Book - Stoehr's Histology 2

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Lewis FT. Stoehr's Histology. (1906) P. Blakiston's Son & Co., Philadelphia.

Stoehr's Histology 1906: 1 Microscopic Anatomy | 1-1 Cytology | 1-2 General Histology | 1-3 Special Histology | 2 Preparation of Specimens | Figures | Histology | Embryology History
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Part II. The Preparation and Examination of Microscopical Specimens

The following directions are limited to those of fundamental importance which are likely to be employed by students who are beginning their histological studies. Further information may be obtained from "The Microtomist's Vade-mecum" by A. B. Lee (3d ed., 1903, Blakiston, Philadelphia) and from Mallory and Wright's "Pathological Technique" (3d ed., 1904, Saunders, Philadelphia). The latter is particularly adapted to the needs of medical students.


Fresh Tissues

Certain transparent tissues may be studied advantageously in a fresh condition. They are merely spread in a thin layer upon a clean glass slide, and after a drop of tap water and then a clean cover glass have been placed upon them, they are ready for the miscrocope. (The glass slides and covers are to be washed with water, using soap if necessary, and sometimes alcohol or strong acids, but all trace of these must be removed. Linen cloths, because of their small quantity of lint, are the proper towels for drying the glassware. Covers and slides as received from the dealers are never ready for use, and some which remain hazy after thorough washing are worthless.) The fresh tissue is spread upon the sUde with needles, being 'teased' into small fragments or drawn out into a thin film. Pure water causes some swelling of the tissue so that dilute solutions of common salt are preferable. A 0.6 per cent, solution has recently been found to cause less distortion than the somewhat stronger solutions formerly recommended. The tissue having been spread in the center of the sUde and a drop or two of salt solution placed upon it, the cover glass is lowered so that air bubbles are not caught beneath it. Especially with the larger slides which are to be preserved permanently this should be done as follows. The square or oblong cover glass is held over the specimen and its left edge is first brought in contact with the slide; a needle held in the left hand keeps this edge in position. Another needle held in the right hand with its point beneath the right edge of the cover enables one to have perfect control of it while it is being lowered. The contact between the cover and the mounting medium (salt solution in this case) spreads gradually from left to right as the cover is lowered, expelling the air as it advances. If bubbles are caught in the medium,^ the cover may be alternately raised and lowered a little imtil they escape, but once the cover is flat upon the specimen it should not be lifted.


Connective tissue, medullated nerves, fat, desquamated epithelial cells and blood should be examined in the fresh state by every student as showing certain features better than the preserved specimens. Chorionic villi may be identified in this way, and the cells in urine are studied unstained. A drop of acetic acid (from i to 5 per cent.) placed upon connective tissue causes the white fiber to swell and disintegrate, but renders the elastic tissue and the nuclei more distinct. . A few drops of stain may be placed upon the tissue for some minutes and then washed off in order to bring out the nuclei. Methylene blue (i per cent, aqueous solution) and methyl green (i per cent, solution in 20 per cent, alcohol) or the haematoxyline solutions may be used for this purpose. If sections are overstained a more dilute solution or shorter appUcation is indicated, but if the section is pale, prolonged staining or stronger solutions are required. Thus the time limits given with the various dyes are only approximate as the response of different tissues is not uniform, and different samples of a given solution vary in their staining capacity.

Isolation

Some tissues cannot properly be separated into their elements in the fresh condition but may be shaken or teased apart after preliminary treatment. Epithelial cells become separable after remaining from 5 to 24 hours in 33 per cent, alcohol (40 cc. of 95 per cent, alcohol and 60 cc. of water). The pieces of epithelium used should be small (5-10 mm. square). The same treatment prolonged for one or two weeks is employed in isolating the nerve cells of the spinal cord. Muscle cells may be pulled apart after remaining some hours in a fresh 35 per cent, solution of potassium hydrate. The muscle fibers should be examined in a few drops of the same solution, since they disintegrate if it is diluted. They may however be transferred to solutions of potassium acetate which neutralizes the potash and prevents further maceration. The elements of nails may be scraped off from fragments boiled in a test tube containing a concentrated solution of potassium hydrate. Immersion in cold concentrated sulphuric acid is recommended for the same purpose.

Another solvent for the intercellular subtances of muscle is a saturated solution of potassium chlorate in nitric acid. (About 5 gr. of potassium chlorate should be added to 20 cc. of nitric acid.) The muscle fibers should be separable in from i to 6 hoiurs. They should be washed in distilled water for an hour or a few days so as to remove the acid, and then may be examined in water or in glycerine.

Other macerating fluids are 10 to 20 per cent, nitric acid, diluted either with water or with salt solution; ^-J-j- to ^ of i per cent, of chromic acid; and water, by which the pulpy portion of organs may be removed from the coimective tissue framework. Complex but valuable methods for demonstrating the connective and reticular networks have been described by Mall and Flint. They involve digestion of the tissues with pancreatic extract.

Sectioning Fresh Material

Since the cutting of freehand sections of fresh tissue held between pieces of pith is no longer practised, the most rapid method for obtaining sections is by means of the freezing microtome. Small blocks of fresh tissue not over 5 mm. thick are moistened with water and placed upon the carrier of the microtome, where they are frozen by a jet of carbon dioxide proceeding from a cylinder of the liquefied gas. Sections 10-15 A* thick may be chiselled from the frozen tissue and placed in a dish of water, in which they unroll. Then they are floated upon a slide and may be stained by ordinary methods. Frozen sections may be made from tissue hardened in formaline as well as from fresh material. In some cases this method is of special value in studying normal tissue; for rapid diagnosis of pathological conditions it is indispensable.


