1. _Introduction._—Many of the most interesting objects with which the
microscopist has to deal, cannot be made to reveal their beauty or
minute structure until they have been cut into slices or _sections_, of
such a degree of thinness as to render them transparent, and thus permit
of their examination by transmitted light, with objectives of varying
power. Unfortunately, however, very few of the objects of this class
are, in their natural condition, in a suitable state to be submitted to
this method of procedure. Some are of such a soft and yielding nature
that any attempt to cut them is an utter failure, for in place of a
perfect section being obtained, nothing remains upon the knife but a
mass of diffluent pulp; others, again, are of such density as to resist
the action of any cutting instrument.
It is evident, therefore, that nothing can be done with such refractory
materials until, by subjection to appropriate methods of preparation,
they shall have been reduced to such a consistence as to render them
suitable for cutting. How this is to be accomplished will depend
entirely upon the physical and chemical nature of the substance to be
operated upon. As the various objects differ so widely from each other
in these respects, so must the methods of preparation suitable to each
also vary. It is clear, therefore, that no general directions for
attaining this end can be given which would be of any practical value.
It is possible, however, and very convenient, to arrange the various
objects into groups or classes, to the treatment of each of which
certain general rules are applicable; but there will still remain a
comparatively numerous series of objects whose individual peculiarities
of structure will demand for them correspondingly special methods of
preparation. When such objects come to be spoken of, the particular
treatment most suitable to each will also be noticed.
2. _On Cutting unprepared Vegetable Tissues._—There are some few
substances, however, which may with more or less success be cut into
sections whilst in their natural condition. Such objects are to be found
in the vegetable world in certain kinds of leaves and allied structures,
whilst in the animal kingdom they are principally represented by the
various internal organs of man and the lower animals. Special directions
are given in text-books for the preparation of sections of leaves and
similar substances. For instance, it is recommended to lay the leaf,
etc., on a piece of fine cork, and with a sharp knife to shave off thin
slices, cutting down upon the cork. Another plan is to place the leaf,
etc., between two thin layers of cork, and cut through the mass. No
method, however, is at once so simple and successful as the process of
imbedding in paraffine. To do this, it is necessary to make a paper
mould by twisting a strip of stout writing paper round a ruler, and
turning-in the paper over the end of the ruler. This mould, the height
of which may vary from an inch to an inch and a half, should now be
about half filled with melted paraffine mixture (§ 11), the leaf or
other object plunged into it, and held in position by small forceps till
the paraffine has become sufficiently solidified to yield it a support.
More of the paraffine mixture is now poured in until the specimen is
thoroughly imbedded; the whole is to be put away in a cold place for an
hour or so, when the mass will be found sufficiently firm to be cut with
ease. Sections may be made with a razor kept constantly wetted with
water, or, if the preservation of colour be no object, methylated spirit
may be employed for the purpose. As the subsequent treatment of such
sections in no wise differs from that required by those cut in the
microtome, we shall defer its consideration until that method of section
has been described (§ 12).
3. _On Cutting unprepared Animal Tissues._—For the cutting of fresh
_animal_ tissues several plans may be followed. Thus, if a section of
only very limited area be required, it may be obtained by snipping a
piece off the tissue with a pair of bent scissors, which, for this
purpose, are so made that the blades are _curved on the flat_
(Carpenter). If this be carefully performed it will be found that a
large portion of the section (particularly at the circumference) so
obtained will be sufficiently thin for examination. If a larger section
be desired, an attempt may be made to cut it with a very sharp scalpel
or razor, the blade of which whilst in use must be kept _flooded_ with
water, or spirit, the latter of which is to be preferred. Recourse may
also be had to _Valentin’s_ knife. This consists of two long, narrow
blades, running parallel to each other, the distance at which the blades
are held apart, and which, of course, determines the thickness of the
section, being regulated by means of a fine screw passing through both
blades. A milled head attached to this screw gives a ready means of
opening or closing the blades, so as to bring them to the desired degree
of approximation. The method of using the knife is very simple. After
having “set” the blades at the desired distance apart by means of the
milled head, the tissue to be cut is held in the left hand immersed in a
basin of water. The knife is now steadily and with a rapid motion
_drawn_ through the tissue, care being taken that the cut is made in
such a manner that the blades move from heel to point. By slightly
separating the blades and gently shaking them in the water, the section
at once becomes disengaged. After use, the blades must be thoroughly
dried, when they may be smeared with some oil which does not readily
oxidize. For this purpose, a very suitable oil is that known as
“Rangoon.”[1] Though it has been deemed advisable briefly to describe
the preceding methods of cutting unhardened tissues, it will be found
that for the purposes of the ordinary microscopical student sections so
obtained are of very little value. They are always of very limited
dimensions, seldom of uniform thickness, and often so extremely friable
as to render it very difficult and frequently impossible to submit them
with safety to such further treatment as is necessary to fit them for
being mounted as permanent objects. This method of section-cutting,
however, is not without its uses, for by its means the medical
practitioner is provided with a simple and ready method of roughly
investigating the structure of morbid tissues, whilst to the general
student it furnishes an easy means of making a cursory examination of
certain substances, in order that he may determine whether it be worth
his while to subject them to some of those various processes of
hardening hereafter to be described.
Footnote 1:
Note A.
4. _Preparation of Vegetable Tissues._—Let us now pass to a brief
consideration of the methods usually adopted for preparing the various
objects for easy section. In the case of _vegetable_ tissues, not only
do we, as a rule, find their texture of too great density to be readily
cut in their natural condition, but they also contain much resinous and
starchy matter, of which it is highly desirable to get rid. In order to
do this we first cut the substance (say a stem or root) into small
pieces, which are to be placed in water for three or four days, by which
time all the soluble gummy matters will have disappeared. The pieces are
now transferred to a wide-necked bottle, containing methylated
spirit,[2] which, in the course of a few days, will dissolve out all the
resin, etc. Many kinds of woody tissue are by these processes reduced to
a fit condition for immediate cutting; others, however, are so hard as
to render it necessary to give them another soaking for some hours in
water, to bring them to a sufficient degree of softness to cut easily.
If the wood (as in some few refractory cases will happen) be still too
hard for section, a short immersion in warm, or if necessary, in boiling
water, will not fail effectually to soften it. The treatment of such
members of the vegetable division as require peculiar methods, will be
found described in future pages.
Footnote 2:
Note B.
