Book - A textbook of histology, including microscopic technic (1910) microscopic technic
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Böhm AA. and M. Von Davidoff. (translated Huber GC.) A textbook of histology, including microscopic technic. (1910) Second Edn. W. B. Saunders Company, Philadelphia and London.
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- 1 Introduction to Microscopic Technic
- 1.1 I. The Microscope and its Accessories
- 1.2 II. The Microscopic Preparation
Introduction to Microscopic Technic
I. The Microscope and its Accessories
A detailed description of the microscope and its accessory apparatus hardly lies within the scope of this book. If, notwithstanding, a few points be touched upon, it is done only that the beginner may have a working knowledge of the different parts of the instrument which he must use. A more intimate knowledge of the theory of the microscope may be acquired by studying such works as those of Dippel, A. Zimmermann, and Carpenter.
Histologic specimens are examined with the aid of the microscope, an instrument which magnifies the objects by means of its optic apparatus. For this purpose simple microscopes, consisting of one or more converging lenses or lens systems may be used, though they generally do not give sufficient magnification to be of much service in the study of histologic specimens ; they give an erect image of the object observed. When greater magnification is desired, it is necessary to use a compound microscope, consisting generally of two or more lens systems, giving an enlarged, inverted, real image of the object observed. The lens system of a compound microscope may be changed according to the needs of the case, and thus a variation in the magnification of the object obtained. The rest of the instrument consists of a framework called the stand, the lower portion of which consists of a footplate or base. From the base rises the column or pillar, to which the other parts of the microscope are attached. From below upward come the movable mirror, the stage and substage with diaphragm and condenser, and the tube with pinion and fine adjustment. One side of the mirror is concave, and serves to concentrate the rays of light in the direction of a central opening in the stage. The other side is plane. If the objects are to be examined by direct illumination, and not by transmitted light, the mirror is so placed that the rays are reflected away from the opening in the stage. The specimen to be examined is placed on the stage, over the central opening. If the light be too strong, the opening may be diminished in size by means of a diaphragm. In some instruments these diaphragms are placed in the opening of the stage, and consist of plates with different sized apertures. A better form is composed of one large disc containing several apertures of different sizes. This is fastened to the under surface of the stage in such a way that by revolving the disc the apertures may be brought one after the other opposite the opening in the stage. A much better diaphragm, constructed on an entirely different principle, is the socalled iris diaphragm. Although its opening is not exactly circular, yet it has the advantage of being easily enlarged or contracted by manipulating a small handle controlling the metal plates sliding over one another.
The tube, which is contained in a close-fitting metal sheath, is attached to the upright of the microscope. In the simpler forms of microscopes the tube is raised, lowered, or twisted by hand. In more complicated instruments the upward and downward movements are accomplished by means of a rack and pinion coarse adjustment. A micrometer screw fine adjustment situated at either the upper or the lower end of the upright, controls the fine adjustment. The tube possesses an upper and a lower opening, into which lenses may be laid and screwed. The ocular, into the ends of which lenses are inserted, fits into the upper opening. The upper is called the ocular lens, the lower the collective lens. The objective system, which is a combination of several lenses or lens systems, the lowest and smallest of which is known as the front lens, is screwed into the lower opening of the tube.
Fig. I. Microscope.
All larger instruments possess several oculars and objectives, which together give different magnifications according to the combinations used. For most objects a magnification of 500 diameters is all that is required, but to obtain this and still have a clear and bright field the ordinary lenses are hardly sufficient. The greater the magnification, the darker is the field. To avoid this, illuminating mechanisms (condensers, Abbe's apparatus) have been constructed, by means of which the rays of light are concentrated and controlled. This arrangement is absolutely necessary for delicate work.
Even with the aid of such an apparatus the dry objective systems are not sufficient. With them the rays of light must pass through different media having various indices of refraction. The rays pass from the object through the cover-slip, and then through the air between the latter and the objective system. They are thus deflected in different directions a defect which would be avoided if the rays were made to pass through a single medium. This latter condition may be practically brought about by placing between the objective and the cover-glass a drop of some fluid having about the same refractive index as the glass. The lens is then lowered into the fluid. As this invention has proved useful, so-called immersion lenses have been made during recent years. There are thus twcr kinds of lens systems the dry and the immersion lenses. The latter are divided into two groups lenses with water and those with oil immersion. As oil has a greater index of refraction than water, and one more nearly approaching that of glass, the oilimmersion lenses are at present the best objectives that we possess; Karl Zeiss, of Jena, and other microscope makers, have in late years made lenses from a special sort of glass which reduces to a minimum the chromatic and spheric aberration of the rays of light in their passage through the objective (apochromatic lenses).
The rays of light reflected from the mirror and passing through the object are refracted by the objective system in such a way that they are focused in a so-called real image at a point about half-way up the tube. This picture is an inverted one, the right side of the microscopic field being at the left of the real image, and the upper portion below. The picture is, in other words, rotated 1 80 degrees. By means of the ocular the real image is again magnified virtual image but no longer inverted, although to the eye of the microscopist the field actually appears inverted. To shut out the rays of light, which cause a diffused picture, diaphragms are sometimes introduced into the tube as well as into the ocular. (See Fig. 2.)