Descriptions of the freezing and other microtomes with full directions for their use will be found in Mallory and Wright's "Pathological Technique." The use of the instruments, however, is seldom learned except by personal demonstration in the laboratory.

Fixation

The fixation of tissues is the process by which post mortem changes are prevented, mitosis, for example, being checked at once and the mitotic figiu-e permanently preserved. The hardening of the tissue is completed subsequently by immersion in alcohol. Small blocks of the desired tissue (about I cc. in volume and preferably less than i cm. thick) should be dropped without handling into a considerable quantity of the fixing fluid. Contact between the fingers and the peritonaeum is sufficient to destroy the thin mesotheUum. It is often advisable to place a piece of absorbent cotton beneath the tissue so that the fixing fluid may have access to its lower surface. Tubular organs should be cut open before being put in the fluid, and their contents together with blood upon the surface of the block may be washed away with salt solution. Membranes may be kept flat and smooth by being tied across the end of a short tube or a detached bottle neck. After being used once the fixing fluids should be thrown away, except alcohol, which can be put to other uses. The following mixtures are those most frequently used.

Zenker' s Fluid is kept m stock as glacial acetic add and the following solution, in preparing which the water is heated and the ingredients are stirred with a glass rod. (Metal instruments should not be put in Zenker's fluid.)

Bichromate of potassium 25 gr.

Sodium sulphate 10 gr.

Mercuric cnloride (corrosive sublimate) 5° gr.

Water 1000 cc.

Shortly before using, Zenker's fluid is to be completed by adding 5 cc. of glacial acetic add to 100 cc. of the solution. The blocks of tissue placed in it should be from 4 to 6 mm. thick; after remaining in the fluid from 10 to 24 hours they are to be placed in running water (or in water frequently changed) for the same length of time. Then they are transferred to 80 per cent, alcohol.

The transfer of tissues from water to alcohol or vice versa is one of the commonest procedures. The abrupt change from water to strong alcohol causes violent diffusion currents which may distort the tissues; therefore graded percentages of alcohol are used, 50 per cent., 70 per cent., 80 per cent., 95 per cent., and absolute alcohol being always at hand. (Sometimes 90 per cent, also is used.) These may be prepared from the commercial 95 per cent, alcohol by adding water in the following proportions:

Ninety per cent., — 475 cc. of 95 per cent, and 25 cc. of distilled water. Eighty per cent,— 425 " " " " 75 "

Seventy per cent.,— 370 " " " "130 "

. Fifty per cent,— 265 " " " " 235 "

Tissues may generally be transferred between water and 50 per cent, alcohol without injury. In passing from 50 per cent, to absolute they may be placed successively in 70 per cent., 80 per cent., and 95 per cent., remaining in each only long enough to become saturated. Stains may be rated according to the alcohol they contain; the transition from 80 per cent, to an aqueous stain should be graded as from 80 per cent, to water. It is a general prindple that all these transfers should be gradual for the best results. Nevertheless abrupt transitions are often made, and ordinarily the tissue preserved in Zenker's fluid and washed in water is next immersed in 80 per cent alcohol.


The chief fault of Zenker's fluid is its tendency to form a precipitate of mercuric chloride (corrosive sublimate) within the tissue. The precipitate is dissolved out by the addition of enough tincture of iodine to the 80 per cent, alcohol to give it a mahogany color. The color fades as the iodine combines with the subUmate and it should be renewed until for two days there is no perceptible change in its color. This may require a week or more. Then the tissue is transferred to 80 per cent, alcohol which is renewed as long as it becomes discolored by the iodine. In 80 per cent, alcohol the tissue may remain for months but it gradually deteriorates. The prolonged action of iodine causes some loss in staining capacity; nevertheless the treatment with iodine is an essential routine part of this method of fixation, and it should be thorough enough to remove the precipitate. The latter appears in sections as dark blotches resembling pigment. They may be dissolved after sections have been cut and attached to the slide by immersing the slide in the iodine solution and then rinsing it in 80 per cent, alcohol TeUyesnizcky's Fluid is employed Uke Zenker's fluid but since it contains no mercuric chloride, the after-treatment with iodine is unnecessary. This fluid is a 3 per cent, aqueous solution of bichromate of potassium to which glacial acetic acid should be added shortly before using (5 cc- of acetic acid to 100 cc. of the solution). Tissues may remain in it for two or more days. The reagent is washed out in nmning water, and the tissue is transferred to 80 per cent, alcohol.

Formaline is a 40 per cent, aqueous solution of formaldehyde gas. Ten per cent, aqueous solutions of formaline, which are 4 per cent, solutions of formaldehyde, are used for the preservation of small embr}'os and of various tissues. Small human embryos obtained by practitioners should be placed at once in 10 per cent, formaline and forwarded to an embryological laboratory. Tissues should remain in the 10 per cent, formaline for 24 hours or somewhat longer, and then are transferred to 80 per cent, alcohol in which they generally shrink. (Frozen sections may be made from the material taken directly from formaline and rinsed in water.) Instead of transferring the tissue from the formaline to 80 per cent, alcohol, some histologists recommend placing it at once in absolute alcohol for 2 days, after which it is immersed in 80 per cent. Formaline is used as a fixing agent in many solutions, especially the following.