5. _Preparation of Animal Tissues._—_Animal_ tissues differ from one
another so greatly, both in consistence and in chemical composition, as
well as in their degree of natural hardness, that no general rules can
be given which would be applicable to the preparation of the whole
class. Such as are of any considerable degree of hardness, as horn and
kindred structures, must be treated much in the same manner as the
denser varieties of wood, viz., by more or less prolonged immersion in
water—cold, hot, or boiling. Those which are of extreme hardness, as
bones and teeth, can be cut only by following certain special methods,
full details of which will be found in the Second Part of this work (§
26). Many, and indeed the vast majority of animal tissues, offer a
direct contrast in point of hardness to those we have just been
considering. All the internal organs of the body are, when freshly
removed, of much too soft a nature to permit, when in their unprepared
condition, of easy or perfect cutting. It is upon bringing them to that
critical degree of hardness, which is often so difficult to attain, that
the chief secret of successful section-cutting depends; for unless the
hardening process has been carried up to, but not beyond, a given point,
which varies with different tissues, the operator, however dexterous,
will fail to obtain satisfactory sections. For, if the hardening has
fallen short of this critical point, he is, to some extent, in the same
position as if he were dealing with unhardened tissues; whilst, if this
point has been exceeded, the tissue will have become so brittle as to
crumble before the knife. For the purpose of hardening animal tissues,
the student has at his command two principal agents, namely, alcohol and
chromic acid, each of which possesses advantages of its own, but the use
of each of which is also attended by its own inconveniences. Thus, by
the use of alcohol, there is very much less risk of overhardening the
specimen than if chromic acid had been employed. Alcohol, however,
though a capital indurating agent in some instances, does not answer so
well in many others. Chromic acid is, therefore, to be preferred for
general use. It is, however, a very delicate agent to manage, for unless
the greatest care be taken it is exceedingly likely to overharden
tissues submitted to its action, and when this happens the specimen
becomes utterly useless for cutting, as there is no known means of
removing the extreme brittleness which it has acquired. By taking the
precautions now to be given, this overhardening may generally be
avoided. Let us harden a portion of some viscus, say the kidney, for
instance. Suppose we cut from the organ five or six small pieces (from
half to three-quarters of an inch square, _not larger_). These must be
placed in a mixture of equal parts of methylated spirit and water for
three days, at the end of which period they may be transferred to a
solution of chromic acid, made by dissolving twenty grains of the pure
acid in sixteen ounces of distilled water. The solution should be kept
in a wide-necked bottle furnished with a glass stopper. At the
expiration of seven days, pour off the solution and replace it by fresh.
At the end of another week, carefully examine the immersed tissues, and
by means of a sharp razor see if they have acquired the necessary degree
of hardness to allow of a section of _moderate_ thinness being made. If
so, remove the pieces and put them into a stoppered bottle containing
from six to eight ounces of methylated spirit. If, however, the
hardening be found not to be sufficiently advanced, the chromic acid
solution is to be poured off and again replaced by fresh. It will now be
necessary to examine the tissues at intervals of about two days, until
they are found to be sufficiently hard, when they must be transferred to
the spirit. Under no circumstances, however, should they be permitted to
remain in the chromic acid longer than the end of the third week, and
though they should at this time appear not to have undergone sufficient
induration, yet it will be advisable to transfer them to the methylated
spirit, which in a short time will _safely_ complete the process of
hardening, without any risk being run of the tissue becoming ruinously
brittle. It will be noticed, that when the specimens have been
transferred to spirit, the latter will in a day or two become of a deep
yellow color, whilst a thick flocculent deposit falls to the bottom of
the bottle. The tissues should then be removed, the bottle emptied and
well washed, and, being refilled with clean spirit, the preparations are
again to be replaced. This may occasionally be repeated, until the
spirit becomes and remains perfectly bright and clear. The specimens are
then ready for section.
6. _Special Methods of Hardening._—The brain (§ 27), spinal cord (§ 43),
liver (§ 34), and several other organs, etc., require special methods of
hardening, details of which will be found in the paragraph devoted to
each. In the case of _injected_ preparations, the best plan is to harden
them in alcohol from the outset, beginning with weak spirit, and
gradually increasing the strength as the hardening proceeds. When the
object has been injected with Prussian blue, a few drops of hydrochloric
acid should be added to the alcohol to fix the color.
It may here be observed, that specimens of _morbid tissues_ require, as
a general rule, a shorter immersion in chromic acid solution than
healthy tissues do. A very small degree of overhardening speedily
renders them brittle and useless. They should, therefore, be removed
from the acid medium at the end of ten days or a fortnight, and their
further hardening carried on by means of alcohol.
7. _Cutting Hardened Tissues by Hand._—Our material being now reduced to
a fit condition for cutting, let us proceed to consider the several
methods by which this may be effected. The readiest and most simple
plan, if the piece be large enough, is to hold it in the left hand, and,
having brought the surface to a perfect level by cutting off several
rather thick slices, endeavor to cut a thin section by the aid of a very
sharp razor, the blade of which must be kept well _flooded_ with spirit.
As in the use of _Valentin’s_ knife, so here, great care must be taken
steadily to _draw_ the blade across the tissue, every effort being made
to avoid _pushing_ the knife, else the section will be _torn off_,
instead of being _cut_. Though this method[3] is of very great
importance for many purposes, yet a considerable degree of manipulative
skill is required to enable the operator to obtain anything like perfect
sections by its means, and, unfortunately, this skill is acquired by
very few persons indeed, even after much practice. If the piece which it
is desired to cut be too small to be conveniently held in the hand, it
may be imbedded in paraffine in the manner already described (§ 3). A
very simple imbedding agent, and one of the greatest practical value, is
a strong solution of gum arabic, which, upon being dehydrated either by
ordinary drying or the action of alcohol, soon acquires such a degree of
hardness as to permit it (with the imbedded tissue) to be cut with ease.
As this method of imbedding, however, is most frequently resorted to
where, by its means, special difficulties have to be overcome, a full
description of the process (§ 35) will be deferred until such special
cases come to be spoken of.
Footnote 3:
Note C.
8. _Microtome._—Although the preceding plans may be sufficient to answer
all his requirements, if the student wishes to obtain only one or two
sections of small dimensions, of a given object, if he requires a number
of such sections he will find these methods fail him, for even though by
practice he may have attained to considerable aptitude in the use of the
knife, it will still unquestionably happen that the vast majority of his
sections will be more or less imperfect. If, therefore, it be desired to
procure a number of perfect sections, of equable thickness and large
area, it is absolutely necessary to resort to the use of some form or
other of microtome, or section-cutter. This instrument, in its simplest
form, merely consists of a stout brass tube closed at one end, and being
by the other fixed at right angles into a smooth plate of metal. A plug
or disk of brass, accurately fitting the interior of the tube, is acted
upon by a fine threaded screw piercing the base of the tube, and by
means of which the plug, and any object it may support, can be elevated
at pleasure. The object by this means being made gradually to rise out
of the tube, sections are cut from it by simply gliding a sharp knife
along the smooth cutting-plate, and hence across the specimen. Any
intelligent worker in brass would make an instrument of this kind at a
very small cost, and although perhaps it might lack the finish of an
instrument bought at the optician’s, it would, if accurately made, do
its work as well as the most complicated and expensive. If, however, the
student resolves to purchase a microtome, there are a variety of forms
in the market from which he may choose. A few hints may perhaps be of
service in enabling him to make a judicious selection. At the outset we
may say that unless the student intends to devote himself solely to the
production of sections of wood, etc., he ought not to procure one of
those forms of microtome known as wood section-cutters, in which the
object to be cut is held in position in the tube by means of a binding
screw which pierces its side.[4] Although these machines are all that
can be desired for cutting hard bodies, they are not so suitable for
soft ones. The chief points to be attended to in selecting a microtome
are, (1) that the cutting-plate of the instrument be made of glass, or
in default of this, of very hard metal of the most perfect
smoothness;[5] (2) that the diameter of the tube be neither too large
nor too small—it ought not to be less than 5/8-in., or greater than 1
inch;[6] (3) that the screw, which should be _fine_ and well cut, be
provided with a graduated head; (4) that there be some kind of index by
which fractional portions of a revolution of the screw may be measured;
and (5) that the plug fit the tube of the microtome so accurately that
when melted paraffine, gum, or other imbedding agent be poured into it,
it may not find its way between the plug and side of the tube (§ 18). It
often happens in cutting tissues imbedded in paraffine, that the
pressure of the knife causes the cylinder of the imbedding agent to
twist round in the tube of the machine, and so cause considerable
difficulty and annoyance. This evil is usually met by running a deep
groove across the upper surface of the plug, and into this the paraffine
sinks, and so is prevented from rotating. It will be found, moreover,
that another difficulty of a kindred, though much more serious
character, will frequently be encountered. During section the paraffine
has a tendency not only to rotate, but also to become loosened from the
subjacent plug, and to _rise_ in the tube of the microtome. When this
happens the power to cut sections of uniform thickness has completely
gone, for some will now be found to be many times thicker than others;
in fact, the irregularity in this respect soon becomes so monstrous as
to render it useless to prolong the sitting. In the ordinary run of
microtomes no provision seems to have been made to meet this difficulty,
and for this reason many instruments, of otherwise great merit, have
their efficiency seriously impaired. Fortunately, this imperfection is
easily remedied, all that is required being that the upper surface of
the plug should be furnished with some kind of projection, having at its
summit a table-like expansion, as shown at A in the figure. The
imbedding paraffine, by penetrating beneath and around this, becomes
firmly attached to the plug, and thus all risk of its rising is
effectually avoided. If the student wishes to secure a really
first-class instrument, none can be so confidently recommended as the
freezing microtome of Professor Rutherford. In addition to its being the
best instrument for carrying out the freezing method (§ 18), this
machine is equally effective for cutting tissues imbedded in paraffine,
or any of the other agents used for that purpose; indeed, whatever work
a microtome _can_ do, _this one_ will perform.