The objects to be examined are placed upon a glass plate called a slide. ' Microscopic slides are of different sizes, and are usually oblong in shape. Those in most common use are three inches long arid an inch wide. The object is covered by a very much smaller and thinner glass plate the cover=slip. The whole
preparation is then placed upon the stage in such a way that the coverslip is upward and immediately beneath the end of the tube. The mirror of the microscope is now so adjusted as to concentrate the rays of light on the preparation, illuminating it as much as is necessary. By means of the rack and pinion, or coarse adjustment, the whole tube is now slowly lowered toward the cover-slip until the bare outlines of the object are dimly seen in the white field. From this point on, the micrometer screw, or fine adjustment, is used in bringing the front lens down to its proper focal distance from the preparation. The object is now seen to be clear and well defined. By turning the screw to the right or the left, different parts of the specimen are brought more clearly into view, this result being due to the fact that not all points in the preparation are in the same plane.
In studying objects it is always well to draw them, using a sharpened pencil and smooth paper. The beginner soon finds that with constant practice he can sketch the different parts .of the field in nearly their proper relationship. This by no means easy work is facilitated by the use of a drawing apparatus called the camera lucida. The best of these is that devised by Abbe. It is fastened to the upper end of the tube, above the ocular. The apparatus is so made that both the preparation and the drawing surface are seen by the same
eye. The microscopic field is seen directly, while the drawing surface is made visible by means of a mirror. When the apparatus is in place and the drawing commenced, it appears to the one sketching as if his pencil were moving over the preparation itself.
Fig. 2. .Diagram showing the principle of a compound microscope with the course of the rays from the object (a b] through the objective to the real image (b l a 1 ), thence through the ocular and into the eye to the retinal image (a* i> 2 ), and the projection of the retinal image into the field of vision as the virtual image (b 3 a 3 ). (Fig 21, Gage, The Microscope, eighth edition. )
Sections Of Fresh Tissues
Outlines are reproduced on paper with great exactness both as to form and size ; finer details must of course be sketched in free hand.
Every preparation should first be examined with a low power, and only after the student has studied the specimen as a whole and found instructive areas should the higher powers be used.
II. The Microscopic Preparation
In many cases the making of a microscopic preparation is a very simple procedure, especially when fresh objects are to be examined. A drop of blood, for instance, may simply be placed upon a slide, covered with a cover-slip, and examined. Other objects, as the mesentery, thin transparent nerves, detached epithelia, spermatozoa, etc., need no further preparation, but may be examined at once.
Portions of larger organs are often studied after having been teased, which may be done by means of two needles fastened in handles. If the objects be composed of fibers running in parallel directions, one needle is thrust into the substance to hold it in place, while the other is used to tear the fibers apart. This method is used in examining muscles, nerves, tendons, etc.
Some tissues are so constituted that they can only be investigated by means of sections, which permit a study of their elements and the relationship of the same to each other. In this method an ordinary razor, moistened in some fluid, may be employed. As a rule, it is not the size of the section, but the thinness, which is important. This latter is obtained only by practice. Every microscopist ought to become accustomed to making free-hand sections with the razor. It is the simplest of all methods, is very rapid, and is especially useful in the quick identification of a tissue. In cutting fresh so-called parenchymatous tissues, such as liver and kidney, an ordinary razor is not sufficient. Here a double knife is necessary. This consists of two blades, which are so placed one above the other that their distal ends touch, while their proximal ends are slightly separated. The distance of the blades from each other is regulated by a screw. If this be removed the knives may be separated for cleaning. In making sections, only those portions of the blades are of importance which are very close together but do not actually touch. Sections are cut by drawing the moistened instrument quickly through an organ, as, for instance, a fresh liver. As the organ is cut in two, a very thin section of the tissue remains between the blades. This is removed by taking out the screw and separating the blades in normal salt solution. Organs of a similar consistence can be frozen and then cut with an ordinary razor the blade of which has been cooled. Sometimes good results may be obtained by drying small pieces of tissue, as, for instance, tendon.
As sections or small pieces of fresh tissue would soon become dry when placed on the slide, they must be kept moist during examination. They are therefore mounted in so-called indifferent fluids (placed on the slide and immersed in a few drops of the indifferent fluid and covered with a cover-slip). These have the power of preserving living tissues for some time without change. Such fluids, for instance, are the lymph, the aqueous humor, serous fluids, amniotic fluid, etc. Artifi. cial indifferent fluids are much used and should always be kept in stock. Of this class, the following are useful :
1. Physiologic saline solution: A 0.75% solution of sodium chlorid in distilled water.
2. Schultze's iodized serum: A saturated solution of iodin or tincture of iodin in amniotic fluid.
3. Ranvier's solution of iodin and potassium iodid : A saturated solution of iodin in a 2% solution of potassium iodid.
4. Kronecker's fluid : Distilled water, 100 c.c. ; sodium chlorid, 5 gm.; sodium carbonate, 0.06 gm.
5. Solution of Ripart and Petit : Copper chlorid, o. 3 gm. ; copper acetate, 0.3 gm. ; aqua camphorae, 75 c.c. ; distilled water, 75 c.c. ; and glacial acetic acid, i c.c. After mixing, this solution is yellow, but clears up within a few hours, and should then be filtered.