Orth's Fluid is Milller^s Fluid with the addition of formaline. Miiller's fluid is a slow fixing solution, in large quantities of which objects may be left from i to 6 weeks; after washing 4 to 8 hours in nmning water they are put through graded alcohols in which the tissue is hardened; or the tissue may be both fixed and hardened by remaining in the fluid for six months.


It is a solution of 30 grains of sodium sulphate and 60 grams of potassium bichromate in 3000 cc. of water. To make Orth's fluid, 10 cc. of formaline are added to ico cc. of Miiller's fluid shortly before using. Small blocks of tissue should remain in it for 3 or 4 days, when, after washing thoroughly in running water, they are put in 80 per cent, alcohol.


Alcohol. The higher grades of alcohol are important fixing fluids, although for most purposes inferior to Zenker's fluid or formaline. Tissue may be put directly into 95 per cent, or absolute alcohol, a piece of absorbent cotton being under it. The alcohol should be changed after 3 or 4 hours, and after 3 or 4 days the tissue is transferred to 80 per cent. Some histologists recommend passing the fresh tissue through graded alcohols before putting it in absolute; this causes less shrinkage but is said to fix imperfectly. One may begin with 80 per cent.


Specimens should be kept in 80 per cent, or 90 per cent, alcohol after they have been preserved. They macerate in the weaker alcohols and lose their staining capacity in those which are stronger.

Decalcification

Specimens which contain bone or calcareous material cannot be sectioned until they have been decalcified, which can be done only after they have been fixed, and hardened for a few days in alcohol. They are then placed in considerable amounts of dilute nitric acid (3 to 5 cc. of concentrated nitric acid in 100 cc. of water). This should be renewed for 3 or 4 days, until the bone can be cut with a scalpel or be penetrated easily with a needle. The acid is removed from the tissue by immersion in running water for a day, and the block is returned to the alcohol.


Phloroglucin is sometimes added to the decalcifying fluid to protect the tissue, and the nitric acid may be diluted with alcohol. The following solution has been recommended:

Phloroglucin i

Nitric add 5

Alcohol • 70

Water 30

A slight addition (i or 2 per cent.) of nitric or hydrochloric acid to 80 per cent, alcohol may be used in decalcifying email embryos.

Imbedding

Imbedding is the process by which blocks of fixed, hardened, and decalcified tissues are prepared for sectioning. Sometimes the tissue is stained before being imbedded, as will be described later; often all the staining is done after the sections have been cut. Imbedding consists in surrounding and infiltrating the tissue with a firm substance which can readily be cut into thin sections. Celloidin and paraffin are used, each having its peculiar advantages.


To imbed in celloidin one needs graded alcohols, a mixture of equal parts of ether and absolute alcohol, a thin and a thick solution of celloidin, and vulcanized fiber blocks of such size as can be clamped in the carrier of the microtome.


Thick celloidin consists of 30 grams of Schering's dry granular celloidin dissolved in from 200 to 250 cc. of an equal mixture of ether and absolute alcohol. It has a thick syrupy consistency and becomes constantly denser as the ether evaporates. It should be kept in a tightly closed preserve jar. Thin celloiden contains twice as much *' ether and absolute" as the thick.

The piece of hardened tissue is trimmed to the size and shape desired and is put successively in 95 per cent., absolute, and absolute and ether, remaining 24 hours in each. Then it is immersed in thin celloidin and finally in thick celloidin, in each of which it stays from 24 hours to a week or even longer. The success of the process depends largely upon the thorough penetration of the celloidin into the tissue. The time required in the celloidin varies with the penetrability of the tissue and the size of the piece. After remaining in the thick celloidin long enough the tissue is taken out with a mass of adherent celloidin and is pressed gently against the roughened surface of a block of vulcanized fiber. The celloidin should cover the tissue and spread out at its base upon the block. As soon as a film has formed over its siuiace, the block and attached specimen are dropped into 80 per cent, alcohol in which the mass becomes firm. It is ready for sectioning in 6 hours. While the block is clamped in the sliding microtome ¥dth which sections from 10 to 15 fi should be cut, it is kept moistened with 80 per cent, alcohol; the knife also should be wet with the same. Sections are immediately transferred to a dish of 80 per cent, alcohol in which they unroll, and where they remain until it is desired to stain them. Each section is surrounded by celloidin which it is not desirable to remove; the sections would then be too fragile. Therefore they are not to be placed in absolute alcohol. In case the tissue was not properly imbedded it may be retupjed to ether and absolute, and again be put through the celloidins.


To imbed in paraffin the block of hardened tissue is immersed for from 6 to 12 hours in the following fluids successively: 95 per cent.; absolute; a mixture of equal parts of chloroform and absolute; chloroform. Then it is transferred to chloroform saturated with parafl&n, which may be kept warm by placing on top of the paraffin bath; in this mixture it remains about 4 hours and then is put in melted paraffin. Hard parafl5n which melts at 50° is ordinarily used, but if this is brittle when cut into the microscopic sections at the temperature of the room, a grade with a lower melting point should be used. The melted paraffin should be in a paraffin bath or in a thermostat maintained at a temperature but sUghtly above the melting point of the paraffin. The tissue should not remain in hot paraffin longer than is required; it is generally left 2 hours in one cup and then is transferred to another in which it remains for two hours longer. The purpose of this transfer is to free the tissue from chloroform, most of which remains in the first cup.


