staining
specimens of blood, for studying karyokinesis, and for investigations
on the supposed parasitic bodies found in cancer cells.
It is prepared by mixing saturated aqueous solutions of the following
aniline dyes, slowly and with constant agitation:--
Solution of Orange G 100 parts
" Rubin S 20 "
" Methyl Green OO 50 "
finally add
Distilled water 70 "
Filter from the copious precipitate which forms. The solution must be
made up frequently as it does not keep well.
Sections may be stained rapidly for half an hour or an hour, but better
results are obtained by diluting the fluid with twenty volumes of
water, and staining all night. Sections should be washed in water and
then passed rapidly through absolute alcohol and xylol and mounted in
Canada balsam.