The examination of fresh tissues comes far from revealing all the finer details of their structure. This is partly due to the fact that the indices of refraction of the different elements of the tissues are too nearly alike, in consequence of which the outlines are somewhat dimmed ; and also, that changes occur, even during the most careful manipulation of the tissues, which result in pictures somewhat different from the normal. With many tissues and organs while yet fresh it is also somewhat difficult to obtain a separation of their constituent elements. It is therefore generally necessary to subject tissues or organs to special methods of treatment before they may be studied microscopically with any degree of profit. Certain of these methods, such as have proved by experience to possess reliability, shall receive consideration in the following pages.
Methods Of Maceration
The reagents employed for the maceration of tissues have in general the property of softening or removing, partly or completely, certain constituents of the tissues, while they at the same time harden or fix other tissue elements. Generally the ground-substance or intercellular substance is softened or removed while the cellular or other constituents undergo fixation. Tissues thus treated when subjected to teasing, crushing, shaking, or brushing with a camel' s-hair brush, are readily broken up into their constituent elements, giving useful and instructive preparations.
1. Alcohol, 30% (Ranvier). Dilute one volume of alcohol
with two volumes of distilled water. Small pieces of tissue are macerated in this solution in twenty-four hours to fortyeight hours. It is often advantageous to fix the pieces thus macerated for about an hour in -|% to i% osmic acid. Useful for macerating epithelia.
2. Dilute solutions of chromic acid, i% to -^% Small pieces of tissue remain in this solution one to several days. Useful for macerating epithelia.
3. Concentrated aqueous solution of caustic potash. Small pieces of tissue are macerated in fifteen minutes to an hour. They are then transferred to a saturated aqueous solution of acetate of potassium, which interrupts the action of the macerating fluid. Useful for macerating epithelia and involuntary and heart muscle.
4. Hydrochloric acid, 20% to 30% aqueous solution. Macerates small pieces of tissue in twelve to twenty-four hours. The pieces are then thoroughly washed in water. Useful for isolating the uriniferous tubules and macerating glands.
5. Nitric acid, 10% to 20% aqueous solution or made up with normal salt solution. Macerates small pieces of tissue in twentyfour to forty-eight hours. Wash thoroughly in water. Useful for macerating involuntary and voluntary muscle.
6. J. B. MacCallum ("Contributions to Medical Science," Baltimore, 1900) recommends the following nitric acid mixture for isolating heart -muscle fibers of embryos and adults : Nitric acid, i part; glycerin, 2 parts ; water, 2 parts. The hearts remain in this fluid from eight hours to three days, according to their size, and are then transferred to a 5 % aqueous solution of glycerin. This method is especially useful for obtaining preparations showing the arrangement of the heart -muscle fibers.
7. Nitric acid and chlorate of potassium (Schulze). Powder the chlorate of potassium and add sufficient nitric acid to make a thin paste. Embed the tissue to be macerated in this paste, in which they remain from one to several hours. They are then washed in water. Useful for isolating the branched, voluntary muscle -fibers of the tongue of a frog.
8. Concentrated sulphuric acid. Useful for isolating the cornified cells of the epidermis, nails, and hair.
The fixing fluids most used for general purposes are the following : Alcohol. Alcohol is frequently used as a fixing fluid. It is at the same time a hardening fluid, as the water of the tissues is withdrawn and their albumin coagulated. Small or thin pieces are put immediately into absolute alcohol, in which they remain for from twelve to twentyfour hours. The period required for fixation may be greatly shortened by changing the absolute alcohol at the end of one or two hours. In the case of larger pieces, a successive immersion in gradually increasing, strengths of alcohol (50%, 70%, 90%) is the method chosen. Pieces i c.c. in size remain for twenty -four hours in each grade of alcohol, larger pieces for a proportionately longer time. Alcohol used in this way is a hardening fluid rather than a fixing fluid. Carney's Acetioalcohol Mixture.
Glacial acetic acid I part.
Absolute alcohol 3 parts.
Fixes very rapidly. Pieces of i centimeter in thickness are fixed in one-half hour to one hour. The after-treatment is with absolute alcohol, which should be renewed at the end of twenty-four hours.
Carney's Acetic Acid-alcohol=chloroform Mixture.
Glacial acetic acid I part.
Chloroform 3 parts.
Absolute alcohol 6 "
Fixes very rapidly, even larger pieces in from one-half to -one hour. The after-treatment is with absolute alcohol.