The imbedding frame in which the paraffin is to be cooled, is a box the Cx bottom of which is made by a glass plate and the sides of which are of metal in two L shaped pieces. By sliding the latter back and forth in relation to one another, the size of the space which they enclose can be varied. Before using the frame the inside surfaces of the metal pieces together with that surface of the glass on which they rest are rubbed with glycerine and the frame is warmed by placing it for a few minutes on the top of the paraffin bath . Melted paraffin is then poured into it, and the tissue, removed from the cup by means of a spatula, is added. It sinks to the bottom and may be placed in any desired position by means of needles warm enough to prevent the paraffin from solidifying over their surface. The paraffin is then n^irlcly (yviIpH hy lowering the frame into a basin of cold water so that the latter surrounds the metal pieces. Water must not reach the upper surface of the paraffin until it has solidified; then the frame is placed under water and in a few minutes the glass plate and metal pieces may be detached from the solid paraffin. As soon as it is thoroughly cool it may be sectioned.

Before the imbedded object is attached to a block of vulcanized fiber, superfluous paraffin is cut away leaving the tissue rising from a broad base of paraffin and completely surrounded by a thin layer. The base is placed upon a heated spatula which rests upon the fiber block. When the paraffin has melted somewhat, the spatula is withdrawn and the base is pressed down upon the block, to which it adheres securely when the paraffin has solidified again. The fiber block is then clamped in a precision" microtome. If a rotary microtome is used the paraffin is attached to a metal disc in place of a fiber block. Sections should be from 6 to 10 // thick, but under favorable conditions they may be made 2 ft thick. If the paraffin on both sides of the tissue is trimmed parallel with the knife blade, the successive sections adhere to one another by their edges forming ribbons. Thus the sections may easily be kept in order. The first one cut is attached to the upper left hand comer of the slide, and the others follow like lines upon a printed page. Sections mounted in this way are called serial sections. Paraffin sections, as they are taken from the microtome, are laid in shallow boxes. Before being stained they must be attached to slides as follows.


To aUach paraffin sections to a slide^ a mixture of equal parts of glycerine and white of egg is used, which may conveniently be called albumen. Its two ingredients should be stirred together thoroughly and filtered, after which a small lump of camphor is added as a preservative. It is kept in a capped bottle with a glass rod for a dropper. A drop or two are placed upon a clean slide and rubbed evenly with the finger over all that area upon which sections may be placed. It should be free from bubbles and should make a layer thick enough to allow the finger to glide easily over the surface of the slide. Then a few drops of water are placed upon it, forming a layer over the albumen deep enough to float the parafl&n sections, strips of which are placed upon the water. The shiny side of the ribbon should rest upon the water. The slide is then held for a moment over the flame of an alcohol lamp so that the water is heated and the sections become flat and smooth. The paraffin must not be melted. This manipulation with a large slide bearing several rows of serial sections, requires some skill; the water should not come in contact with the fingers holding the slide and if the albumen layer ends abruptly before reaching the border of the slide, the water will not spread beyond it. Surface tension is such that enough water can be put upon the slide to float the sections freely. After the flattening process the water is cautiously drained off by a moist sponge held at the comer of the slide. The sections settle down upon the albumen and may be arranged in straight lines with needles applied to the paraffin but not to the sections themselves. After this the slide is held vertically in contact with filter paper to drain off any water which may remain. The slide is then placed in a drying oven which is not warm enough to melt the paraffin. It is well to let the sections remain there over night but a few hours may be sufficient to dry them thoroughly.

In preparing large numbers of slides, each bearing only one or two paraffin sections, fragments of the ribbon containing the desired number of sections are floated in a basin of water warm enough to flatten but not to melt them. Slides rubbed with albimaen are dipped into the water beneath the sections which are held in place upon them with a needle. The slides are drained and dried in the usual way.

Staining and Mounting

The staining of paraffin sections is accomplished by placing the slides to which the sections have been attached, in pairs back to back, in tubelike vials containing stains. One should have a dozen such vials containing various alcohols, xylol, stains, etc., the sections being passed from one to the other. The reagents are kept tightly corked and can be used for some time before being renewed. The separate stains are to be described in the following section. For staining large numbers of paraffin sections pans have been made with vertically grooved sides, resembling wooden slide boxes. In these 25 or 50 slides may be stained at once, one fluid being poured out of the pan and another substituted. Staining solutions can be used repeatedly and are not to be thrown away.


Before parafiin sections are stained, the paraffin is to be removed by immersing the slide in xylol; it is then transferred in turn to a mixture of equal parts of xylol and absolute alcohol, then to absolute, 95 per cent., and through graded alcohols to that which corresponds with the solvent of the stain. After being stained the sections must be dehydrated, cleared and mounted. They are dehydrated in 95 per cent. , and then in absolute alcohol. They are transferred to the mixture of xylol and absolute, and then into xylol in which they should become perfectly clear. Since the sections are thin and easily penetrated, they need to remain only a few minutes in each of these reagents. After the section has been cleared the xylol is drained ' from the slide, the borders of which (up to the specimen) may be wiped dry; the section itself should not become dry before a drop or two of damar is placed upon it and the cover glass is lowered as described under fresh tissue. The sUde may be used at once although the damar does not become solid for some time.


Damar is a resin derived from trees of the genus Damara; for mounting microscopic objects is should be dissolved in xylol and filtered. The solution should be perfectly clear and nearly colorless. By evaporation of the xylol it thickens, but it may be diluted at any time by adding more xylol. When ready for use it should have the consistency of rather thin syrup. Damar is preferable to balsam since the latter gradually becomes yellow after it has been used.