Footnote 4:
Note D.
Footnote 5:
Note E.
Footnote 6:
Note F.
[Illustration: SECTION OF MICROTOME-TUBE SHOWING ARRANGEMENT (A) TO
PREVENT “RISE” OF PARAFFINE.]
9. _Æther Microtome._—A word here as to freezing microtomes, where the
agent used is æther. Such as have fallen under our notice have not
answered the expectations we were justly entitled to form of them. That
it is possible to freeze a piece of tissue by their use is undeniable,
but it is, as a rule, at an expenditure of such a quantity of æther
(only the very best of which must be used) as to constitute it a very
expensive proceeding. Another serious disadvantage they possess is, that
if the supply of æther be intermitted for only a very short time, the
already frozen tissue thaws with great and most inconvenient rapidity.
10. _Section-Knife._—Of not less importance than the microtome is the
section-knife, to be used in conjunction with it. How perfect soever the
former, and whatever the dexterity of the operator, unless he be
provided with a suitable and well-made knife, he will never succeed in
obtaining satisfactory results. As to the most desirable _size_ of the
knife, much difference of opinion seems to exist, section knives varying
in this respect from a blade of extreme shortness to one which fell
under our observation, in which the portentous length of _thirteen_
inches was attained. What advantages were to be expected by prolonging
the blade to this extravagant length, must remain an inscrutable mystery
to all save its designer. Concerning the _shape_ of the knife, it is
frequently advised that the surface which has to glide along the
cutting-plate of the microtome should be ground _flat_. A most
unsuitable arrangement, as a very little actual experience of
section-cutting will speedily demonstrate. After many unsuccessful
attempts to obtain a really good and reliable section-knife, we
determined to have one specially made, which, as it has proved
everything that could be desired, merits a brief description. It is of
the utmost importance that the blade be made of good and well-tempered
steel, not only that it may be capable of receiving an edge of the most
exquisite keenness, but also that it may _retain it_. The knife of which
we speak (and which was made by Mr. Gardner, of South Bridge, Edinburgh)
is furnished with a blade _four inches_ long, and 7/8-inch broad, set
into a square handle of boxwood, also four inches in length. The
thickness of the blade at the back is not quite 1/4-inch, while _both_
of its surfaces are slightly hollow ground. It is essentially necessary
that the back and edge of the blade be strictly parallel to each other,
otherwise the knife, when in use, will have such a tendency to tilt over
as to render its management extremely difficult. It is very easy to
discover if this condition be fulfilled, for if on carefully laying the
flat of the blade upon a piece of level glass, every portion of both
back and edge are found to be in close contact with it, the knife may in
this respect be considered perfect. Every student who aspires to be a
successful section-cutter should provide himself with a good Turkey
oilstone, _and learn to use it_. He should also possess a razor strop,
as it will be in constant requisition.[7] It may here be remarked that
though _razors_, as a rule, are unsuitable for use with the microtome
from want of uniformity in the thickness of their blades, yet, if only a
small object is to be cut—for instance, a thin root or stem—very good
results may be obtained from their use, especially if one of the
old-fashioned make, having a thick back and slightly _concave_ surfaces,
be employed.
11. _Imbedding in Paraffine for Microtome._—Having described at some
length the various instruments necessary for section-cutting, we will
now consider how they are to be used. Let us endeavor to cut some
sections—say of a piece of kidney—and in so doing we will adopt the
“paraffine” method of imbedding. Ordinary paraffine, however, when used
alone, is rather too hard for our purpose. In order, therefore, to bring
it to a suitable consistence, it must be mixed with one-fifth its weight
of common unsalted lard, a gentle heat applied, and the two thoroughly
stirred together. A quantity of this should be prepared, so that it may
always be ready when wanted—it is very conveniently kept in an ointment
pot or preserve jar, the top of the latter being well covered, to keep
out the dust. When it is intended to use this mixture for the purpose of
imbedding, only just about the quantity required should be melted; for
in doing this it is advisable to use as low a degree of heat as
possible, not only to prevent injury to the tissue to be imbedded, but
also that the paraffine when cooling may not undergo such an amount of
contraction as to cause it to shrink from the sides of the
microtome-tube. It is therefore a good plan to effect the melting in a
water-bath, a simple kind of which, something after the fashion of a
glue-pot, would be made for a few pence by any tinman.
Footnote 7:
See Note G.
The kidney which we are about to cut has, of course, gone through the
process of hardening already described (§ 5), and is now preserved in
spirit. A small piece, say half an inch square, is selected, removed
with forceps, and placed on a bit of blotting paper, when the surface of
the tissue will rapidly become dry (_only the surface_ must be allowed
to dry). It is the usual plan now to proceed at once to imbed it in the
melted paraffine. This is a most undesirable step, and gives rise at a
later stage of our proceedings to a great amount of trouble and
annoyance, for after sections have been cut from a tissue so imbedded it
will be found that portions of paraffine adhere to their edges with such
tenacity that in the case of many of them there is no effectual method
of removing the paraffine, short of soaking the sections in warm æther;
a very objectionable proceeding, for though the æther will undoubtedly
remove the paraffine, it will also dissolve out any fatty matters which
the section itself may naturally contain. All this annoyance may be
prevented by subjecting the tissue to a simple preparatory treatment
before it is imbedded in the paraffine. For this purpose prepare a very
_weak_ solution of gum arabic in water—twenty grains to the ounce. Into
this, by means of the forceps, dip for a few moments the already
_surface-dried_ tissue, taking special care not to squeeze it, or the
pressure will cause the spirit from its interior to remoisten the
surface, which would prevent the gum from adhering. We shall see the
value of this a little later on. Remove the tissue from the solution on
to blotting paper, when the superfluous gum will speedily drain off, and
in two or three minutes the _surface_ will have become quite glazy and
dry. Having melted some paraffine mixture in the water-bath, the tissue
held in the forceps must be plunged for an instant into the heated
liquid and immediately withdrawn, when the crust of paraffine with which
it is enveloped will promptly harden. Whilst this is taking place we may
make ready the microtome. Having by means of the milled head or handle
depressed the plug in the tube so as to leave a free opening about an
inch deep at its upper end, we must pour in the melted paraffine, which
by this time will have become a little cooler, until the cavity be about
half filled. The prepared tissue must now be introduced, care being
taken to place it in such a position that the sections may be cut in the
desired direction. The tissue must, if necessary, be held in position
with forceps or a needle point, till the imbedding material becomes hard
enough to give it due support. It is here to be remembered that it will
not be advisable to place the tissue in the centre of the tube—it will
be much more easily cut if placed rather nearer to that edge of the tube
which is situated next the operator in the act of cutting. More
paraffine is to be slowly added, until the tissue is completely covered;
even after this still more should be added, for it will be found that in
cooling the paraffine shrinks so as to leave a cup-shaped depression in
its centre, whereby portions of the tissue which were previously covered
are again laid bare. The best method of preventing this is to use the
paraffine at as low a temperature as possible, and to use plenty of it.