Osmic acid is a reagent that kills quickly, fixes protoplasm exceedingly well, but nuclei not so well, and colors certain tissues. Only small pieces can be fixed in this fluid, as it does not easily penetrate the tissues. It is ordinarily used in a \ c / c to \ c fo aqueous solution, the objects remaining immersed twenty-four hours. They are then washed in running water for the same length of time, after which they are transferred to 90% alcohol. Very small objects may be treated with osmic acid in the form of vapor (vaporization). This is done as follows: A very small quantity of osmic acid solution is put in a small dish. The object is then suspended by a thread in such a way that it does not come in contact with the fluid. The dish should be covered with a well-fitting lid.
Flemming's Solution. A solution with a similar action, but fixing nuclear structures better than osmic acid, is the chromic-osmic-acetic acid solution of Flemming :
Osmic acid, I c / c aqueous solution . . 10 parts.
Chromic acid, I c /o aqueous solution ... 25 " Glacial acetic acid, \/o aqueous solution .10 " Distilled water 55 "
Small pieces are fixed in a small quantity of the fluid for at least twenty- four hours, sometimes for a longer period, extending even to weeks. They are then washed for twenty-four hours in running water and passed through 50%, 70%, and 80%, each twenty-four hours, into 90% alcohol.
Flemming also recommends a stronger solution, which is made as follows :
Osmic acid, 2^ aqueous solution .... 4 parts. Chromic acid, I f /o aqueous solution ... 1 5 " Glacial acetic acid ." I part.
Fol's Solution. Fol has recommended the following modification of Flemming's solution :
Osmic acid, I % aqueous solution .... 2 parts. Chromic acid, I c /c aqueous solution ... 25 " Glacial acetic acid, 2 c fo aqueous solution .5 " Distilled water 68 "
The after-treatment is the same as for Flemming's solution.
Hermann's Solution. Very good results sometimes follow the use of the platinum-acetic-osmic acid solution of Hermann (89, i). It is employed as is Flemming's solution :
Osmic acid, 2 ^ aqueous solution .... 4 parts. Platinum chlorid, \c/ aqueous solution . .15 " Glacial acetic acid i part.
After fixing with this solution, Flemming's solution, or any other osmic mixture, the subsequent treatment with alcohol may be followed by crude pyroligneous acid. The objects are placed for from twelve to twenty-four hours in the latter and then again immersed in alcohol. The result is a peculiar coloring of the specimen which often makes subsequent staining (see below) unnecessary (Hermann).
Corrosive Sublimate. An excellent fixing fluid is made by saturating distilled water or a physiologic saline solution (see p. 22) with corrosive sublimate. Small pieces, about 0.5 cm. in diameter, are immersed in this fluid for from three to twenty-four hours, are then washed in running water for twenty -four hours, and then transferred into 70% alcohol. After twenty-four hours the tissues are placed in 8o c / c for the same length of time, and then preserved in go f / c alcohol. It often occurs that after changes in temperature crystals of sublimate are formed on the surface or in the interior of the object. For their removal a few drops of a solution of iodin and potassium iodid are added to the alcohol (P. Mayer). It is a matter of indifference whether the jo c / f , 8o ( /c or 90% alcohol is thus iodized. In the further treatment of the object, as well as in sectioning, any such crystals of sublimate will not be found to be a hindrance. Indeed, in the case of very delicate objects it is often more advantageous to undertake their removal after sectioning by adding iodin to the absolute alcohol then used.
Acetic Sublimate Solution. This is an excellent fluid, and at present much used for embryonic tissues and for organs containing only a small quantity of connective tissue. To a saturated aqueous solution of sublimate, 5^ to 10% of glacial acetic acid is added. After remaining two or three hours or more in this solution, the objects are transferred to 35% alcohol, after which they are passed through the higher grades of alcohol.
Picric Acid. Small and medium-sized objects (up to i c.c.) are fixed in twenty-four hours in a saturated aqueous solution of picric acid (about 0.75%), although an immersion lasting for weeks is not detrimental, especially if the objects be of considerable size. The tissues are transferred to 70% or 80% alcohol, in which they remain until the alcohol is not colored by the picric acid. They are then preserved in 90% alcohol.
Instead of a pure solution of picric acid, the picrosulphuric acid of Kleinenberg or the picric=nitric acid of P. Mayer may be used. The first is made thus : i c.c. of concentrated sulphuric acid is added to 100 c.c. of a saturated aqueous picric acid solution. This is allowed to stand for twenty-four hours, then filtered, and diluted with double its volume of distilled water. The picric-nitric acid solution is made by adding 2 c.c. of pure nitric acid to 100 c.c. of a saturated picric acid solution. Filter after standing for twenty-four hours.
Rabl's Solutions. C. Rabl (94) recommends the following mixtures, especially for embryos : ( i ) Concentrated aqueous solution of corrosive sublimate, i vol. ; concentrated aqueous solution of picric acid, i vol. ; distilled water, 2 vols. (2) i per cent, aqueous solution of platinum chlorid, i vol. ; concentrated aqueous solution of corrosive sublimate, i vol. ; distilled water, 2 vols. In both cases, after being washed twelve hours in water (in the first preferably in alcohol) the specimens are transferred to gradually increased strengths of alcohol.