The staining of celloidin sections is performed in a series of small shallow staining dishes. The sections are taken from 80 per cent, alcohol and transferred through graded alcohols to water or the solvent of the stain. Then they are immersed in the stains, washed in alcohol or water, dehydrated, cleared, and moimted. They are transferred from dish to dish with bent metal or glass needles. Because celloidin is dissolved in the strongest alcohols, the sections are dehydrated in 95 per cent. Since this extracts stains the sections are passed through it rapidly and are placed in the clearing fluid, either oil of bergamot or oil of origanum {oleum origani cretici). In this they should quickly become clear; if opaque spots remain, the section may be returned to 95 per cent, for further dehydration. The clearing oils may be used repeatedly and are not to be thrown away; the alcohol cannot be used twice. The section is mounted by taking it frpm the oil upon a spatula, and transferring it to the center of a slide upon which it should be spread out flat. The oil around it is wiped away and several layers of filter paper are placed directly upon the section; the finger is rubbed over them so that the section is further flattened. Remove the filter paper, and mount in damar as with paraffin sections.


The handling of large numbers of celloidin sections is facilitated if they are placed in a perforated cup which fits into another ordinary cup. The ordinary cups contain the various reagents and the sections are transferred from one to the other in the perforated cup. The latter may be obtained as Hobb's tea infusers, and the solid lemonade cups are of proper size to receive them.

General Stains

Haemaloxyline and eosine. Haematoxyline is a dye obtained from logwood, which stains nuclear structures blue. Eosine is an aniline dye staining protoplasm red. This and all aniline dyes used in histological stains should be prepared by Grubler in Germany.

There are many solutions of heamatoxyline among which is the following:

  • Haematoxyline crystals 1 gr.
  • Saturated aqueous solution of ammonia alum l00 cc.
  • Water 300 cc


Dissolve the crystals in the water, which may be heated, and add the alum solution. Put the mixture in a bottle and drop in a crystal of thymol to prevent the growth of mould. A loose plug of cotton is used for a stopper and in this condition the solution is kept in the light for 10 days to ripen. It changes color during this process of oxidation, after which it is ready for use and is kept tightly stoppered. It deteriorates in a few months. If a strong solution is desired the amount of water may be reduced.


Another haematoxyline solution in common use is Delafield^s. It is made by dissolving 4 gr. of haematoxyline crystals in 25 cc. of 95 per cent, alcohol, and then adding 400 cc. of a saturated aqueous solution of ammonia alum. This is kept unstoppered for 3 or 4 days and then is filtered. 100 cc. each of 95 per cent, alcohol and of glycerine are added. It should not be used imtil it has become dark colored by remaining in the light for several days. Then it is to be filtered and tightly stoppered.


Eosine is sold in two forms, one soluble in water, the other in alcohol. In connection with the haematoxyline stain, a ?? per cent, aqueous solution may be used; or a 1 per cent, solution of alcoholic eosine, made in 60 per cent, alcohol.


To stain with haematoxyline and eosine the sections are placed in the haematoxyline solution from 2 minutes to an hour. They are then placed in water changed repeatedly for half an hour or longer (they may remain in it over night). As seen imder the microscope the nuclei should be deeply stained but the protoplasm should be nearly free from color. Stain in eosine for 1 to 5 minutes; dehydrate, clear, and moimt. For parafl&n sections this means treatment with 95 per cent., absolute and xylol, xylol, and damar. For celloidin sections, 95 per cent., oil of origanum, and damar.


Methylene blue and eosine is highly recommended, especially for tissues fixed in Zenker's fluid and sectioned in paraflSn. Stain in a 5 or 10 per cent, aqueous solution of eosine for 20 minutes or longer, overstaining the tissue since the eosine is partly lost in the subsequent treatment. Wash out the excess of stain in water, and transfer to Unna's alkaline methylene blue diluted with three or four times as much water. Unna's blue is made by dissolving 1 gr. of methylene blue and 1 gr. of potassium carbonate in 100 cc. of water. Sections should be stained in the diluted solution for 10 to 15 minutes. Then they are washed in water and dehydrated and decolorized in 95 per cent, alcohol, moving the section about so that the stain may be washed out evenly. The pink color returns and when, as seen imder the microscope, the blue is limited to the nuclei the section is cleared in xylol and mounted in damar.


Borax carmine and Lyons blue is perhaps the best general stain for embryos. Dissolve 4 gr. of borax in 100 cc. of hot distilled water. When cool stir in 6 gr. of the best carmine and then add 100 cc. of 70 per cent, alcohol. After 24 hours, filter. The Lyons blue may be used in i per cent, alcoholic solution, made with 50 per cent, or 95 per cent, alcohol. Generally it is desirable to dilute it somewhat with alcohol before using.


Before imbedding the tissue, it is stained in borax carmine from 24 to 48 hours, larger blocks of tissue requiring more time than small ones. After being placed in water for 5 minutes (a step which some omit), the tissue is transferred to acid alcohol (0.5 cc. of hydrochloric add in 100 cc. of 70 per cent, alcohol). In this the excess of stain comes out but the tissue acquires a deeper color. After remaining in the acid alcohol from 15 minutes to an hour the tissue is washed thoroughly in 70 per cent, alcohol and is imbedded and sectioned in parafiin in the ordinary way. After the sections have been attached to the slide they are stained in Lyons blue, rinsed in alcohol, dehydrated, cleared and mounted.

Special Stains

An attempt to present all of the important histological stains would exceed the desired limits of this book. The four modifications of Golgi's method, the very important but complex Weigert stain for myelin, and the iron haematoxyline stain for cytological details are omitted with many others. Since they are so well described in Mallory and Wright's Technique, which the medical student who intends to understand bacteriological and histological methods should possess, it seems best to limit this account to the stains which the beginner may employ.


