SECTION CUTTING.
*Embedding of sections.*--Before sections are made the tissues require
to be embedded in some fluid, which will permeate their interstices,
and is capable of being rendered firm so as to support the most
delicate parts when the knife passes through the tissue.
The most generally useful substances are:--
(1) gum, (2) celloidin, (3) paraffin or wax.
*Gum.*--_Picked_ colourless gum arabic 2 parts, cold water 3 parts.
Leave with frequent stirring until dissolved. Add ten drops of carbolic
acid to each ounce of the mucilage.
Specimens are thoroughly freed from all trace of the hardening fluid by
washing in water, and the tissue is then placed in the gum solution for
at least twelve hours, or if enough carbolic acid be added, it may be
left there for an indefinite time.
When frozen, gum forms a firm non-crystalline mass, which supports the
tissue on all sides. It must not be frozen too deeply, or it becomes
hard and rather brittle and is apt to injure the razor. If this have
occurred the surface can be softened sufficiently by breathing gently
on it.
After cutting in gum, the sections are gently removed from the knife
into distilled water by a soft camel’s hair brush, and left there for
an hour or two, until the medium is entirely dissolved out. They may
then be stained and mounted, or they may be put away in spirit for an
indefinite time, and then stained and mounted.
*Celloidin* is for many purposes almost an ideal embedding medium. (1)
It has great penetrating power; (2) it can be made of an admirable
consistence for cutting purposes; (3) after sections are made it allows
them to be very freely manipulated without fear of injuring them: (4)
and being perfectly transparent and homogeneous in thin sections, it
does not require to be removed from a section before mounting. It is
insoluble in water, and in weak spirit; slightly soluble in alcohol of
more than 90 per cent. strength, and very readily soluble in ether, or
in a mixture of alcohol and ether. The last solvent is the one commonly
employed.
The embedding solution is made thus:--
Take some pure celloidin (“Schering’s,” sold in boxes containing an
ounce of shavings, is very good) and pour on it about eight times its
volume of a mixture of equal parts of absolute alcohol and ether. Allow
this to stand all night until the celloidin is dissolved. The solution
should be made about the consistence of ordinary mucilage.
It is also convenient to have a thinner solution made by using double
the proportion of alcohol and ether. Both solutions should be kept in
wide mouthed stoppered bottles, and the stopper should be well greased
with vaseline as an additional obstacle to the evaporation of the
volatile ether.
Before embedding a specimen it is necessary to dehydrate it thoroughly
for twelve to twenty-four hours in absolute alcohol. It should then
be placed in a mixture containing alcohol and ether for an hour or
two, and afterwards transferred to the thin solution of celloidin for
twenty-four hours, and then to the thick solution for the same period.
The celloidin penetrates slowly and in the case of nerve tissues and
other delicate structures it is wise to give the full allowance of time
for the different steps. When the tissue has been thoroughly permeated
by the celloidin, it is gently removed from the celloidin and placed
in position on a piece of cork of suitable size for clamping in the
holder of the microtome. Celloidin is painted round the object so that
it is supported on every side. It is then left exposed to the air
until the surface has become firm, when the cork is placed, with the
tissue downwards, in methylated spirit. The cork floats but the tissue
and celloidin remain submerged. At the end of twenty-four hours the
celloidin will have become semi-opaque and opalescent, and of the same
consistence as hard boiled white of egg. When it is impossible to wait
so long, rapid hardening of the celloidin may be secured by immersing
it in methylated chloroform in place of spirit, but the slower method
gives more uniformly satisfactory results.
Pieces of tissue embedded in celloidin may also be cut on a freezing
microtome. After the celloidin has become firm by immersion in
methylated spirit, the tissue with the celloidin round it may be cut
off the cork, washed in water to remove the alcohol, and then soaked
for an hour or two in gum, placed on the plate of an ether spray
microtome, frozen and cut in the usual way.
Subsequent staining operations are conducted in the same way as for
sections cut by hand or in gum. As celloidin is only slightly stained
by hæmatoxylin, alum carmine, borax carmine, &c., it is not necessary
to remove it from the sections, but it exhibits so intense a staining
reaction with aniline dyes that it is necessary to remove it by
treatment with alcohol and ether either before or after the staining
operation.