Vom Rath's Solutions. O. vom Rath (95) recommends, among others, the following two solutions: (i) Picric=osmic=acetic acid solution. Add to 1000 c.c. of a cold saturated picric acid solution i gm. of osmic acid, and after several hours 4 c.c. of glacial acetic acid. Objects are fixed, according to their size, in four, fourteen, and fortyeight hours, and then transferred to T$ c /c alcohol. (2) Picric=sub= limate=osmic acid solution. A mixture of 100 c.c. of a cold saturated aqueous picric acid solution with 100 c.c. of saturated sublimate solution is made, into which is poured 20 c.c. of a 2 c / ( osmic acid solution. 2 c.c. of glacial acetic acid may also be added. Tissues fixed by either of these fluids may be treated with pyroligneous acid or tannin. The crystals of sublimate must be removed by iodized alcohol.
Nitric Acid. Small objects may be fixed in about six hours in 3% to 5% nitric acid (sp. gr. 1.4). A longer immersion is injurious, as certain nuclear structures are affected. After washing thoroughly in running water, the tissues are treated as usual with alcohols of increasing concentration.
Chromic acid is used in a Y3% to i% aqueous solution. Small pieces are fixed for twenty-four hours, larger ones for a longer time, even weeks. The quantity of the fixing fluid should be at least more than fifty times the- volume of the tissues to be fixed. The objects are subsequently washed in running water and run through the ascending alcohols. This last should be done in the dark.
Two or 3 drops of formic acid may be advantageously added to each 100 c.c. of chromic acid solution (C. Rabl).
Potassium bichromate 2 to 2.5 gm.
Sodium sulphate I "
Water loo c.c.
With this solution it requires several weeks for proper fixation, and the process must be conducted in the dark. During the first few weeks the solution should be changed every few days, and later once a week. According to the results desired, the pieces are either washed out in running water and subsequently treated in the usual manner with alcohol, or they are placed directly in 70%, which is later replaced by 80 % and goj/o alcohol. It is important that all these procedures should take place in the dark.
The use of Erlicki's fluid (potassium bichromate, 2^ gm.; cupric sulphate, 0.5 gm., and water, 100 c.c.) is quite similar to that of Miiller's, except that it acts much more quickly. A temperature of 30 C. to 40 C. shortens the process in both cases considerably, Miiller's fluid fixing in eight and Erlicki's in three days.
Tellyesnicky's Fluid. This solution gives better nuclear fixation than Miiller's fluid.
Potassium bichromate 3 gm.
Glacial acetic acid 5 c - c Water 100 "
Small pieces of tissue remain in this fluid for one or two days. Larger pieces may also be used, but require a longer period of fixation. Wash thoroughly in flowing water. Dehydrate in graded alcohol, beginning with 15%.
Potassium bichromate 2.5 gm.
Sodium sulphate I "
Corrosive sublimate 5 "
Glacial acetic acid 5 c - c Water 100 "
It is advisable to add the glacial acetic acid in proper proportion to the quantity of the solution to be used, and not to add it to the stock solution. The tissues are allowed to remain for from six to twenty-four hours in this mixture, in which they float for a short time. They are then washed in running water for from twelve to twenty-four hours, and transferred to gradually concentrated alcohols. Crystals of sublimate which may be present are removed with iodized alcohol. Zenker's fluid penetrates easily, and fixes nuclear and protoplasmic structures equally well without decreasing the staining qualities of the elements.
Formalin (Formol). Of recent years formalin, which is a 40 % solution of the gas formaldehyd in water, has been much used as a fixing fluid. It is best employed in the form of a solution made by adding 10 parts of formalin to 90 parts of water or normal saline solution. Small pieces of tissue remain in this solution for from twelve to twentyfour hours, larger pieces or organs a number of days or weeks, and are then transferred to 90% alcohol.
Potassium Bichromate and Formalin.
Potassium bichromate, 2% to 3% aqueous
solution 90 parts.
Formalin 10 "
Tissues remain in this fluid from several days to several weeks, depending on their size. Wash thoroughly in water and dehydrate in alcohol. Useful for fixation of central nervous system.
We have attempted to give only the fixing and hardening fluids commonly employed for general purposes. There are numerous other fluids used for special purposes ; these will be noticed under the headings of the corresponding tissues and organs.
Infiltration And Imbedding
Few tissues have a consistency, even after fixation, which enables them to be cut into sections thin enough to be studied under high magnification, without being especially prepared for this purpose. To admit of sectioning, it is generally necessary to imbed them in media which offer no resistance to the knife, while giving them firmness, and do not obscure the structure of the sections when cut, or which may be removed from the sections by methods which are not harmful to them. The media used for imbedding may be classed under two heads : ( i ) Such as are fluid when warm, and may in this state be caused to penetrate the tissue, and are solid when cold ; ( 2 ) such as are fluid when in solution, and in this state will penetrate tissues, but which become solid on the evaporation of the solvent. The best example of the former class of substances is paraffin ; and of the latter, celloidin (collodion or photoxylin).
i. PARAFFIN IMBEDDING.