Elastic fibers are stained dark purple or almost black with Weigert's resorcin-fuchsin. Other parts of the tissue should be nearly colorless. The stain is prepared by heating imtil it boils, in an evaporating dish, 2 gr. of fuchsin and 4 gr. of resorcin in 200 cc. of water. Then 25 cc. of liquor fern sesquichlorati are added and the mixture is boiled for 5 minutesIt is cooled and filtered in order to collect the precipitate. The dish in which the boiling took place is dried, together with whatever precipitate remained in it, and after the precipitate upon the filter paper is also dry it is placed with the paper in the dish. 200 cc. of 95 per cent, alcohol are added and boiled to dissolve the precipitate; the paper is removed. When the solution has cooled it is again filtered to collect the filtrate. 4 cc. of hydrochloric acid, and enough 95 per cent, alcohol to make up 200 cc. of stain, are added.


In this solution paraffin or celloidin sections may be stained from 20 minutes to an hour; then they are washed in alcohol, dehydrated, cleared, and mounted. If the stain has affected other parts of the tissue than the elastic fibers, the sections should be washed in alcohol containing a few crystals of picric add, " or in alcohol containing 1 per cent, of hydrochloric add.


White fibers of connective tissue may be stained by Mallory's aniline blue. Fibrils of connective and reticular tissue, amyloid, and mucus stain blue; nuclei, protoplasm, musde, nerves and neuroglia fibres stain red; red corpuscles and myelin stain yellow. Paraffin or celloidin sections of material fixed in Zenker's fluid are stained 5 minutes or longer in a ^V per cent, aqueous solution of acid fuchsin. They are transferred directly to a stain consisting of 0.5 gr. of aniline blue soluble in water, and 2 gr. of orange G, dissolved in a 100 cc. of a i per cent, aqueous solution of phosphomolybdic add. In this they remain 20 minutes or longer. They are washed in several changes of 95 per cent, alcohol, deared, and mounted.


Fat may be stained red in frozen sections of fresh material or of that hardened in formaline, by means of a saturated solution of Scharlach R. in 70 per cent, alcohol The frozen sections are transferred from water to the stain, which has been filtered and is kept tightly stoppered, since evaporation of the alcohol causes a precipitation of the stain. The sections remain in the stain from 15 minutes to over night; then they are washed in water, stained with haematoxyline and mounted in glycerine which clears them. They are not dehydrated in alcohol since strong alcohol dissolves the fat and its stain.


Osmic acid in i per cent, aqueous solution stains fat in fresh tissues dark brown or black; myelin responds like fat both to osmic acid and Scharlach R. The fat is blackened in tissues preserved in a mixture of 2 parts of Miiller's fluid (p. 404) and i part of the i per cent, osmic add solution. Tissues should remain in it for about a week, after which they are transferred to dilute alcohol (50-70 per cent.) for a few days. They may then be imbedded in paraflSn in the usual way, since the stained fat is rendered insoluble in alcohol; it dssiolves in xylol however, so that the sections should be cleared in chloroform and mounted in damar dissolved in chloroform.


Blood may be stained for the study of leucocyte granules and blood plates with Wright's stain which should be prepared as follows: After 0.5 gr. of sodium bicarbonate has been completely dissolved in 100 cc. of distilled water, add i gr. of Griibler's methylene blue (either the form called BX, Koch's, or Ehrlich's rectified). "The mixture is next to be steamed in an ordinary steam sterilizer at ico® C. for one hour, counting the time after steam is up. The heating should not be done in a pressure sterilizer, or in a water bath, or in any other way than as stated." After the steaming the mixture is taken from the sterihzer and allowed to cool, the flask being placed in cold water if desired. When cold it is poured into a large dish or flask. To 100 cc. of the mixture add about 500 cc. of a yV P^r cent, solution of Grtibler's yellowish eosine soluble in water. The amount of the eosine solution should be determined by the appearance of the mixture which it forms, the whole being stirred if in a dish, or shaken if in a flask, while the eosine is added. The color changes from blue to purple, and a yellowish metallic scum forms on the surface, "while on close inspection a finely granular black precipitate appears in suspension." The solution is then filtered and the precipitate is allowed to become perfectly dry upon the filter paper. The stain is made by dissolving 0.5 gr. of the precipitate in 100 cc. of pure methyl alcohol. The stain need not be filtered, and like the precipitate it keeps indefinitely. If by evaporation of the alcohol it becomes too concentrated, as is shown by the formation of precipitates when it is used, it should be filtered and a small quantity of methyl alcohol added.


Blood is obtained usually from a needle puncture in the lobule of the ear. Two cover glasses, perfectly clean and dry, should be at hand. When the blood is flowing freely, the center of one of the covers is touched to a small drop as it emerges, and is then immediately inverted and dropped upon the other cover. The blood should spread evenly between the two cover glasses, forming a film which caimot be too thin. The covers are then drawn rapidly apart, sliding over one another, and the blood dries from exposure to the air. It remains stainable for weeks.


To stain the blood film, the cover glass may be held in the forceps devised for this purpose (cover-glass forceps), with the film uppermost. Stain sufficient to cover it is poured upon it, and after one minute several drops of distilled water are added to the stain, until a delicate metallic scum forms upon the surface. The stain should not be so diluted as to become transparent. After two or three minutes, the stain is washed oflF. The preparation appears blue. Distilled water is placed upon it to extract the excess of stain and the color changes to orange, or pink if the decolorization proceeds further. The general color of the specimen is due to that of the the red corpuscles which at first are blue. When they have become orange or pink as is desired, the water is removed by applying several layers of filter paper, and the preparation is mounted in damar. The process of decolorizing may be watched through the microscope by placing the cover glass (with the film side up) on a slide. Thicker portions of the film which remain blue when the thinner parts are orange, should be disregarded. The leucocytes are figured on page 147.


