A fine, long cambric needle should be passed through the tis-
sue, and then (the mould being placed in position) the point of the
needle is to be pushed through the bottom into the table beneath.
Then the mixture of the liquid wax and oil, which has been
heated to the point of melting and no more, should be poured
slowly into the mould, so as to slightly cover the specimen.
During the process of hardening, minute bubbles of air will be
liberated from the tissue ; they will escape more rapidly, and
the embedding material will harden more quickly and thor-
oughly, if the microscopist blows gently and continuously on
the surface of the liquid. Just at the moment when the mass
is no longer liquid, the needle should be suddenly withdrawn.
As soon as it is hard throughout, the tin-foil mould may be
torn off by breaking the edge at any point with the finger.
The foil tears like paper.
When moulds are not at hand, an excellent substitute may
be made with ordinary writing paper. Some confectioners
make them of pressed paper.
Embedding in glycerine and tragacantJi. Mr. John Ste-
venson's plan is as follows: He takes two drachms of glyce-
rine and mixes them with one drachm and a half of powdered
gum tragacanth. The tissue to be cut is then placed in a small
pill-box, and the mixture poured in. The box is then laid
away in a cool place from eight to twelve hours, when sections
may be made with the knife. In case the specimen is to be
preserved for a longer time, the bottom of the box may be
taken off, and the side slit up. The specimen will now be
found embedded in a solid elastic cake, and may be slipped
into alcohol until required. When it is to be kept in spirits
less than twenty-eight hours, the mixture should be glycerine,
2 drachms ; powdered tragacanth, 1 drachm ; gum arabic, 15
grains. Tissues that have lain in spirit should be steeped in
cold water a few hours before embedding.
The hand section-cutter is used by some microscopists. It
is simply a cylinder which is designed to receive the object
and the material in which it is embedded. A plunger, which
is driven up from beneath by the revolution of a screw, pushes
THE ORDINARY METHODS OF PREPARING TISSUES. 17
up the specimen so that it may be sliced off by an ordinary
knife. For some purposes it is very useful.
Freezing section- cutters. Of these there are many in use,
and they have certain advantages. In conjunction with Dr.
J. H. Hunt, of Brooklyn, I have devised a modification of the
ordinary instrument. 1 (Fig. 9.)
FIG. 9. Freezing section-cutter : B, metallic box ; S, cylinder; a, well; c, c, frame for holding knife
A, A ; G, indicator ; D, milled head ; F, F, plugs ; E, E, tubes to fit in well ; H, H, covers to metallic
box ; K, binding screw attaching box to table.
It consists of the brass cylinder, S, made of rather large
size, and placed in the centre of a metallic box, B. The
length of the cylinder, with driver, D, is about five inches. The
diameter of the well, a, measures If inch. Fitted round and
about the cylinder is a plate of glass which from its smooth-
ness permits the knife to sweep it easily.
The knife, A, A, is large, measuring 13 inches in length, in-
cluding handle ; in breadth, If inch. It is fitted into a brass
frame, c, c, 7J inches in length and 3i in breadth. Two strong
brass springs, and two sliding clamps, hold it in place. The
knife is slightly concave on both sides.
The well is so large that it will hold an ordinary kidney
after hardening, or at least so much of it that a transverse sec-
1 Made by Miller Bros., 1213 Broadway, New York city.
18 MANUAL OF HISTOLOGY.
tion may be made of the whole organ at one sweep of the knife.
The knife and frame are modifications of those devised by Dr. E.
Curtis of this city, and the section-cutter and box are not dif-
ferent in any essential particulars from those in common use.
They are larger, however, and the indicator, Gr, enables the
observer to determine with accuracy the thickness of his sec-
tions. Thus, in my own instrument thirty-one turns of the
milled head drives the plug forward one inch.
Each revolution consequently drives the specimen forward
^ T inch. Now, the circumference of the milled head is marked
off into thirty divisions.
When the indicator marks that the plug has been driven
forward one division, the distance traversed will be -fa inch.
It is easy, therefore, to determine the thickness of any sec-
tion with considerable accuracy.
When it is desirable to put the instrument in use, the
plug that is to be used is well oiled, as also the thread of the
driver, and the metallic box is filled with a mixture of ice and
It is necessary to be particular and oil the bearings
thoroughly, else they will bind and the instrument will be
clogged while the freezing process is going on. The usual
plan is to soak the tissue (as Dr. Pritchard suggests) in a
thick solution of gum, which cuts like cheese when frozen.