In describing the method of paraffin infiltration and imbedding it is assumed that the tissues have been previously fixed and hardened and are in alcohol ready for further manipulation. From the hardened tissues small flat pieces are cut with a sharp knife or razor. If possible, they should be square, rectangular, or triangular in shape, their surfaces not exceeding i^ square inch, and their thickness from ^ to ^ of an inch. Pieces of larger size may be imbedded, if desired, provided the requisite care be exercised. The pieces selected are placed in absolute alcohol, in which they remain until thoroughly dehydrated. From the latter they can not be passed directly into paraffin, as alcohol dissolves only a small percentage of paraffin, and, consequently, the preparation would not be infiltrated with the imbedding mass. The pieces of tissue are therefore first placed in some fluid which mixes with absolute alcohol and at the same time dissolves the paraffin. There are many reagents which have this property, such as xylol, toluol, chloroform, and a number of oils (oil of turpentine, oil of cedar, oil of origanum, etc.). Of these xylol may be recommended for general use. In the xylol the tissues remain for from two to twelve hours, the time depending somewhat on the size of the pieces and on the density of the tissue. When thoroughly permeated by the xylol, they are transparent. From the xylol (toluol, chloroform, or oils) the tissues are placed in melted paraffin. Two kinds of paraffin are generally used, one having a melting point of 38 to 40 C. soft paraffin and another with a melting point of 50 to 58 C. so-called hard paraffin. The paraffin should always be filtered before using. This is best done by using a hot-water filter. It is essential that melted paraffin have a constant temperature while the tissues are being infiltrated. This is attained by placing the receptacle containing the paraffin in a paraffin oven regulated by means of a thermostat to a temperature about two degrees above the melting point of the hard paraffin.
Filtered hard and soft paraffin may be kept in suitable glass beakers in respective compartments in the paraffin oven. After the tissues are thoroughly permeated with the xylol, this is poured off and melted soft
paraffin added, and the dish replaced in the paraffin oven. In the soft paraffin the tissues remain from one to four hours, at the end of which time the soft paraffin is poured off and hard paraffin added, and the dish again placed in the oven. In the hard paraffin the tissues remain from Fig. 3,-Box for imbedding tissues. two to twelve hours, depending on the size of the pieces. They are now ready to be imbedded. Two metallic L's are placed together on a glass or metal plate in such a way as to make a rectangular box. (Fig. 3.) This is filled with melted hard paraffin taken from the oven. Before the paraffin cools, the piece of tissue to be imbedded is taken from the hard paraffin in the oven and placed with one of its flat surfaces against one end of the box. If several pieces of tissue are to be imbedded, a piece may thus be placed in each end of the box. While transferring the tissues from the hard paraffin to the imbedding box they should be handled with forceps, the blades of which have been warmed in a flame. As soon as the paraffin in which the tissues are imbedded has cooled sufficiently to allow the formation of a film over the melted paraffin, the imbedding box is placed in a dish of cold water. This cools the paraffin quickly and prevents its becoming brittle. A stay of from five to ten minutes in the cold water hardens the paraffin so that the L's may be removed, and the paraffin block containing the imbedded tissue may be taken from the plate. It is well to place the paraffin block thus obtained back into the cold water for a short time, so that it may become hard all the way through. As the paraffin often adheres closely to the glass or metal plate and the L's, it is advisable to cover these parts with a very thin layer of glycerin before imbedding. There is then no difficulty in separating them from the paraffin block.
If a large number of small pieces of tissue are to be imbedded, it is often more convenient to imbed them in a small flat dish of suitable size. The dish to be used is covered on its inner surface with a thin layer of glycerin and partly filled with hard paraffin and the several pieces of tissue to be imbedded transferred to it and arranged on the bottom of the dish. As soon as a film forms over the paraffin the dish is placed carefully in cold water and the paraffin allowed to harden. The large piece of paraffin thus obtained may then be cut into several smaller pieces, each containing a piece of the imbedded tissue.
On transferring an object from one fluid into another, so-called currents of diffusion occur, which produce, especially in such tissues as contain cavities, shrinkage and tearing. This often results in totally changing~"the finer structure of the tissues. It is therefore necessary to proceed with greater caution than in the method above indicated. Mixtures containing different percentages of alcohol and the intermediate fluid (xylol, toluol, chloroform) may be prepared, and the object, according to its delicacy, passed through a greater or smaller number of such solutions. In ordinary cases a single mixture of alcohol and the intermediate fluid is sufficient, the object remaining in the solution for a length of time varying with its size before being passed into the pure intermediate fluid. This part of the treatment may of course be slowed or hastened according to the number of such mixtures, each succeeding one containing more and more of the intermediate fluid. After the object has been passed into the pure intermediate fluid it should be just as carefully passed into the infiltrating fluid. If paraffin is to be used and the object be delicate, the following method is advisable : The object is placed in a glass vessel half filled with the intermediate fluid, into which a few pieces of soft paraffin are dropped. The vessel is then covered and allowed to remain at the temperature of the room. When the paraffin is dissolved the cover is removed and the vessel placed in a paraffin oven kept at a temperature corresponding to the melting point of the paraffin. The volatile intermediate fluid evaporates gradually, and in a few hours the object is infiltrated with an almost pure soft paraffin. It may now be transferred into pure melted hard paraffin. In this the tissue remains for a longer or shorter time, according to its size.