The microtome, with its contents, must now be removed to a cool place,
when the paraffine will soon become solidified. Whilst this is being
accomplished we may make our further preparations. The first thing we
require will be a large basin, full of freshly-filtered water, and
provided with a cover. A small beaker of methylated spirit, with a
dipping rod or pipette, will also be necessary. We must now see that the
section-knife is in thorough order, to ensure which it will be advisable
to give it a few turns on the strop. An ordinary razor will also be of
service.
12. _Employment of Microtome._—The paraffine being sufficiently hard, we
will clamp the microtome on to the table, and seat ourselves on a chair
of convenient height before it. To our right stand the basin of water,
razor, and section-knife; the beaker of spirit to the left, and a cloth
on our knee. A few turns of the microtome screw having brought the
paraffine to the surface, a thick slice is to be cut off, and this
repeated until the imbedded tissue comes into view. This preliminary
work had best be done with the razor, as it is needless to subject our
section-knife to unnecessary wear and tear. By a fractional revolution
of the screw the tissue is now slightly elevated, and with the pipette
held in the left hand, a large drop of spirit is to be let fall upon its
surface. The section-knife, grasped firmly but lightly in the right
hand, is to be laid flat upon the cutting-plate of the machine, so as to
occupy the diagonal position shown in the figure. Two fingers of the
left hand are now laid gently upon the back of the blade, so as to give
it an equable support, whilst the knife with a rapid motion is pushed in
the combined direction of _forwards_ and to the _left_, so that the
blade in cutting the tissue will pass through it from point to heel.
Thus it will be observed that the stroke of the knife is _from_ the
operator—a far easier and more effective mode of cutting than the
reverse plan. The blade of the knife, having the section just cut,
either floating in a small pool of spirit on its surface or adhering
thereto, must now be immersed in the basin of water, when by a little
very gentle agitation of the knife the section will be floated off. And
now we shall find the great practical value of immersing the tissue in
gum before imbedding, for no sooner is the section disengaged from the
knife than the thin film of gum which separates the paraffine from it
becomes dissolved, and the section will be observed gradually to subside
to the bottom, leaving the paraffine floating upon the surface. After
carefully wiping the knife from all shreds of paraffine, the microtome
screw must again be partially revolved, more spirit applied to the
tissue, and another section being cut, it must be transferred to the
water as before, and so on, until a sufficient number of sections have
been obtained. As to how thin the sections should be cut, no general
directions can be given; each case must be regulated by its own
conditions. The denser the tissue, the thinner should the section be;
whilst certain substances of loose and spongy texture do not require the
sections to be particularly thin—it may be said, however, in a general
way that sections, and especially animal ones, _cannot be cut too thin_
so long as they remain perfect and entire. If Professor Rutherford’s
microtome (as made by Gardner) be employed, the head of the screw will
be found to be graduated into divisions of slightly unequal value; the
sections will therefore be marked by corresponding variations of
thickness, so that amongst a number cut, there must be many of the exact
thickness to meet the requirements of any individual case.
[Illustration: DIAGRAM SHOWING DIAGONAL POSITION OF KNIFE IN
COMMENCING TO MAKE A SECTION.]
13. _Staining Agents._—Before proceeding to mount the sections which
have just been cut, it will be very advisable that they should be
submitted to the action of some staining fluid, in order to render more
clear and distinct their minute structure. Organic substances possess
the property of being able to absorb various colouring matters from
their solution, and to incorporate such colour into their own texture.
This power of attraction is not, however, possessed by all substances
indiscriminately, or to an equal extent. Some possess it in a high
degree, while others appear to be nearly, if not entirely, devoid of
such power. Hence it follows, that if we immerse an organic tissue (one
of our sections, for instance) of complex structure, in a suitable
staining fluid, the tissue will not become stained in an even and
uniform manner throughout, but the several portions of it will receive
varying depths of colour in accordance with the varying attractive power
of its several constituents. By this means we are enabled in stained
sections to discriminate by their difference of shade, minute and
delicate structures, which in the unstained condition it would be
difficult and often impossible to differentiate. For the purpose of
section-staining there are many agents in use, the most generally
suitable being carmine, logwood (§ 19), and aniline blue (§ 27); whilst
for special purposes chloride of gold (§ 28), pyrogallate of iron (§
28), and several others are all of much value.
14. _Carmine Staining._—In the case of animal sections, carmine is, as a
rule, to be selected, giving as it does most satisfactory and beautiful
results. Tissues may be stained with carmine by two different plans: in
the first, a strong solution is used, and the tissue subjected to its
action for a very short period only, whilst in the latter only very weak
solutions are employed, the time of immersion being considerably
prolonged. The rapid method, however, is not to be recommended, for the
strong carmine acts so powerfully upon the tissue as to give the various
elements comprising it no time, as it were, to exercise their power of
quantitive selection, but involves the whole in one uniform degree of
shadeless colour. By adopting the gradual method much better results are
obtained, each portion of the tissue being now at liberty to acquire its
own particular shade. Amongst the various formulæ for the preparation of
carmine fluid, none can be so safely followed as that devised by Dr.
Lionel Beale. It runs thus:—Place ten grains of the finest carmine in a
test tube, add thirty minims of strong liquor ammonia, boil, add two
ounces of distilled water, and filter; then add two ounces of glycerine,
and half an ounce of rectified spirit—this solution ought to be kept in
a well stoppered bottle. The best vessels in which to stain sections are
small jars of white porcelain, capable of holding about two fluid
ounces, and furnished with lids—they are much preferable to beakers or
watch glasses, for owing to the white background which they afford it is
very easy to watch how the staining is proceeding. The carmine solution
which we have just described is both too strong and of too great density
to be used in its pure state. It will, therefore, require to be diluted
with distilled water before use—the most useful degree of dilution being
attained by adding one part of stain to seven of water. Sections may be
placed in this solution for twenty-four hours, in which time they will
usually be found to have acquired a sufficient depth of colour. If,
however, the tissue be unusually difficult to stain, the time of
immersion may be doubled, or still further prolonged, without detriment
to the section.
[Illustration: SECTION SPOON.]