The soaking should continue for a number of hours, say until
the next day.
When the tissue is ready, a thick solution of the guru
should be poured into the well and the tissue held until it is
fixed by the ice. Some non-conductor is to be placed over
the well as soon as fixation has commenced, in order that ac-
cess of heat may be prevented.
If ice is used it should be ground up finely and then packed
tightly about the well ; snow is better. The whole process
takes only ten or fifteen minutes. The freezing section-cutter
is of use when we are desirous of making a rapid examination
of fresh tissues.
It is obvious that they are seen under more natural circum-
stances than when they have passed through the bichromate
or chromic acid solutions, or alcohol, all of which cause more
or less change in such delicate substances.
It has been hoped that by the freezing method we should
THE ORDINARY METHODS OF PREPARING TISSUES. 19
learn much that is new about the finer structures of the brain
and the character of the corpuscular elements of the body, bub
as yet it has not reached our expectations.
Hailes' s microtome. A very ingenious and excellent instru-
ment (Fig. 10) has been devised by Dr. William Hailes, Pro-
fessor of Histology and Pathological Anatomy at the Albany
Medical College. Objections to it will be mainly on the ground
Dr. Hailes uses it as a simple instrument or as a freez-
ing microtome, arranged either for ice and salt, ether-spray,
The employment of ice and salt (coarse) is preferred, be-
cause it costs but little and freezes the mass solidly and
quickly, and, if desired, 500 or 1,000 sections can be obtained
in a few moments, depending, of course, upon the rapidity
and skill of the operator.
The time of freezing is about seven minutes, except in
very warm weather, when it requires a few moments longer.
The instrument does not work quite so satisfactorily in very
warm weather, owing to the rapid melting at the surface of the
It is absolutely necessary that the mass should be frozen
solid, or the sections cannot be cut smoothly.
An extra freezer may be employed, and while one specimen
is being cut the other is being frozen ; by exchanging cylinders
(they being interchangeable) no delay is- necessary.
The art of cutting is readily acquired. Two hundred or
two hundred and fift}^ sections have been made in a minute,
and of a uniform thickness of TY Vo- f an inch. It is not
necessary to remove the sections from the knife each time,
but twenty or thirty may be permitted to collect upon the
blade. They lie curled or folded up upon the knife, and when
placed in water, straighten themselves out perfectly in the
course of a few hours. The knife employed is an ordinary
long knife from an amputating case.
Perfectly fresh tissues may be cut without any previous
preparation, using ordinary mucilage (acacia) to freeze in, but
most specimens require special preparation.
If preserved in Miiller's fluid, alcohol, etc., they require to
be washed thoroughly for several hours, and then, according
to the suggestion of Dr. David J. Hamilton, F.R.C.S., etc., of
MANUAL OF HISTOLOGY.
the University of Edinburgh, Scotland, the specimen is placed in
a strong syrup (sugar, two ounces ; water, one ounce) for twenty-
four hours ; it is then removed to ordinary mucilage for forty-
eight hours, and finally is cut in the freezing microtome.
These sections may be kept indefinitely in a preservative
FIG. 10. Poly-microtome (without freezing apparatus) : A, small well fitting on pyramidal bed-plate ;
B, pyramidal bed-plate containing different sizes; C, micrometer screw; D, ratchet-wheel attached to
screw ; E, lever actuating the micrometer screw by means of a pawl engaging in teeth of ratchet-wheel ;
F, arm carrying a dog, which prevents back motion of screw ; G, regulator for limiting the throw of
lever, and consequently governing the micrometer screw ; H, lever-nut for fixing regulator ; I, index, with
pointer and graduated scale, from 1/2400 inch to 1/200 inch ; K, knife for cutting sections ; L, knob to
turn micrometer screw direct when pawls are detached ; M, table clamp ; T, table of microtome, with
glass top to facilitate cutting.
FIG. 11. (Very much reduced in size). A, B, tube containing specimen which is surrounded by freez-
ing mixture in tin receiver C, D ; B, F, revolving hopper with wings, W, W, for stirring the ice ; G, out-
let for melted ice.
fluid recommended by Dr. Hamilton:
destil., aa. f iv. ; acid, carbolic., gtt. iij.
addition of alcohol, ij., is advisable.
$. Glycerin., aquae
Boil and filter. The
STAINING FLUIDS. 21
TJie Vincent microtome. This instrument, which was de-
vised by Dr. Vincent, of New York city, is a flat piece of steel
(Fig. 12) 12 inches long by 2 2 inches wide, with a bevelled
cutting edge, 6 inches long. The handle is simply the rounded
and smoothed extremity of the knife.