If intercellar cement spaces and the boundaries of endothelial cells may be blackened by a ?? to i per cent, solution of silver nitrate, which acts chiefly upon free surfaces. The fresh tissue should be kept flat, the mesentery for example being tied over a bottle neck, while it is immersed in the solution for from i to 10 minutes. Then it is placed in distilled water and exposed to direct simlight. As soon as it becomes brown (usually in 5 or 10 minutes) it is washed in dilute salt solution and slowly hardened in graded alcohols. Larger blood vessels may be injected through glass tubes with the silver solution, and after sections have been made and exposed to the lighty the endothelial cell outlines become dark.


The courses of blood and lymphatic vessels and of ducts are studied by means of injections. Colored fluids, usually such as harden by cooling or otherwise, are forced into them by pressure from a syringe. The sjrringe is connected by a short rubber tube with a tapering glass tube or cannula; the latter is inserted into the vessel which is then tied securely around it. Pressure may also be obtained by having the injection mass in a receptacle which connects with the cannula by a long flexible tube; pressure is increased by elevating the receptacle. The organs to be injected must be fresh; they may be left within the body or removed and injected separately. To avoid imdue distension of the vessels and to allow the injection to flow more readily, the efferent vessels may be cut, so that the blood escapes. Sometimes the vessels are washed out by a preliminary injection of salt solution. The efferent vessels may be tied to cause the smaller side branches to be filled. After the injection has been finished, the tissues may be hardened in alcohol or Miiller's fluid, and sectioned in the usual way; thick sections are necessary in order to follow the course of the vessels.


Solutions of Berlin blue or India ink are the simplest injection fluids. Carmine may be prepared by dissolving i gr. in the required amoimt of ammonia and adding 20 cc. of glycerine. The solution is completed by adding i gr. of common salt dissolved in 30 cc. of glycerine (or 20 drops of hydrochloric acid in 20 cc. of glycerine). The second solution is to neutralize the first solution, since the ammoniacal fluid tends to spread through the vessel walls.


Gelatin injection masses are used while warm and fluid, and the tissues which receive them must be kept warm in a water bath. .Clean sheets of the best French gelatin are soaked in water for several hours, imtil soft and swollen. Then they are melted over a water bath and an equal quantity of an aqueous solution of Berlin blue, saturated or dilute as desired, is stirred in. The mass is filtered through flannel wrung out in hot water, and is injected while warm.


A carmine mass may be prepared by dissolving from 2 to 4 grs. of the best carmine in the required amount of ammonia. The solution is filtered and stirred into filtered melted gelatin prepared as already described. The amoimt of gelatin may be from 10 to 50 grs. Twenty-five per cent, acetic acid is then added drop by drop, until the mass becomes bright red and loses its ammoniacal odor. If too much acetic add is added a precipitate forms and the mass is spoiled. During the process the mixture is kept warm over a water bath and is constantly stirred. It is filtered through warm flannel and may be used at once or allowed to cool and heated when needed.


Prepared injection masses are sold by Griibler.

Many ingenious injection methods have been devised, such as the injection of small living pig embryos by allowing ink to enter the umbilical vein and be distributed through the body by the heart's action; or the injection of vessels with milk and staining the frozen sections with Scharlach R.

The Microscope

It is trnfortimate that the price of a microscope is prohibitive to many medical students, and that some who would otherwise purchase instruments at the beginning of their work, wait until an official position entitles them to a discount. The price of microscopes is not always quite as high as is listed, and sometimes when several students buy microscopes at one time they may secure lower rates by having one of their number act as agent.


Within the past ten years the cost of a good instrument has been so reduced that an increasing proportion of students can enjoy the advantage of having a microscope of their own.

Lewis1906 microscope.jpg


Microscopes of a certain grade are required, and if they cannot be afforded, no instrument should be bought. The necessary equipment, as shown in the figure, is a stand with fine and coarse adjustment ("micrometer screw" and "rack and pinion) and a large square stage. The more expensive round and mechanical stages are not necessary. There should be an Abb condenser (with iris diaphragm), a triple revolver, a high and a low eye-piece or ocular, and the following objectives: a f inch and a J or I inch, which must be parfocal, together with a iV oil immersion for cytological and bacteriological work, and a 2 inch (very low power) for embryological work. The ^ oil immersion is an expensive objective, and its purchase may be postponed. The 2 inch is a cheap objective which is very useful in obtaining a view of an entire section, and for embryological reconstructions it is essential. The price of such an outfit, including the oil immersion objective, is from $70.00 to $90.00.


Satisfactory microscopes of American manufacture are made by the Bausch & Lomb Company. A sample submitted by the Spencer Lens Company to the Harvard Embryological Laboratory is also quite satisfactory. The Leitz microscopes, made in Germany, are preferred by some to the American instruments just described; they are not much more expensive. All agree that the Zeiss microscopes (German) are the best (and most expensive). It is undoubtedly true that any of these instruments will fill the requirements of medical students and physicians. If the microscope is purchased by a student unfamilar with its use, it is well to have the lenses examined by a disinterested microscopist.


For a description of the nature and use of the microscope, the student is referred to the 9th edition of " The Microscope, " by Professor S. H. Gage, (Comstock Pub. Co., Ithaca, N. Y.).