Having prepared and filtered some of this dilute solution, say an ounce,
let us proceed to stain with it those sections which we left in the
basin of water (§ 12). Here we are at once met by a practical
difficulty. How are the sections to be transferred from one vessel to
the other? This is ordinarily effected by means of a soft camel’s-hair
pencil. It is a method, however, open to grave objections, for the
sections so curl around the brush, and get entangled amidst its hairs,
that, notwithstanding every care, valuable sections not unfrequently
become torn during transit. Every difficulty at once vanishes if we
substitute for the brush a small implement, which any one can readily
make for himself. All that is necessary is to take a strip of
German-silver, or copper, of the thickness of stout cardboard, and about
seven inches in length by five-eighths of an inch in breadth. The sharp
angles are to be filed off and the edges carefully smoothed, whilst at a
distance of five-eighths of an inch from each extremity the end must be
turned up so as to form an angle of about 35°. One end must be left
plain, whilst the other, with the aid of a punch or drill, is to be
pierced with five holes about the thickness of a stocking needle[8] (see
Figure). If we now dip the perforated end of this spoon into the water
containing the sections, and gently agitate it, the sections will rise
from the bottom and float about. The spoon is now brought under one of
them, and being steadily lifted up the water flows downwards through its
apertures, and the section smoothly spreading itself out upon the spoon,
may be gently lifted out of the water, and on the spoon being dipped
into the staining fluid the section at once floats off. By this simple
means sections, however large, thin or delicate, may with ease be
conveyed from one fluid to another, with the utmost certainty of their
not being injured during the process. The sections having been in the
carmine fluid for about twenty-four hours, as much of the liquor as is
possible must be gently poured off, and its place supplied by a
freshly-filtered mixture of five drops of glacial-acetic acid to one
ounce of water, when in a few moments the carmine will become
permanently _fixed_ in the tissue, and the process of staining be
complete.
Footnote 8:
Dr. Klein describes a kind of “lifter,” made by bending some
German-silver wire, but as no drawing accompanies his description, it
is not easy to form a clear idea as to the form of this instrument. In
the recent and philosophical work of Schäfer, a lifter is figured,
which consists of a wire stem, having attached to its end a spade-like
blade. It will be observed that the spoon described in the text
differs from this lifter in having one end perforated, and in this
consists the real value of the implement.
15. _Mounting Media._—The further treatment of the stained sections will
entirely depend upon the nature of the medium in which it is intended to
mount them. There are a variety of fluids in use for this purpose, the
principal being dilute alcohol (§ 26), dammar, or Canada balsam (§
22-23), and glycerine. These, however, cannot be used indiscriminately,
each possessing certain special properties which render it suitable for
use with particular classes of objects only. Thus, weak spirit, having
no tendency to increase the transparency of objects, can advantageously
be used with such only as are already perfectly transparent. It is also
more suitable for the preservation of vegetable tissues (when the
retention of colour is no object) than animal, since with the latter it
has a tendency after a while to cause a kind of granular disintegration,
which ultimately destroys much of the usefulness of the preparation.
Dammar and Canada balsam, on the other hand, possess very great
refractive power, so that they are of great service in mounting objects
which require their transparency to be much increased. For this reason
they are not well adapted to the preservation of very delicate or
transparent tissues (unless previously stained), the minute details of
which become almost entirely obliterated when mounted in them. The chief
advantage possessed by these resinous media is, that tissues mounted in
them undergo no alteration, even after the lapse of many years.
Glycerine, in respect of its clarifying powers, occupies an intermediate
position between spirit and balsam, being much more refractive than the
former, infinitely less so than the latter. It is, therefore, of very
great value for the preservation of such tissues as possess a medium
degree of transparency, and which would become obscured if mounted in
spirit, or have their outlines rendered indistinct if preserved in
balsam. It is of the utmost value for mounting unstained anatomical
sections which, when put up in this medium, reveal such minute details
of structure as would readily have escaped observation had any other
agent been employed. It may also be used with stained sections, but in
this case the sections should be of extreme thinness, otherwise the
refractive power of the glycerine will be insufficient to render them
thoroughly transparent. The great drawback to the use of glycerine is
the extreme difficulty experienced in preventing its escape from beneath
the covering glass, for it unfortunately possesses such great
penetrating power that no cement hitherto devised can be thoroughly
depended upon for withstanding its solvent action for any considerable
length of time.[9] Attention to the instructions presently to be given
(§ 16) will, however, reduce this risk of leakage to a minimum. In the
use of glycerine Dr. Carpenter’s caution must ever be borne in mind,
viz., that, as carbonate of lime is in time dissolved by glycerine, this
agent ought never to be employed for the preservation of objects
containing such salt.
Footnote 9:
Note H.
16. _Mounting in Glycerine._—To illustrate the method of using this
medium we will mount our present sections in glycerine. In the first
place we shall require a deep watch-glass, which is to be half filled
with glycerine diluted with an equal amount of distilled water. By means
of the spoon, one or more sections may be transferred into this, either
directly from the acetic acid solution (§ 14), or if, since cutting,
they have been preserved in spirit, they should first undergo a short
immersion in a large vessel full of water. The watch-glass should now be
covered with an inverted wine-glass, and put away for some hours, in
order that the sections may become thoroughly saturated with the dilute
glycerine. When this has been accomplished, a slide must be cleaned, and
one of the sections, with the aid of the _unpierced_ end of the spoon,
be transferred to its centre.[10] As the kind of section with which we
are now dealing is, or ought to be, of extreme thinness, no cell (§ 26)
is necessary. After tilting up one end of the slide, so as to drain off
as much of the weak glycerine as possible, a drop of Price’s best
glycerine must, with a glass rod or pipette, be allowed to fall gently
upon the section, so as to avoid the formation of air-bubbles. If any of
these, however, should be produced, they must be removed with the point
of a needle set in a wooden handle,[11] and the slide then covered with
a small bell-glass (or wine-glass). A circular cover is now to be
cleaned with a soft handkerchief, and after gently blowing from it any
adhering fibres of lint, etc., it will be advisable to hold the side of
the glass which is to come into contact with the preparation close to
the mouth, and breathe upon it, so as to cover it with moisture. The
cover held between the thumb and forefinger of the left hand must now be
applied by its edge near to the margin of the preparation, and the
surface of the cover directed in an inclined manner over it. Beneath the
overhanging edge of the cover the point of the needle, held in the right
hand, is now to be inserted (see Figure). By gently lowering the needle,
the cover will come into gradual contact with the slide, driving before
it a minute wave of glycerine, in which any air-bubbles that may have
become developed are usually carried off. A very considerable degree of
tact, however, is required to perform this little operation, simple as
it may appear, for the retreating wave of glycerine not unfrequently
floats out the section, either wholly or partially, from beneath the
cover. Air-bubbles, also (the _bêtes noires_ of this process), are
exceedingly likely to arise. When this happens the best plan to adopt
is, by means of the needle point, gently to raise and remove the cover,
apply another drop of glycerine to the section, and cover _with a fresh
piece of thin glass_. It will now be necessary to remove any superfluous
glycerine which may have collected around and near the cover. The great
bulk must be wiped away by means of a camel’s-hair pencil, slightly
wetted between the lips, any remaining stickiness being removed with a
bit of blotting paper which has been slightly damped. With a very small
camel’s-hair pencil, charged with solution of gelatine, a ring must be
made round the margin of the cover, of sufficient breadth to take in a
small tract of both cover and slide. As this cement is perfectly
miscible with glycerine, it readily unites with any of that fluid which
may ooze from beneath the cover, and which, in the case of any of the
ordinary varnishes, would act as a fatal obstacle to perfect adhesion.
