A cat, dog, rabbit, or Guinea pig will furnish parts from which sections
can be cut for the study of histology. Whichever animal is selected
should be young and well developed. Put it under influence of chloroform,
and open into the cavity of the chest; make an incision into the right
ventricle, and allow the animal to bleed to death; cut the trachea and
inject the lungs with a solution of one and a half drachms of chromic
acid in one quart of water, care being taken not to overdistend the lung.
Tie the severed end to prevent the escape of the fluid, and carefully
remove the lung. It is a difficult thing to do this without rupturing it,
but with care and patience it can be done. Place the lungs in a solution
of the same strength as used for injecting; after fifteen or twenty hours
change it to a fresh solution, and allow it to remain for about a month,
and then change it to rectified spirits, in which it may remain until
required.
Cut the tongue into several transverse and longitudinal pieces, also the
small intestines, and put them into a solution of fifteen and one-half
grains chromic acid, thirty grammes bichromate of potash, and three pints
of water; change the solution the next day, and let them remain two weeks
and then place in spirits. Cut longitudinal and transverse portions of
the stomach and large intestines, wash in a weak solution of salt and
water, and put them in the same solution as used for the lungs, and treat
similarly.
Cut the kidneys longitudinally and transversely, and put them in a
solution of six and one-half drachms bichromate of potash, two and
one-half drachms sodium sulphate, one quart of water; change the solution
the next day, and at the end of four weeks transfer to alcohol. Wash the
inner surface of the bladder with salt and water, and after cutting it
longitudinally and transversely, put the sections in a solution of three
drachms bichromate of potash in a quart of water. Cut the liver into
small parts, and place in the same solution as used for the kidneys;
change the solution after a day, and let them remain four or five weeks,
then change to spirits. The spleen and portions of the thin abdominal
muscles may be placed in a solution of three drachms chromic acid to one
quart of water, and transferred to alcohol after three or four weeks.
Carefully remove an eye and divide it behind the crystalline lens, put
the posterior portion in a solution made by dissolving fifteen grs.
chromic acid in five drachms water, and slowly adding five and one-half
ounces alcohol; change to spirits in two weeks. The lens should be put in
the same solution, but should remain a few days longer. Open the head,
remove the brain, and place transverse and longitudinal sections of it
in spirits for eighteen hours, then transfer to a solution of one drachm
chromic acid in a quart of water, and let it remain until hard enough to
cut. Place the uterus in a solution of one and one-half drachms chromic
acid in one quart of water, change to a new solution the next day, and at
the end of a month transfer to alcohol.
The bones from one of the legs should be carefully cleaned of its
muscles, cut into several pieces, and placed in a solution of fifteen and
one-half grains chromic acid, one-half drachm nitric acid, and six ounces
water. Change the fluid frequently until the bones are sufficiently
softened, and then change to alcohol.
_Section cutting_ machines for cutting sections can be procured of the
dealers, but a very simple and effective one can be easily made if one
does not wish to go to the expense of buying an instrument.
A strip of wood twelve or fourteen inches long and about two inches wide
has attached to its center a bridge-shaped piece of wood, a, Fig. 1. This
is covered with a brass plate, c, pierced with a hole one-half of an inch
in diameter. This hole extends through the wood, and is fitted with a
piston. Two long narrow inclined planes of nearly equal inclination, b,
b, grooved to slide on each other, are placed under the bridge; the lower
is to be fastened to the board; the end of the piston rests on the upper
one. The object from which we desire to cut a section is placed in the
hole, in the piston. If the upper plane be pushed in, the piston will be
forced upward, and with it the object. As the inclination of the plane is
very gradual, the vertical motion will be very slight as compared with
the horizontal.
When the object is raised a little above the brass plate, a keen edged
razor, thoroughly wet, is pushed over the hole, cutting the object. This
gives the section a smooth surface, and even with the plate; now push the
plane forward one-eighth to one-quarter of an inch, and cut again; this
will give a thin section of the object. The thickness of the section
depends, of course, on the distance the wedge is pushed.
With a little practice, much better sections can be cut by the hand than
by any machine; this does not apply of course to large sections. A razor
of good steel, with a blade thin and hard, are the most essential points
in an instrument for hand cutting. For ordinary purposes it is not
necessary to have the blade ground flat on one side, although many prefer
it. The knife should always be thoroughly wet, in order that the cut
tissue may float over its surface. Water, alcohol or salt and water may
be used for this purpose.
[Illustration: FIG. 1.]
_To out a section by hand_, hold the object between the thumb and first
two fingers of the left hand, supporting the back of the knife by the
forefinger. The knife is to be held firmly in the right hand, and in
cutting should never be pushed, but drawn from heel to point obliquely
through the tissue. The section should be removed from the knife by a
camel's hair brush.
When the object is too small to hold, it is usually _embedded_ in some
convenient substance. A carrot is sometimes very useful for this purpose.
A hole rather smaller than the object is cut out of the middle. Put
whatever is to be cut into this, and cut a thin section of the whole. The
carrot does not cling to either the knife or the section, and the knife
is wetted at every slice by it.
Paraffin is the agent usually employed for embedding purposes. Melt it,
and add a little lard to soften it; the addition of a little clove oil
renders it less adhesive.
Melt the paraffin at as low a temperature as possible, and pour it into a
paper cone. Dip the object into this and remove immediately; as soon as
the layer of paraffin surrounding it becomes hardened, replace it in the
paraffin; this prevents overheating the tissues.
