HISTOLOGICAL METHODS.
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.
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.