To make the cement, take half an ounce of Nelson’s opaque gelatine, put
in a small beaker, add sufficient cold water to cover it, and allow the
mixture to remain until the gelatine has become thoroughly soaked. The
water is now poured off, and heat applied until the gelatine becomes
fluid, when three drops of creosote should be well stirred in, and the
fluid mixture transferred to a small bottle to solidify. Before use,
this compound must be rendered liquid by immersing the bottle containing
it in a cup of warm water. When the ring of gelatine has become quite
set and dry (which will not take long), every trace of glycerine must be
carefully removed from the cover and its neighborhood, by gently
swabbing these parts with a large camel’s-hair pencil dipped in
methylated spirit. After drying the slide a ring of Bell’s microscopical
cement may be applied over the gelatine, and, when this is dry, another
coat is to be laid on. If it be desired to give the slide a neat and
tasteful appearance, it is a very easy matter, by means of the
turn-table, to lay on a final ring of Brunswick black or white zinc
cement (§ 24). Every care has now been taken to render our preparation
permanent; but, to make assurance doubly sure, it will be well to follow
Dr. Carpenter’s advice, and, every year or so, to lay on a thin coating
of good gold-size.[12]
Footnote 10:
The appearance of a slide is vastly improved if the preparation be
placed _exactly_ in its centre. This may readily be done in the
following manner:—Take some very finely-powdered Prussian blue, and
rub it up in a mortar with a little of the weak gum solution (§ 11),
so as to form a thin blue pigment. A quantity of this should be made,
so as always to be at hand. A slide having been cleaned, the _best
surface_ is to be selected, and on the _reverse_ side, by means of the
self-centring turn-table, a small circle is to be drawn with a
camel’s-hair pencil, charged with the pigment. In the centre of this
ring, but on the opposite side of the slide, the section is to be
placed, when it of course will occupy a position exactly central. When
the slide comes to be finished, the blue ring may easily be removed
with a wet rag.
Footnote 11:
A _crochet-needle_ holder made of bone, and which may be bought at
the smallware dealers’ for about sixpence, makes an admirable handle
for microscopical needles. At one extremity there is a small cavity,
closed with a cap, for the storage of reserve needles, whilst the
other end terminates in a metal tip, provided with a crucial slit and
central perforation for the reception of the needle in actual use, and
so arranged that, by means of a small screw-nut, needles of various
sizes may be firmly held in position.
Footnote 12:
If square covers be employed, they may be fixed to the slide by a
simple method much in vogue in Germany. A thin wax taper is to be
lighted, and being partially inverted for a few seconds, the wax
surrounding the wick will become melted. After the slide has been
freed from excess of glycerine, a drop of this heated wax is allowed
to fall upon each corner of the cover, and a line of the melted wax
run along the margins of the cover between these points, so as
perfectly to surround it. If a good coat of white zinc cement be
subsequently laid over the wax a very durable, and not unornamental,
line of union will have been formed.
[Illustration: METHOD OF APPLYING COVER.]
17. _Use of Freezing Microtome._—Our preceding consideration of the
method of employing the microtome in conjunction with paraffine as an
imbedding agent (§ 11), will have formed a very suitable introduction to
the study of the somewhat more complicated process of imbedding the
tissue in gum, for section in the freezing microtome. This method is of
the utmost value to the practical histologist, for by its means he is
enabled with ease to possess himself of perfect sections of several
structures, the cutting of which, before the introduction of this
process, was always a matter of difficulty and anxiety. The freezing
microtome is especially valuable for the section of such substances as
from their extreme delicacy are liable to be injured by being imbedded
in paraffine—for instance, the delicate villi of the intestines becomes
very frequently, by the use of paraffine, denuded of their epithelium,
and the villi themselves not seldom become torn off or otherwise
damaged. The great value of the method is also very well seen in the
treatment of those tissues which, like the lung, are of such loose and
spongy texture as to offer insufficient resistance to the knife unless
their interstices have previously been filled up with some solid yet
easily cut material. As the space at our command is strictly limited, we
are precluded from entering as fully into this branch of section-cutting
as the importance of the subject demands and our own inclination would
lead us. To those who wish to become thoroughly conversant with the full
value of this method we cannot do better than recommend the perusal of
Professor Rutherford’s _Practical Histology_, 2d edition, than which, on
the whole subject of physiological microscopy, no treatise with which we
are acquainted is at once so plain, practical, and profound.
18. _Employment of Freezing Microtome._—A very suitable object with
which to demonstrate the method of using this form of microtome will be
afforded us by a portion of intestine, say of the ileum of a cat or dog.
Suppose we have some of this in methylated spirit—let us select a piece
of about half an inch in length. Our first care will be to deprive this
of its spirit; for so long as the tissue remains impregnated with
alcohol it would, of course, be impossible to freeze it. We will,
therefore, throw it into a large basinful of water, and leave it there
for twenty-four hours, during which time it would be as well to change
the water once or twice. We shall now require a strong solution of gum.
This, which should have been made some time previously, may be prepared
by placing a quantity, say three or four ounces, of ordinary gum arabic
in a glass beaker, and adding sufficient water to cover it—the mixture
must be stirred occasionally with a glass rod until solution has taken
place, which will be in a few days. If _necessary_ a little more water
may be added, but so long as the gum will pour from vessel to vessel, it
cannot well be made too strong. Mucilage, by keeping, is very apt to
become sour and mouldy—this may be prevented by adding to each ounce of
the water with which it is prepared about half a grain of salicylic
acid. We now pour some of this mucilage into a small vessel—an egg cup
will answer very well—and into it transfer the piece of ileum from the
water. Here we must allow it to remain for a time sufficient to permit
of its becoming thoroughly saturated with the gum, for which purpose
some hours will be necessary. When this soaking has been accomplished we
will prepare the microtome, which we will assume to be Rutherford’s. In
the first place it will be necessary to remove the plug—which is to be
done by turning the handle connected with the screw until the plug rises
so high in its tube that it may be grasped with the fingers and removed,
when it is to be well smeared all over with sperm oil and replaced. This
is done to prevent any unpleasant adhesions taking place whilst the
freezing is going on. We must next depress the plug, so as to convert
the upper part of the tube into a kind of “well” of sufficient depth to
hold our specimen. It will now be very advisable to look carefully into
this _well_ and observe whether the plug fits accurately into the tube
(§ 8) for if there be any interval between the two it will give rise to
much subsequent annoyance, as the gum penetrating this interstice will
there become firmly frozen into irregular patches, which will so
interfere with the even gliding of the plug within its tube as to cause
the former to ascend in such an irregular and jerky manner as to be
utterly destructive of all accuracy in the cutting. If this defect be
observed, it may be at once remedied by dropping a small quantity of
gently heated paraffine into the _well_, which will effectually close up
any fissures. The microtome, by means of its clamping arrangement, must
now be firmly attached to the table, and a suitable vessel be placed on
the floor beneath it, so that it may catch the water which will issue
from the waste-pipe of the apparatus. The next requirement is a supply
of block ice and finely-powdered salt. A lump of the ice must be wrapped
in a towel, and crushed into small pieces; these, by means of a large
mortar, are to be further reduced to a very _fine powder_. Any attempt
to hurry over this troublesome part of the operation will lead to future
disappointment, for unless the ice be used in a very fine powder great
delay (at least) in the freezing will be the result. With the aid of a
small spoon the ice and salt are in alternate spoonsful to be conveyed
into the freezing-box of the machine, great care being taken that the
cavity under the cutting-plate and around the tube be thoroughly packed,
after which the uncovered portion of the box should also be well filled.