The sections after staining may be mounted in Farrant’s solution
(p. 59), or in Canada balsam (p. 61). If the latter medium is employed,
the section should be clarified, after dehydration in alcohol, by means
of oil of bergamot, or oil of origanum, instead of oil of cloves, as
the latter dissolves out the celloidin and causes the section to break
up.
Celloidin is most useful for cutting sections of the coats of the eye,
of the internal ear, and of bone marrow. It should always be used for
the Weigert-Pal hæmatoxyline method of staining the nervous centres, as
it protects the section from being injured by the transference from one
fluid to another which is repeatedly required during the process. The
stain is completely discharged from the celloidin by the decolourising
solution used (p. 90).
_Paraffin._--Paraffin is a very convenient embedding medium for
delicate structures, as very thin sections can be obtained and the
paraffin need not be removed from the section until the latter is
safely on the slide. It is unsuitable for large sections. Staining
operations are not easily carried out after cutting in paraffin, and
it is better to stain the blocks of tissue in bulk before embedding.
The best stains for penetrating are borax carmine (p. 75), alum carmine
(p. 76), and Kleinenberg’s hæmatoxyline (p. 70). The tissue must be
left in them for four to ten days.
Various kinds of paraffin are employed. It is usual to keep two kinds,
one “soft,” melting at 110° F., and another “hard,” melting at 140° F.
A mixture of two parts of the hard and one of the soft will be found
most generally useful. In winter a large proportion of the soft variety
and in hot weather a larger proportion of the hard may be required. A
paraffin mass which is always available has been suggested recently by
Dr. F. E. Batten, who employs an ordinary white candle, composed of
paraffin and wax. If the mass is found to be too hard, it can easily be
made of a suitable consistence by adding a little paraffin with a low
melting point.
To prepare a piece of tissue for embedding in paraffin, it should be
stained, washed in distilled water, and as much moisture as possible
removed by blotting paper. The block is then dehydrated, first in
methylated spirit for several hours, finally in absolute alcohol. It
is taken carefully by means of forceps from the alcohol and placed
in xylol for an hour or two according to size. Superfluous xylol is
removed from the surface, and the tissue placed in the melted paraffin.
This will set round the cold tissue at once, but soon melts again and
must be kept at a temperature just above melting point for one to
four hours, according to size. The tissue is then transferred to a
mould (which can be easily made of paper), about half an inch cube,
and melted paraffin poured round it until the mould is full. The mould
may be made by folding a piece of paper to form a box about half an
inch cube, or a small pill box may be used. Another convenient method
is to place two L-shaped pieces of lead in contact with each other so
as to enclose a space of suitable size as in the diagram (fig. 3). The
tissue is now hermetically sealed, and can be kept indefinitely if it
is not convenient to cut it at the time. To prepare it for cutting,
all superfluous paraffin is trimmed away with a warm knife, and the
block is fixed on a piece of wood, cut so as to suit the clamp of the
microtome, by melting the lower end of the paraffin block with a hot
needle or wire and pressing it down on the wood.
[Illustration: Fig. 3.]
When sections are cut they may be transferred singly to the slide
(which should be lightly smeared beforehand with a saturated solution
of celloidin in oil of cloves), or they may be cut so that the back of
one section of the paraffin block adheres to the front of the next,
and in this way a continuous delicate ribbon of serial sections is
obtained. The ribbon is broken up into lengths of about two and a half
inches and transferred to the slide, on which several ribbons may be
placed side by side, and so a large number of sections kept in the
order in which they are cut. A mark should be made on the slide to
indicate where the series begins, and each slide should be numbered, so
that the exact position of each section in the series can be recognised
at once.
Before mounting, the paraffin must be removed from the sections. This
is easily done on the slide in the case of single sections and of
ribbons. If the sections are curled, a little warmth will make them
unbend and lie flat. The slide is warmed over a spirit lamp until the
paraffin just melts. The sections will keep their places owing to the
celloidin beneath. Xylol is then allowed to flow over the slide from a
pipette, until the paraffin has been completely dissolved, which can
be ascertained by glancing at the sections under the low power of the
microscope. The slide is placed in an almost vertical position to let
the xylol drain off, excess is wiped off from the edge of the slide
with blotting paper, a drop of Canada balsam solution (p. 61) is run on
the slide, and a cover-glass of suitable size is applied.