For the sake of emphasis it may be said that the microscopist works with his right hand upon the fine adjustment and his left hand upon the slide. As the latter is moved about, bringing diflFerent fields into view, the focussing is done with the adjustment and not with the eyes. Both eyes should be open (as will be natural after becoming, accustomed to the instrument). Often one acquires the habit of using only the right or the left eye for microscopic work, but it is better to learn to use both.


Always examine a specimen first with a low power and then with a high power objective. In focussing the microscope, have the objective drawn away from the slide and focus down. This should be done cautiously, with a portion of the specimen actually beneath the lens; if there is only cover glass and damar there, the objective will probably be driven down upon the slide. Unless one is sure that stained tissue is in the field, the slide should be moved back and forth as the objective is being lowered.


In working with the Abb^ condenser the flat surface of the mirror should be uppermost.

The objectives must never be scratched. Lens paper or fine linen should be used to wipe them. If they are soiled with damar they should be wiped with a cloth moistened with xylol. Since the lenses are mounted in balsam, xylol must be applied to them cautiously.

In lifting the microscope it should never be taken by any part above the stage; the pillar should be grasped below the stage.

Drawings

Drawings should be made of all the significant structures observed; the structure should be observed however, before any drawing is attempted. In other words a thorough study of the specimen should precede the drawing. The nondescript character of many drawings seems due to the fact that the student had nothing definite in mind to portray. It is true nevertheless that the repeated observation made while a careful drawing is in progress, reveals many details which would otherwise be overlooked.

The drawings should be simple but exact, made and shaded with a hard (6 H) lead pencil having a sharp point. They should not be encum' bered with surrounding circles. The parts are to be labelled in one's plainest handwriting (not printing); and the terms should be explicit. A line proceeding from a mass of chromatin within a cell nucleus ought not to be labelled either cell or nucleus but chromatin. Some knowledge of drawing is very desirable although perspective is scarcely involved in histological work. The hghtly colored structures should be made lighter and the dark ones darker than they appear, to preserve the contrasts of the stains. The lines should be few and made with assurance, — ^not pieced out as if one were feeling his way. Every line should correspond with some structure; if a cell has no wall, the even or granular shading representing its protoplasm should end abruptly, but without a bounding h'neT^

Reconstructions

There is an important arrangement of mirrors (Abba's camera lucida) for drawing the outlines of sections. It is attached to the microscope so that the image of the section beneath the objective appears spread upon the drawing paper. The paper is on the table beside the base of the microscope. On looking through the camera into the microscope one can see the pencil point, as it is made to trace the outUne on the paper. In this way a succession of serial sections may be drawn with uniform magnification. The magnification is determined by substituting a stage micrometer for the shde of sections. The micrometer is a slide upon which 1 mm., with subdivisions into twentieths or hundredths, has been marked oflF by scratches in the glass; the subdivisions may be drawn with the camera under the same conditions as the sections, and the enlargement of the subdivisions may then be measured.


From the camera-drawings of serial sections, wax reconstructions adult glands or embryonic organs may be made. If the sections are 10 um thick and alternate sections have been drawn, magnified 50 diameters, then on the scale of the drawings these alternate sections are i mxn. apart. Wax plates imm. thick are therefore to be made, either by rolling the wax, or by spreading a weighed amount of melted wax in a pan of hot water. It floats and spreads in an even layer, solidifying as the water cools. The outlines of the drawings are then indented upon the wax plates, and the desired portions are cut out and piled up to make the model. In this way reconstructions like those of the ear (p. 380) may be made. The details of the process should be learned from demonstrations in the laboratory.


Graphic reconstrtictions are usually side views of structures, made from measurements of their transverse sections. Fig. 161, p. 138, is from such a reconstruction. A camera drawing of the side of an embryo (or other structure) is made before it is sectioned. The outline of this drawing is enlarged, and parallel lines equally spaced are ruled across it, corresponding in number and direction with the sections into which it was cut. Often only every other section or every fourth section is used for the reconstruction, and the number of lines to be ruled across the drawing is correspondingly reduced. Camera drawings of a lateral half of every section used in the reconstruction are made, and across each drawing two lines are ruled. The first follows the median plane of the body; and the second is at right angles with it, being drawn so as to touch the dorsal or ventral surface of some structure to be included in the reconstruction. Provided that the camera drawings and side view have been enlarged to the same extent, the perpendicular distance from the middle of the back to the jimction of the two lines is marked off on the side view, on the line corresponding with the section in question. The perpendicular distances from the second line to the dorsal and to the ventral surfaces of all structures to be reconstructed are also marked off upon the line in the side view. The same is done in the following section, and the points belonging with a given structure are connected from section to section. Thus the outlines of the organs are projected upon the median plane; two dimensions are accurately shown but the third is lost.


Often it is undesirable to attempt to make the magnification of the sections and of the side view identical; the measurements may be enlarged or reduced as they are transferred for plotting, by means of the draughtsman's proportional dividers an indispensable instrument forthis method of reconstruction. The corrections for unequal shrinkage of the sections in parajfin and other details can best be explained in the laboratory with the drawings at hand.



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Stoehr's Histology 1906: 1 Microscopic Anatomy | 1-1 Cytology | 1-2 General Histology | 1-3 Special Histology | 2 Preparation of Specimens | Figures | Histology | Embryology History

Cite this page: Hill, M.A. (2019, September 18) Embryology Book - Stoehr's Histology 2. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Stoehr%27s_Histology_2

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