The _well_ is now to be filled with the strong gum to within a little
distance of its top, and a piece of sheet gutta-percha (such as shoe
soles are made of) being applied over the well, and kept in position by
a weight, we must wait until the freezing commences. In a short time we
shall notice that the gum has acquired a thick muddy appearance. The
tissue must now, by means of the forceps, be transferred to the well,
and there placed in such a position that the sections, when cut, shall
run in the desired direction. After more gum has, if necessary, been
added, so as completely to cover the tissue, the well is again to be
covered, and attention given to the freezing-box. As the mixture which
this contains becomes melted, it must constantly be renewed, care being
at the same time taken that the mouth of the discharge-pipe be kept
quite free, otherwise water accumulating in the box, the freezing
mixture will degenerate into a useless puddle. When the gum becomes
sufficiently hard to cut, this must be done much in the same manner as
if paraffine had been used (§ 12). In this case, however, no fluid will
be required, or must be used, to wet the knife with, and especial care
must be taken that in disengaging the sections from the knife into the
water they be not torn. These sections often adhere very tenaciously to
the blade, but if a little patience be exercised the water will soon
float them off in safety—much more safely than if any attempt be made to
liberate them prematurely. There is one circumstance connected with the
use of the freezing microtome which is rather annoying. The moisture of
the breath and atmosphere is apt to become condensed on the
cutting-plate, and here, mixed with accidental smears of gum, it becomes
frozen into a jagged and irregular sheet of ice, which not only
seriously interferes with the smooth play of the knife, but also
constitutes a real peril to its edge. As this evil cannot be avoided,
all we can do is, by constant wiping, to keep the cutting-plate clean
and free from this accumulation. This is best done with a bit of soft
rag _just moistened_ with spirit, but this must not come into contact
with any portion of the cylinder of frozen gum, else it will instantly
thaw it. When using the freezing microtome it is always advisable to
wear an apron, otherwise our clothes may receive considerable damage
from the constant splashing of the salt water, as it falls from the
waste-pipe into the vessel beneath it. After use, the microtome must be
well washed in plenty of cold water till every trace of salt be removed,
for if any of this remain it will quickly corrode the brass-work of the
instrument. The plug and screw, as also the section-knife, should be
well smeared with Rangoon oil before the machine is put away.
19. _Logwood Staining._—The employment of logwood as a staining agent is
now becoming very general. It acts much in the same manner as carmine,
but the violet color which it produces is by many thought to be of a
more soft and agreeable character than that due to the action of
carmine. A valuable and very convenient property also which it possesses
is that it stains tissues very rapidly, and this without interfering
with that differential kind of coloration (§ 14) upon which the chief
value of all staining processes depends. A simple method of preparing
the logwood fluid is to mix an aqueous solution of extract of logwood
with a solution of alum (1 to 8) till the deep impure red colour has
become violet, and then to filter the mixture (Frey). This will stain
sections in about half an hour. This stain, though here mentioned for
the ease with which it may be made is, as a rule, very inferior to a
fluid prepared directly from hæmatoxylon, the alkaloid or active
principle of logwood. As, however, it is difficult and troublesome to
make the solution in this manner,[13] it will be advisable for the
student to purchase, ready prepared, such small quantity of the dye as
he may require. Small bottles may be obtained for a few pence of Mr.
Martindale, 10, New Cavendish Street, London, and from repeated trials
of this solution we can recommend it as producing excellent results. It
is a very strong fluid, and requires to be diluted before use. The
degree to which the dilution must be carried cannot, however, be very
accurately indicated, for all staining fluids of this nature possess the
very undesirable property of becoming decomposed by age. After the fluid
has been kept for some time, a portion of the colouring matter is thrown
out of solution, and becomes deposited upon the sides and bottom of the
vessel in which it is contained, hence the older the preparation, the
weaker it will have become. As the time required for staining with
logwood is but short, it is desirable that all the sections should begin
to be submitted to its action at the same time, otherwise some will
become more deeply stained than others. A good plan is to fill a small
porcelain jar (§ 14) with filtered water, and into this transfer the
sections. Whilst they are settling well down to the bottom, a mixture
must be prepared of half a drachm of Martindale’s solution (fresh) to
one ounce of distilled water, and everything got in readiness for its
immediate filtration. The water is now very gently to be poured off the
sections, and if care be exercised this may be done in such a manner as
to leave them undisturbed at the bottom, after removing almost every
drop of water. The diluted logwood fluid must now be _immediately_
filtered upon the sections, so that they may run no risk of becoming
dry. In the present instance the staining may be allowed to proceed for
about thirty minutes, and this will be found a convenient time for the
immersion of the general run of animal sections. If the logwood fluid be
not quite fresh, either a little more of it will have to be added to the
water, or the time of immersion must be prolonged until the desired
depth of color has been produced. It is well whilst the staining is
going on gently to shake the vessel occasionally, so that the sections
may not remain in a heap at the bottom, but all be as fully as possible
exposed to the action of the dye. When the staining is judged to be
complete, the logwood solution must be gently poured off, leaving the
stained sections at the bottom of the jar, when they should be quickly
covered with methylated spirit, which will _fix_ the colour. We shall
now be able to see if the coloration obtained be perfectly satisfactory.
If not deep enough, it is very easy again to submit them to the action
of the dye for a few minutes longer. If on the other hand, and as more
frequently happens, the coloration should be too deep, the excess of
colour may readily be removed by transferring the sections for a short
time into some diluted acetic acid prepared by adding five drops of the
glacial acid to an ounce of water. The action of this should be
carefully watched, and when the colour has been reduced to the desired
tint the sections may be retransferred to the methylated spirit.
Footnote 13:
Should the student, however, determine to prepare this solution for
himself, he will find a good formula for the purpose in Schäfer’s
“Practical Histology,” p 176. Note I.
20. _Absolute Alcohol._—As we purpose mounting the sections which have
just been stained, in Canada balsam, we will briefly consider the
preliminary treatment to which they must be submitted before this can be
effected. The object of this is to abstract from the tissue all its
water, for if any moisture be permitted to remain in the section it
will, when mounted in balsam, become obscured and surrounded by a kind
of opalescent halo, due to the imperfect penetration of the balsam into
the only partially dehydrated tissue. The old-fashioned plan of
dehydration was simple exposure to the air. The method now generally
adopted is to bring about the same result by means of absolute alcohol.
This fluid has such a strong affinity for water that tissues submitted
to its influence are rapidly and effectually deprived of any water they
may contain. Absolute alcohol in small quantity may be obtained from the
druggist at about sixpence per ounce. It will be necessary for the
student to provide himself with a little of this agent, say about two
ounces, the method of using which will very shortly be explained.
Absolute alcohol must be kept in a bottle with a very accurately-fitting
stopper, in order to prevent its absorbing moisture from the air. For
our purpose such a bottle, having a neck _as wide as possible_, is to be
selected.