*Microtomes.*--After a large amount of practice, persons with a fair
amount of manual dexterity may acquire sufficient skill to be able to
cut very satisfactory sections of specimens embedded in paraffin, &c.,
by hand. In the Pathological Laboratory of a large German University,
until quite recently the use of a microtome was prohibited by the
Professor, who is himself a most distinguished histologist. The amount
of time expended before one acquires the necessary skill, and the
cheapness and great convenience of the modern microtome have combined
to throw hand cutting into the background, and some form of microtome
is now almost universally adopted.
[Illustration: Fig. 4.--Cathcart’s Ether Spray Microtome.
A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising
the zinc plate H. J. Ether bottle. L. Tube from air bellows.]
Of these there are a very large number in the market, each having
special advantages, and often special drawbacks. A few of the more
generally useful only will be described. We have microtomes for cutting
in gum frozen by ether spray or ice, and those intended for cutting in
paraffin or celloidin.
Cathcart’s *ether spray microtome* (fig. 4).--This, or its more recent
modifications (_see_ later), is perhaps the most useful and economical
microtome for the purposes of the student. Its prime cost is low, it is
small and portable as well as being clean and inexpensive to work with.
It consists of an oak frame which can be firmly clamped on to a table.
On this frame are two narrow parallel supports about two inches high,
which are covered by strips of plate glass, and serve as smooth rests
along which the razor may glide in making sections. Between them is
a brass well and in this a zinc plate firmly fixed in the horizontal
position, which is almost at the level of the glass runners. It is
capable of being raised or lowered through about 3/8 inch by means of
a screw with a very fine and accurate thread. This screw is turned
by a large milled wheel beneath the microtome. Just beneath the zinc
plate are two small tubes, one connected with an india-rubber bellows,
the other with a bottle at the side which contains ether. As the air
issues from the first tube, it passes over the open end of the second,
and thus draws the ether out and makes it play on the zinc plate, and
at the same time causes it rapidly to evaporate, and so reduces the
temperature of the zinc plate.
In cutting sections with this microtome the tissue is taken out of the
gum and placed on the zinc plate. The bellows are then worked until the
gum on the zinc plate is completely frozen. The plate should be lowered
by means of the screw until the surface of the piece of tissue is on a
level with the glass runners. These and the razor should then be wetted
with water. The razor being held firmly in the hand is pushed along
the glass runners in a rather oblique direction. The plate should then
be raised by turning the screw below through a very small arc, another
section taken off and so on. Sections are carefully removed from the
razor to a vessel of water by means of a soft wet camel’s hair brush.
The needle should never be used for this purpose.
If the specimen is very delicate, and likely to be spoiled by being
curled up on the knife, the latter should be kept cold by frequently
dipping it in a vessel containing lumps of ice in water. The gum will
then remain frozen after cutting, and support the tissue better. Each
section should be at once transferred to a glass slide from the knife,
washing it off with a stream of ice water from a pipette.
The knife that is used may be an ordinary razor, with the edge ground
straight. It requires to be held steadily with both hands. As this is
rather inconvenient, Dr. Sheridan Délépine suggested the employment
of an ordinary plane iron such as is used in a carpenter’s plane.
This only requires one hand, and the other can be kept on the head of
the screw beneath to raise the plate at once after each stroke of the
knife. Its disadvantages are that it is rather heavy for prolonged
working, and that it is less easy to “set” than a razor.
A. Frazer has recently introduced a valuable improvement in the
Cathcart microtome (fig. 5).
In this the brass frame carrying the zinc plate and ether spray tubes
is surrounded by a brass cylinder, in which it fits accurately, and
is pushed up as desired by turning the screw beneath the instrument.