It has been in use at the School of Histology connected with
the Columbia Veterinary College, and has proved to be a very
The mode of action is very simple. The object having been
previously placed in any ordinary hand-cylinder and mounted
in wax, paraffine, or pith, the sections are made by a stroke of
the knife, which is pushed straight forward. As will be readily
seen, the larger the section the wider the knife must be.
The blade is made of the best plate steel, and is easily kept
Ammonia carmine. This is one of the oldest and best
known solutions. Take one part, by weight, of the best car-
mine, which is known as " No. 40," dissolve it in 100 parts of
distilled water, and add one part of aqua ammonise. The pre-
vious dull color now gives place to a most brilliant and deep
red. It is necessary, however, that the carmine be either neu-
tral or very faintly alkaline, else the color will diffuse and the
tissues will not be differentiated. Expose the fluid, therefore,
for some weeks to the air, or evaporate over the water-bath
until the odor of ammonia is no longer perceptible.
The nuclei should be deeply and brightly stained, while the
intercellular substance is in no way affected. If, however,
diffusion has taken place, a great deal of it may be removed
by soaking the section in a saturated alcoholic solution of ox-
alic acid. When a brick-red color has in this way been ob-
tained, the object has been accomplished. Crystals of oxalic
acid are apt to be found in specimens that have been prepared
22 MANUAL OF HISTOLOGY.
in this way. It is therefore desirable, after using the acid, to
wash thoroughly in alcohol or water.
Borax carmine (Arnold's formula). The following method
is given by Dr. M. N. Miller as the one in use by students in
the histological laboratory of the New York University. It
originated with Prof. J. W. S. Arnold. A saturated solution
of borax is prepared in a wide-mouthed pint bottle. The borax
should be in some excess. "No. 40" carmine is now added
to the solution under constant agitation, until after a while it
no longer dissolves, and an excess remains at the bottom of the
vial, mingled with the crystals of borax. After twenty-four
hours the supernatant fluid is decanted. To this clear portion
f. ^ij- of alcohol are added, and f. 3 j. of caustic soda solution
(U. S. P.). The staining solution is now ready. Or, the alco-
hol may be omitted (Arnold), and the liquid evaporated to dry-
ness ; the red amorphous mass is then powdered. Of this,
15 grains are placed in an ounce of water, to which f. 3 j. of
alcohol is added. 1
Sections, after staining, should be washed in alcohol to re-
move the superfluous coloring fluid, and then transferred to a
saturated solution of oxalic acid in alcohol to fix the color.
The oxalic acid is then washed out in alcohol ; finally the sec-
tions are cleared up in oil of cloves, and mounted in balsam or
Double staining by borax carmine and indigo carmine.
Drs. W. T. Norris and E. O. Shakespeare, of Philadelphia,
have recommended a method which is a modification of Mer-
kel's. Two staining fluids are made, one red and the other
blue. The red one contains carmine, gr. 7J ; borax, 3 ss. ; dis-
tilled water, 1 j. The blue contains indigo carmine, 3 ss. ; bo-
rax, 3 ss. ; and distilled water, 3 vij.
After thorough trituration the ingredients are mixed and
left in a vessel ; the supernatant fluid is then poured off. The
sections, if previously hardened in bichromate, picric acid, or
chromic acid, should be well washed ; they then are to be
placed for a few minutes in a mixture (equal parts) of the red
and blue fluids, then transferred, without washing, to a satura-
ted solution of oxalic acid and allowed to remain in it rather
less time than in the staining fluid. When sufficiently bleached
1 [This preparation of borax carmine is the best that I have ever used. T. E. S.]
STAINING FLUIDS. 23
the sections should be washed in water until every trace of ox-
alic acid is removed. .Sections thus prepared may be mounted
in balsam or dammar. Connective- tissue substances are blue,
while the nuclei are red. The osseous lamellae of bone are
blue, the cells in the lacunae red, while the marrow is apple-
Picro-carmine (Miller's). Add one part of a saturated so-
lution of picric acid to two parts of the 15-graih borax carmine
solution (Arnold's). The epithelium of the glands and the
muscles are stained yellow, while the nuclei of the cells and the
connective tissues acquire the carmine color. Sections should
remain in the picro-carmine solution for about twenty -four
hours. Next they are washed quickly in water, then in alcohol,
after which they are transferred to the oil of cloves. (For Ran-
vier's method of making picro-carmine, see the chapter upon
the Histology of the Nervous System.)