21. _Clove Oil._—After being thoroughly dehydrated the sections may, in
special instances (§ 48), be at once mounted in balsam; but, as a
general rule, it will be found necessary (particularly in the case of
animal sections) to treat them with some clarifying agent, in order to
remove the cloudiness and opacity which is (in part) due to their
previous immersion in alcohol. For this purpose turpentine, or any of
the essential oils, may be used: of these, oil of cloves is to be
specially recommended. It is rather expensive, ranging from sixpence to
one shilling per ounce: but, as a drop or two will be sufficient for
preparing each slide, only a small quantity—say half an ounce or an
ounce—need be procured. The most convenient vessel in which to keep the
oil is one of the small test bottles used by watchmakers. These bottles
are provided with a glass cap to exclude dust, and the stopper is
prolonged into a glass rod, which dips into the bottle. The use of this
rod and the method of employing the oil will be explained shortly.
22. _Canada Balsam_, as ordinarily met with, is a thick resinous balm of
great viscidity, but readily rendered perfectly fluid by the application
of heat. Formerly, sections were mounted in this medium in its pure
state, but owing to the annoyance which was so constantly being
experienced from the tenacity with which intruding air-bubbles were held
by the viscous medium, this plan of mounting is rapidly falling out of
use.[14] It is now usual to employ the balsam in a diluted condition,
the two chief diluents being chloroform and benzole. As balsam, however,
often contains more or less moisture, it is desirable to drive this off
before adding the diluent. A very convenient way of doing so is to
expose some pure balsam to the heat of a cool oven for several hours,
when the balsam will be found to have assumed a hard, vitreous
character. It should now be broken into small pieces, these put into a
bottle, and some methylated chloroform added, which in a little while
will completely dissolve the hardened balsam. More chloroform is then to
be added, until a solution is obtained sufficiently thin to run through
filtering-paper. A glass spirit lamp must now be procured, having a
capacity of about two ounces, and provided with a cap. Into the
wick-holder of this (which must be made of porcelain) a hollow glass
tube is to be so fitted that its end dips into the lamp to within about
a third of the bottom. (See Fig.) The thin chloroform-balsam is now to
be filtered into this lamp, very fine filtering-paper—through which a
little chloroform has first been passed—being used for the purpose. When
the lamp is full it must (deprived of its cap) be put in a warm place
until sufficient of the chloroform has evaporated to leave behind it a
fluid of the consistence of thin syrup.
Footnote 14:
Although we cannot too strongly insist upon the use of
chloroform-balsam wherever practicable, yet it sometimes happens in
the mounting of substances of _considerable thickness_, that after all
the chloroform has evaporated an insufficient amount of balsam is left
behind to fill up the cavity between slide and cover. In such cases,
therefore, it is advisable to use pure balsam, which may be done in
the following manner. The object having been previously thoroughly
dehydrated by immersion in absolute alcohol, is to be thence
transferred to a little good turpentine, or benzole, where it should
remain until perfectly transparent. It is now to be placed in the
centre of a slide which has been gently warmed, and a drop or two of
_fresh_ fluid balsam added, the greatest care being taken to prevent
the formation of air-bubbles. Should such arise they must be touched
with the point of a heated needle, which will cause them to burst and
disappear. The chief difficulty of the process has yet to be
encountered in the application of the cover; for it is during this
procedure that the development of air-bubbles is most likely to take
place. This annoyance may, however, be entirely avoided by taking the
simple precaution of dipping the cover into turpentine before it is
applied (§ 16), when it will be found that “you can’t get air-bubbles,
even if you try.” The courtesy of Mr. J. A. Kay, of Chatham, enables
us to give our readers the benefit of this practical “_wrinkle_.”
[Illustration: SPIRIT LAMP ADAPTED TO CONTAIN BALSAM.]
23. _Mounting in Balsam._—Let us now return to our sections which, it
will be remembered, were left in methylated spirit (§ 19). These we will
now mount in balsam, and although, of course, any number may be
proceeded with at the same time, yet to avoid confusion, in the
following directions one section only will be spoken of. This section
then is, with the perforated spoon, to be transferred to the bottle of
absolute alcohol, where it may remain for about an hour—considerably
less time is _actually_ required, but as from constant use the spirit
becomes weakened, it is as well to be on the safe side. It must now be
removed to the centre of a clean glass slip, and here the _plain_ end of
the spoon comes into use. If this be employed for effecting the
transfer, it will be found that when the section is being removed from
the alcohol it will bring along with it a small pool of the spirit. A
slight touch of the needle applied to the edge of the section will cause
it to float from the spoon on to the slide, at the same time carrying
the pool of alcohol with it, in which it will gently spread itself out
upon the slide without the faintest risk of injury. The superfluous
spirit is now to be drained off, and just as the section is becoming
glazed and sodden-looking (_not dry_) we must, by means of the long
glass stopper (§ 21), apply to it a large drop of clove oil. The oil,
however, should not be placed on the section, but be allowed to drop on
to the slide near to its margin. By gently tilting the slide the oil
will gradually insinuate itself _beneath_ the section and slowly ascend
through it to the surface. The slide should now be covered with a
bell-glass (or wine-glass), and about two minutes allowed for the oil
thoroughly to saturate the section. As much as possible of the
superfluous oil must then be drained off, and the remainder removed with
blotting paper. By means of the glass rod a small quantity of
chloroform-balsam is now taken from the spirit lamp which contains it,
and allowed gently to fall upon the section, which must then be covered
with a thin glass circle in the manner previously described (§ 16). When
the object is very fragile, it is a good plan, after draining off the
clove oil, to apply the cover directly upon the section, and then to
place a drop of the balsam near to the edge of the cover. This, by
capillary attraction, will speedily diffuse itself beneath the cover,
flowing over and surrounding the object, without in the slightest degree
disturbing its position. If, during the process of mounting, any
air-bubbles arise, we may view their development with equanimity, being
well assured that as the chloroform evaporates they too will quickly
disappear. When the mounting is completed, the slide should be roughly
labelled and placed on a warm mantel-piece for a few days to dry.
24. _Finishing the Slide._—In the course of two or three days it will be
advisable to take an old penknife, and after heating the blade in the
flame of a spirit lamp, gently to run the point of it round the margin
of the cover, so as to remove any excess of balsam which may have oozed
from beneath it. In a few days more, any remaining balsam may be
carefully scraped away with a cold knife. All remaining traces of balsam
are then to be removed from around the cover by means of a rag _just
moistened_ with methylated spirit, or, what is better, with a mixture of
equal parts of spirit and æther, after which the slide is to be
thoroughly washed in cold water. The slide is now in reality finished,
but, in order to give it a smart appearance, it is usual, with the
assistance of the turn-table, to run a ring of colored varnish round the
covering glass. A very useful varnish for the purpose is the _white zinc
cement_. To prepare this, dissolve an ounce of gum dammar in an ounce of
turpentine by the aid of heat. Take one dram of oxide of zinc and an
equal quantity of turpentine; rub them up together in a mortar, adding
the turpentine drop by drop, so as to form a creamy mixture perfectly
free from lumps or grit. One fluid ounce of the dammar solution
previously made must now gradually be added, the mixture being kept
constantly stirred (_Frey_). The cement, when made, should be strained
through a piece of fine muslin, previously wetted with turpentine, into
a small wide-necked bottle, which, instead of having a cork or stopper,
should be covered with a loose metal cap. Instead of a bottle, the
varnish may be kept in one of the collapsible tubes used by artists; but
though this plan is highly recommended by many, it is not without its
disadvantages. If the varnish becomes thick by keeping, a few drops of
turpentine or benzole well stirred in will soon reduce it to a suitable
consistence for use.
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