This brass frame and with it the zinc plate, &c., can be easily drawn
altogether out of the outer tube, and replaced by a second brass
well, which exactly fits its place and can be raised by the screw
as desired. In this is a small toothed clamp which can be screwed up
so as to hold a piece of wood carrying a piece of tissue embedded in
paraffin. Sections can also be cut in celloidin with this instrument,
but as oblique strokes with the knife cannot be made, it is impossible
to get very thin sections. The combined microtome can be obtained for a
guinea from Frazer, 22 Teviot Row, Edinburgh.
[Illustration: Fig. 5.--Frazer’s Modification of Cathcart’s Microtome.
A. Microtome arranged for ether spray. B. Cylinder with clamp for
holding object embedded in celloidin, &c. to replace ether spray
apparatus.]
There is another modification which is more generally useful, and at
the same time more expensive than the original model. In this, instead
of glass runners to support the knife, there is a flat glass plate
about eight inches square sufficiently large to allow of “Swift’s
plough” (fig. 6) being used for the purpose of cutting sections. This
instrument consists of a triangular brass frame, supported on three
legs, each of which is a screw, tipped with ivory. There is one screw
in front and two behind. Beneath the plate, and held in position by
the posterior screws in front, and a little clamp behind, is a razor
with the edge directed forwards. The edge can be raised or depressed by
turning the anterior screw, on which the frame is supported. Before
sections are cut the edge of the razor should be brought down to the
level of the tissue, taking care that all the legs are equal in length.
The plough should then be firmly grasped with both hands, (the index
finger of one hand being left free to turn the anterior screw) and
pushed rather obliquely through the tissue. The edge of the razor is
then slightly lowered by turning the screw through a very small angle,
and another section made, and so on. With a little practice very thin
uniform sections may be made with great rapidity.
Another useful ether spray microtome is that made by Jung of
Heidelburg. The knife swings round a pivot, and there is an ingenious
ratchet arrangement which works synchronously with each swing of the
knife, to raise the tissue automatically the requisite distance for
the next section to be made. The exact thickness of the sections can
be graduated with great nicety by a simple contrivance. The instrument
can be obtained in this country for about £2. It works satisfactorily,
but, with practice, the student will get equally good results with the
cheaper “Cathcart.”
*Williams’ ice freezing microtome* (fig. 6).
This consists of a round mahogany water-tight box provided with an exit
tube below, and covered with a strong plate glass lid. Firmly fixed in
the centre of the floor of the box is a stout brass pillar surmounted
by a brass disc which fits into a hole in the centre of the glass lid,
so that its surface is on a level with that of the lid.
[Illustration: Fig. 6.--Williams’ Ice Freezing Microtome, with Swift’s
plough.]
To use it, the box is filled with alternate layers of pounded ice and
salt; the lid is then put on and fixed by means of a lateral screw.
The tissue to be frozen is gently removed from the gum and placed on
the brass disc and plenty of gum painted round it. It should then be
covered with a tin cap for a few minutes until frozen. Sections are
made with a Swift’s Plough (p. 44).
[Illustration: Fig. 7.--Schanze Microtome (see text).]
*Schanze microtome* (fig. 7) is the pattern used in the Leipsic
laboratories. It consists of a heavy iron frame with a large base. The
knife is carried in a clamp which slides along the full length of the
instrument, gliding upon two smooth plates of iron which are arranged
at an angle to one another. The knife must be moved very steadily
and gently, as when using a long blade vibrations are easily set up
which prevent good sections being obtained. The surfaces of contact
must be kept scrupulously free from dust, and lubricated with equal
parts of olive oil and castor oil. There are several object holders,
which can be removed and interchanged, one connected with an ether
spray apparatus, another suitable for holding an object embedded in
paraffin, and a third for grasping an object embedded in celloidin.
When celloidin is employed, a specially long knife must be used, and
it must be fixed very obliquely in the clamp. The object holder is
raised by a fine screw worked by a large brass toothed wheel. There is
a ratchet arrangement, by which the object may be raised automatically
any desired distance, after each stroke of the knife. It gives most
satisfactory results with celloidin and paraffin. (Messrs. R. and J.
Beck are the agents). Its cost is about £5.