Where the tissues are too soft to be cut, they may be soaked in a
solution of gum arabic and dried; in this condition they can be readily
cut, after which the gum can be dissolved off. This is an extremely
useful method for cutting the lung or other organs where an interstitial
support is needed. For a very thin object, a cork fitting any kind of a
tube is to be split, and the object placed between the two parts; the
cork is then thrust into the tube, and a sufficient degree of firmness
will be obtained to allow cutting. The sections should always be
manipulated with camel's hair brushes.
Much practice will be required before dexterity is attained.
_Methods of preserving the tissues_.--All water must be removed from the
tissue, either by drying or by immersing it in rectified spirits, and
then in absolute alcohol, and the alcohol driven off by floating it upon
oil of clove or turpentine. The substances used to preserve the tissues
are Canada balsam, Dammar balsam, glycerine, Farrant's solution,
potassium acetate, spirits, naphtha, and creosote.
The section is to be floated on to the slide or placed in position with a
camel's hair brush. It should be spread out, and then examined under the
microscope for the purpose of improving its position if necessary, or of
removing any foreign particles. A drop of the preserving medium is then
placed upon it, and another placed on the cover and allowed to spread
out. The cover is then taken by a pair of pincers and inverted over the
object, and one edge brought to touch the slide at one part of its
margin. The cover is then gently lowered, and the whole space beneath the
cover filled and the tissue completely saturated. If air bubbles show
themselves, raise the cover at one corner and deposit a further quantity
of the medium.
The slide should be set aside for a few days. First, the excess of the
medium must be removed; if it is glycerine, much of it can be removed by
a piece of blotting paper, but the cover must not be touched, for it is
easily displaced; that near the cover can be replaced by a camel's hair
brush. A narrow ring of glycerine jelly should be placed around the edge
of the cover, to fix it before the cement is applied. When this has set,
a narrow strip of cement is to be put on, just slightly overlapping the
edge of the cover and outside the margin of the jelly. Until it has been
perfectly secured, a slide carrying glycerine must never be placed in an
inclined position, as its cover will slide off.
_Preservative media_.--Canada balsam may be prepared as follows: Place
some pure Canada balsam in a saucer, and cover with paper to exclude
dust; dry it in an oven at a temperature of 150°; when it cools, it will
become hard and crystalline. Dissolve this in benzole, and use in the
same way as glycerine.
Dammar is now used as a substitute for Canada balsam. By its use the
tissues are rendered more transparent. To prepare it, dissolve one-half
ounce of Dammar rosin and one-half ounce of gum mastic in three ounces of
benzole, and filter. This may be used to mount unsoftened bone and tooth,
hair, brain, and spinal column, and most tissues that have been hardened
in alcohol or chromic acid, which require to have their transparency
increased.
Glycerine is not adapted for white fibrous tissue or blood vessels,
unless they have been hardened in chromic acid, as it causes the white
fibers to swell up and lose their normal features. Sections of liver,
lung, skin, and alimentary canal show better in glycerine unless they
have been stained.
Farrant's solution may be substituted for glycerine in many instances,
because of its feebler tendency to render the tissues transparent. It
consists of equal parts of gum arabic, glycerine, and a saturated
solution of arsenious acid. In mounting preparations with this medium,
the covered object should be allowed to lie a day before the varnish is
applied, so that the cover may be fixed, and thereby prevented from being
displaced. Rectified spirits may be used for mounting softened bone and
tooth, and naphtha and creosote are useful for preserving urinary casts.
When the section is mounted in Canada or Dammar balsam, no cement is
required, but for all other preservative media the margin of the cover
must be covered with cement. To do this, dry the edges of the cover
thoroughly with bibulous paper, and paint a layer of gold size, allowing
it to overlap the cover an eighth or sixteenth of an inch, then cover
this with white zinc cement.
_Preparation for mounting the different tissues_.--To obtain a section of
bone or tooth requires a grinding down of the tissue until it is so thin
as to be transparent. A section should first be cut as thin as possible
by a fine saw. It should be attached by the flattest side to a piece of
glass, and then ground down by a grindstone or by very fine emery, on a
perfectly flat piece of lead. When sufficiently thin and transparent,
mount in rectified spirits or Dammar. Sections of the tongue may be made
by embedding in paraffin, and mounted in Farrant's solution or glycerine.
Sections of the stomach may also be made by embedding in paraffin, but
better ones can be made by freezing. Farrant's solution makes a good
mounting.
The intestines also give a better section from freezing than by
embedding, as the paraffin injures the villi; mount in the same medium as
the stomach.
The liver may be embedded in paraffin, and the section mounted in
Farrant's solution or glycerine. The kidney may be treated in the same
way. The cornea of the eye can be readily cut by embedding in paraffin,
and the section may be mounted in Farrant's solution. The crystalline
lens and retina may be treated similarly.
The brain and spinal cord should be embedded in paraffin or a carrot, and
the section mounted in Dammar. Sections of the uterus and ovaries are
best mounted in glycerine or Dammar. Sections of lung maybe made by
embedding in gum or by freezing, and mounted in Farrant's solution.
Every slide should be of uniform size, and labeled. The usual size is 3×1
inches, and should be of a good quality of glass, free from scratches or
air holes. They may be labeled either by writing with a diamond, or a
small piece of paper affixed to one end, on which is written what is
required.
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