Hcematoxylin solution (Boehmer' s). Dissolve 20 grains of
hsematoxylin in one-half an ounce of absolute alcohol ; then
dissolve 2 grains of alum in an ounce of water. Some drops of
the first solution are added to the second, which, after a short
time, becomes a beautiful violet. It improves after keeping for
a few days, and should always be filtered before using (Thin).
Hcematoxylin solution (Kleinenburg' s). First make a satu-
rated solution of the chloride of lime in seventy per cent, alco-
hol, and add alum to saturation.
Then make a saturated solution of alum in seventy per
cent, alcohol. Add the first to the second in the proportion of
one to eight. To the mixture add a few drops of a saturated
solution of hsematoxylin in absolute alcohol (Thin).
Hcematoxylin solution (Miller's method). Take a pint bot-
tle, as in the former process, fill with water, and add about an
ounce of common extract of logwood in coarse powder. Allow
this to remain in a warm place for twenty -four hours, with
occasional stirring. After the expiration of this time add pow-
dered commercial alum until the liquid changes from the
muddy brown color given by the logwood to a brilliant purple.
The alum is to be added until no change is produced. An
excess of the salt will do no harm. Add about f. I j. of alcohol,
and after decanting or filtering it is ready for use. One
may omit the alcohol at this stage, and evaporate to dryness
as in the borax-carmine process. The powder thus obtained is
24 MANUAL OF HISTOLOGY.
added to water when required. Three grains to the ounce of
water will give a fluid that will stain alcohol-hardened tissue
in from ten to fifteen minutes. A solution containing ten
grains to the ounce will stain very quickly. If it is desired to
keep the solution, add f. j. of alcohol for each ounce. Hsema-
toxylin stainings are soaked in water for a few minutes to
wash out the alum, then transferred to alcohol, clarified in the
clove oil, and finally mounted in balsam or dammar.
Klein } s formula for Ticematoxylin. Mix in a mortar 5
grammes of the officinal extract of hsematoxylin, with 15
grammes of alum, and pulverize carefully. To this add grad-
ually 25 c.c. of distilled water, and filter. To the residue add
15 c.c. of distilled water and again mix in a mortar, and filter ;
to this filtrate add 2 grammes of alcohol. Now mix the two
filtrates and keep in a glass-stoppered bottle. If the liquid
should at any time become muddy, filter again. Care must be
taken to prevent any acid from intermingling with the fluid.
Acids cause the hsematoxylin to turn red ; for this reason, sec-
tions which have been hardened in chromic acid should be
placed in a watch-glass and covered with distilled water, to
which add a drop or two of a 30 per cent, solution of caustic
potassa ; allow it to remain therein 10 to 15 minutes. To use
the hsematoxylin fluid, add a few drops to an ounce of distilled
water, so as to make a pale violet solution ; allow sections to
remain in this solution for 12 to 24 hours. Or, a stronger so-
lution may be employed which will stain specimens in 10 to 30
minutes, and still give good results. Mount in glycerine, ace-
tate of potassa, balsam, or better, resinous turpentine.
Eosine solution. Eosine, first introduced by Fischer in
1875, is much used in staining fresh preparations. It is cus-
tomary to have a strong solution of one to ten or twenty on
hand. A few drops are then added to a watch-glassful of water
or alcohol. Fresh tissues are both stained and hardened. It
affects the body of the cells, together with the nuclei. It is apt
to diffuse, unless special care is taken, and long soaking, say
for twenty-four hours, is practised.
Double- staining with cosine and other aniline colors.
Schiefferdecker first stains in an alcoholic solution of eosine
and then in a one per cent, watery solution of an aniline color
(dahlia, methyl violet, or aniline green). Care must be taken
not to extract the color when dehydrating the specimen in
STAINING FLUIDS. 25
alcohol according to the usual method ; very deep staining is
Green coloration of the nuclei. To effect this, Tafani em-
ploys a fluid containing three or four parts of a saturated
watery solution of aniline blue to some six or seven parts of a
saturated watery solution of picric acid.
Eosine and Jicematoxylinfor staining bone. Busch recom-
mends eosine and hsematoxylin for double-staining the zone of
ossification in growing bone. The sections of decalcified bone
are first immersed a few days in a one-half per cent, chromic
acid solution, or in a one per cent, solution of the bichromate
of potassium, and then, after washing with water, in a watery
solution of eosine. In young bone, where ossification is pro-
gressing, the cartilage matrix is blue, while the nuclei of the
cartilage-cells adjoining the line of bone are red ; the contents
of the medullary spaces are also bright red, while in the bone
trabecles there is a combination of blue and red.