*Becker’s microtome* is made on exactly the same principles as the
Schanze. The modifications are that the carrier glides on glass plates
instead of iron ones, and that instead of the whole surface of the
carrier being in contact with the plates, there are a few smooth ivory
buttons only. Friction is thus reduced to a minimum, and very uniform
sections can be obtained. The price is the same as that of the Schanze.
Frazer has introduced a “student’s sliding microtome” on the same
principle as the Schanze which costs about £3.
*The Cambridge Rocking microtome.*--This instrument, as made by the
Cambridge Scientific Instrument Company, or the slightly modified form
made by Messrs. Swift (fig. 8), is the best instrument for cutting
sections of small objects embedded in paraffin. Ribbons of serial
sections can be obtained from it with greater ease and certainty than
with other microtomes. This microtome differs from those which have
been previously described in that the knife is fixed, while the
object is moveable. The microtome consists of an oblong heavy metal
stand. A long bar is arranged so that it rides in see-saw fashion on
two strong vertical pillars arising from the frame. One end of this bar
is hollow, and receives the piece of wood carrying the tissue embedded
in paraffin, which is firmly clamped in position. This end is depressed
by means of a strong spiral spring. In order to raise it there is an
arrangement by which the other end of the bar is depressed by a cord
which revolves round a pulley. When the handle is turned, the tissue
is raised, and when the cord is relaxed, the spring pulls the tissue
firmly and steadily down. The razor, which must have a straight edge,
is fixed firmly by screws, with its edge upwards at the end of the
microtome. The object is then adjusted so that in its descent a thin
slice is taken off by the razor. There is an ingenious arrangement
by which the depression of the bar to raise the section pushes it a
little further in the direction of the razor. The distance can be
graduated from 1/500 to 1/3000 inch. The actual working of the machine
is therefore very simple. The position of the block containing the
tissue to be cut having been adjusted so that the razor just cuts it,
the free end is depressed by means of the pulley. This also pushes the
section a little beyond the razor. The strong spring then draws the
tissue steadily past the edge of the razor, and a thin section is left
on the blade. This may be at once transferred to a slide, or if the
paraffin be of the proper consistence, another cut may be made, when
the two sections should adhere by their edges, and so by repeating the
movement a continuous ribbon may be obtained. If there is difficulty in
obtaining a good ribbon, it will usually be got over by taking a little
soft paraffin and attaching it by means of a hot needle to the lower
end of the paraffin block. The cost of the instrument is about £5.
[Illustration: Fig. 8.--Swift’s Modification of the Cambridge Rocking
Microtome.]
*Fresh sections.*--Although these are not so satisfactory as hardened
specimens for accurate histological work, it is often very useful to
make them both in the post-mortem room where an immediate opinion of
the nature of the tumour or diseased organ is desired, and also in
the operating theatre. With a little practice sections may be cut,
stained, and mounted, within ten minutes of the removal of the specimen
from the body. In this way important information may be afforded to the
operating surgeon, and in not a few cases it has caused the proposed
treatment to be entirely altered. Thus, in one case, a supposed chronic
periostitis was shown to be a sarcoma, and the limb was amputated. In
another, a supposed sarcoma of the thigh was found to be a gumma, when
a portion was removed and microscopically examined.
A portion of the specimen should be placed without any preparation on
the zinc plate of the freezing microtome, and some gum painted round
it. It is then frozen. The serum in the tissues is not in sufficient
mass to injure the knife when it is frozen. The knife should be wetted
with, and sections transferred to, either pericardial serum, or 3/4
per cent. solution (70 grains to the pint), of common salt, neither
of which causes the cells to swell up as plain water does. They
should be carefully floated out on a glass slide, an operation which
requires much more patience than in the case of hardened sections, as
fresh sections are less coherent and also more sticky, so that the
edges tend to curl up on the knife, &c. They should then be examined,
one unstained, simply mounted in salt solution; another stained with
picrocarmine and examined in the saline solution; and a third stained
in picrocarmine, mounted in Farrant’s solution, and preserved. The last
usually gives the best results, the picrocarmine staining becoming
quite brilliant after a week. The glycerine, however, is apt to make
the sections shrink a good deal, and the weight of the cover-glass
tends to break up the unhardened section.