It is often of advantage to infiltrate the tissues in a partial vacuum. In this way there is obtained a better infiltration of the tissues with the paraffin, and this seems to obtain a better consistency. Especially is this method to be recommended in imbedding larger embryos or tissue with cavities. A simple and convenient method is as follows : a glass bottle of suitable size is warmed and partly filled with melted hard paraffin and placed at one end of a copper plate, the other end of which is heated by a flame, care being taken to heat the copper plate only sufficiently to keep melted the paraffin in the bottle. The bottle is fitted with a rubber cork with two holes, into which have been inserted two L~ shaped glass tubes, provided, the one with a short rubber tube, which is clamped, the other with a tube of sufficient length to reach to a Chapman water-pump. The tissues are placed in the paraffin, the bottle tightly corked, and the water-pump allowed to play for about half an hour, after which the tissues are imbedded in the paraffin used during this procedure. High temperatures are, as a rule, injurious to tissues. This should always be borne in mind, and the student should aim to keep his specimens at the lowest possible temperature conducive to proper infiltration. If for any reason higher temperatures become necessary, the exposure of the tissues to their action should be as brief as possible. The paraffins most used have a melting point of 40 to 60 C. The kind of paraffin used should depend upon the temperature of the room in which the sectioning is to be done. It is even well to have different mixtures of hard and soft paraffins at hand, so that, if the temperature of the room be low, tissues may be imbedded in a softer mixture, and vice versa.
The process of infiltrating and imbedding in paraffin is represented by the following diagram (instead of xylol, other intermediate fluids may be used) :
t Xylol -<
t Xylol-paraffin (cold)
t Xylol-paraffin (in paraffin oven)
t Soft paraffin -^ f Hard paraffin
The size and density of the tissues must necessarily regulate the length of time necessary for their proper infiltration. It is therefore hardly possible to give any definite figures. In presenting the following table we have taken as a standard any tissue that has the general consistency of liver fixed in alcohol. The time is given in hours, and should in each case be regarded as a minimum. A longer stay in any one fluid will, under favorable circumstances, do no harm.
SMALL OBJECTS UNDER
MIDDLE-SIZED OBJECTS UP
LARGE OBJECTS UP TO 10
VERY LARGE OBJECTS, ALTHOUGH NOT MORE THAN A
I MM. IN
TO 5 MM. IN DIAMETER.
FEW CM. IN Di
Absolute alcohol . . .
For a longer or
shorter time in the fluids, according to the
From now on in par
size of the object.
affin oven :
Hard paraffin ....
The best and most convenient celloidin to use in microscopic work is Schering's granular celloidin, put up in i -ounce bottles. Of this a stock or thick solution is prepared by dissolving 6 gm. of the celloidin in 100 c.c. of equal parts of absolute alcohol and ether. Of this, when required, a thin solution is prepared by diluting a quantity of the stock solution with an equal quantity of the ether and alcohol solution.
The hardened tissues are cut into small pieces, which should not be much more than J^ of an inch in thickness and not have a surface area of more than ^ of a square inch. Much larger pieces of tissue may be imbedded in celloidin. This is not advised, however, unless it is necessary to show the whole of the structure to be studied. The pieces to be imbedded are placed for twenty-four hours in absolute alcohol, and are then transferred for twenty-four hours to a mixture of equal parts of absoute alcohol and ether. Then they go into the thin celloidin solution, where they remain for from twenty-four hours to several days, depending on the size and density of the pieces to be imbedded. The pieces of tissue are then transferred to the thick celloidin solution, where they again remain for from twenty-four hours to several days. If it is desired to imbed large pieces, especially if these be of the medulla or brain, the stay in the celloidin solutions should be lengthened to several weeks. The hardening of the celloidin may now be obtained by one of several methods.