Eosine for permanent specimens. Renaut has employed
eosine to differentiate all forms of protoplasm, whether bodies
or their processes. He either employs a watery solution alone,
or with the admixture of one-third its volume of alcohol.
The coloration is obtained after immersion of the sections from
one-half minute to one minute. They are then washed in
distilled water, and may be preserved in a neutral solution of
glycerine to which one per cent, of chloride of sodium has been
added to prevent the glycerine dissolving the eosine. These
preparations will then remain unchanged for months.
In examining the fixed corpuscles of the subcutaneous tis-
sue, the same author injects beneath the skin a solution of
eosine and water (1-500), and then removes a portion of the in-
filtrated tissue with the scissors. The fibrous fascicles are un-
affected, while the elastic fibres take the color deeply.
The fixed corpuscles appear as red granular plates, while
their nuclei take a very intense color. This reagent, therefore,
is well suited for the study of connective tissues. In special
instances the silver method may be used first, and then the
Preparation of tlie cornea. Klein has adopted the follow-
ing plan for exhibiting this most delicate tissue. He first burns
the centre of the cornea of a kitten with caustic potash, and
then, twenty-four hours later, brushes the surface with nitrate
26 MANUAL OF HISTOLOGY.
of silver, and, half an hour afterward, immerses it in water
acidulated with acetic acid ; after a day or two it is found to
have a glutinous appearance. The lamellae are then easily
stripped off, and in the middle portions, the corneal corpuscles
assume a purplish-brown color while their nuclei are uncol-
ored. The outlines of the lymphatic channels are also sharply
Picro-hcematoxylin and eosine (triple-staining). Wendt
has described a method of double-staining by picric acid and
hsematoxylin. Only the very thinnest sections, however, give
satisfactory results. A strong solution of hsematoxylin is first
employed. In this the sections are allowed to remain about
twelve hours. After washing them in water, they are placed
in a saturated solution of picric acid and carefully watched.
They may be removed from time to time, examined with a low
power, and, when properly stained, put in alcohol and mount-
ed in Canada balsam with as little delay as possible. To ob-
tain triple- staining, eosine may be conveniently combined with
this picro-hsematoxylin method. To insure good results some
amount of practice is necessary.
Double, triple, and quadruple staining. Dr. Gibbes re-
commends for double-staining, immersion first in picro-carmine
and then in logwood, or which is better, immersion first in a
spirituous solution of rosine or aniline violet, and then in an
aqueous solution of aniline blue or iodine green. In obtaining
more than two colors there is considerable difficulty. To ac-
complish it he uses first the chloride of gold or picro-carmine
and then the spirituous and aqueous solutions of the ani-
Staining with BismarJc brown. Make a watery solution of
gr. ij. 3J-, heat and filter; soak in the solution about three
minutes ; set the color with acetic acid (glacial) 4 per cent,
for half a minute. After dehydrating with alcohol mount in
dammar varnish. Weigert prepares the Bismark brown as
follows : he makes a concentrated aqueous solution by boil-
ing in water, filtering from time to time. He also uses a weak
alcoholic solution, and combines with other colors.
[To combine with eosine put the sections in a strong aqueous solution of
Bismark brown ; remove after about two minutes, set in weak acetic acid (four
per cent.), then place in a weak alcoholic or aqueous solution of eosine, and
then again in the acetic acid solution. T. E. S.]
STAINING FLUIDS. 27
Solution of alum-carmine. Grenadier recommends this
fluid : Take a one to five per cent, solution of ordinary alum,
or ammonia alum ; boil with one-half to one per cent, powdered
carmine for twenty minutes. Filter, and add a little carbolic
acid to preserve.
Naphthaline yellow for bone. In sections of the femur
from a foetal pig, three and a half inches in length, the follow-
ing method was found to yield very excellent results :
After immersion for three days in Miiller's fluid, sections
were made, and, after washing in water, immediately dipped in
an alcoholic solution of naphthaline yellow (gr. iv. J j.) ; after
eight to ten minutes the sections were removed, and dipped in
a watery solution of acetic acid of three per cent. ; then they
were immersed for about ten minutes in the ordinary solution
of ammonia-carmine, rendered neutral by exposure to the