A sufficient quantity of the stock or thick celloidin solution to cover well the tissues to be imbedded is poured into a flat dish large enough to allow the pieces to be imbedded to be arranged on its bottom and leave a space of about ^ of an inch between adjacent pieces. The dish is then covered, not too tightly, and set aside to allow the ether and alcohol to evaporate. In one or two days the celloidin is usually hard enough to cut into small blocks, each block containing a piece of the imbedded tissue. The blocks of celloidin are now further hardened by placing them in 80% alcohol. A stay of several hours in this alcohol is usually sufficient to give them the hardness required for section cutting. After the celloidin pieces have obtained the right degree of hardness they are to be stuck to small pieces of pine wood or vulcanized fiber so that they may be clamped into the microtome. This is done in the following way : A piece of celloidin containing a piece of tissue is trimmed with a sharp knife so that only a rim of celloidin about ^ of an inch in thickness surrounds the piece of tissue. It is now placed for a few moments in the ether and alcohol solution. This is to soften the surfaces of the celloidin. One end of a small pine -wood or vulcanized-fiber block about one inch long, the cut end of which has a surface area slightly larger than the celloidin block, is dipped for a few moments into the ether and alcohol solution and then into the thick celloidin. The celloidin block is now taken from the ether and alcohol solution, dipped into the celloidin, and pressed against the end of the wooden or vulcanized-fiber block, which has been coated with the celloidin. The whole is now set aside for a little while to allow the celloidin to harden slightly, and is then placed in 80% alcohol. In the alcohol it may remain indefinitely ; it may, however, be used for cutting as soon as it again becomes hard.
The piece of tissue to be imbedded may be mounted at once on pine-wood or vulcanized-fiber blocks from the thick celloidin solution by pouring a small amount of thick celloidin over one end of the block and placing the piece of tissue from the thick celloidin solution onto the layer of celloidin on the block. In three to four minutes a layer of the thick celloidin solution is poured over the piece of tissue and the end of the block. It may be necessary to do this several times if the piece of tissue is large or of irregular shape. The block is now set aside for about five minutes, and is then placed in 8b% alcohol, where it remains until the celloidin is hard, or until it is desired to cut sections.
The tissues may be imbedded by pouring the thick celloidin, together with the objects, into a small box made of paper. The surface of the celloidin hardens in about an hour (preliminary hardening), after which the whole is transferred to 80% alcohol, in which the final hardening takes place. The paper is then removed, the block of celloidin trimmed to a convenient size and fastened on a block.
While being cut, celloidin preparations are kept moistened with 80% alcohol. Organs consisting of tissues of varying consistency, as well as very dense objects, can be cut with better results in celloidin than in paraffin. On the other hand, celloidin sections can never be cut as thin as paraffin sections, and the after-treatment (see below), fixation on the slide, etc., are much more complicated than in the case of paraffin sections.
The following is a diagram showing the process of infiltration and imbedding in celloidin.
t Abs. alcohol
t Abs. alcohol and ether (in equal parts)
t Thin celloidin solution
f Thick celloidin solution
t Sof alcohol
To combine the advantages which infiltration in celloidin and in paraffin offer, a method of celloidin -paraffin infiltration is recommended. Preparations that have been imbedded in celloidin or photoxylin and hardened in 80% alcohol are placed for about twelve hours in 90% alcohol, from which they are transferred to a mixture of equal parts of oil of origanum and 90% alcohol. They are then immersed for a short time in pure origanum oil, then in a mixture of equal parts of origanum oil and xylol, and finally in pure xylol. From this point the regular method of infiltrating with paraffin is followed, care being taken that the pieces remain for as short a time as possible in the different fluids, in order that the celloidin may not become brittle.
Very thin sections may be obtained by painting the cut surface with a thin layer of a very dilute celloidin solution. This hardens and gives the tissue a greater consistency. This treatment is useful in the combined celloidin-paraffin method, as well as when paraffin alone is used.
The Microtome And Sectioning
Instruments known as microtomes have been devised in order that section cutting may be rendered as independent as possible of the skill of the individual, but more especially t<3 obtain series of sections of uniform thickness. Their construction varies greatly. Some of these instruments, as the so-called rocking microtomes, are so specialized that they only cut paraffin objects when the knife' is transversely placed. Others have a more general function, celloidin as well as paraffin objects being sectioned with the knife in any position. To the latter class belong the sliding microtomes.
In figure 4 is shown an instrument which may be recommended for general laboratory work. This instrument consists of a horizontal base which rests on the table, and a vertical plate (#), and a slide (^) which supports a block (V), to which is fastened a knife by means of a thumbscrew (V). On the other side of the vertical plate is a metal frame (<?), into which are fastened the paraffin and celloidin blocks ; this frame is attached to a slide (/"), which may be elevated or lowered by a feed (g~). This feed consists of a micrometer screw acting on the lower surface of the slide. The micrometer screw is provided with a milled head, divided into a definite number of parts which bear a definite rela
Fig. 4. Laboratory microtome.
tion to the pitch of the micrometer screw. The instrument shown in the figure is further provided with a lever (/), which may be so adjusted as to move the milled head on the micrometer screw i or any given number of notches at each movement of the lever ; and as each notch on the milled head has a value of 5 microns (oinnr ^ an i ncn )> every time the milled head is moved i notch (toward the manipulator) the slide carrying the clamp holding the tissue is elevated 5 microns ; 2 notches would elevate the tissue 10 microns (ToW f an i ncn ) ') 4 notches, 20 microns (j^Vo" f an i ncn )> etc. It is not essential to have a lever attached to the instrument as above described, although this is very convenient ; if not present, the milled head is moved the desired number of notches with the hand.
Minot has recently devised two kinds of microtomes which deserve special mention, and are especially to be recommended for accurate work. One of these (see Fig. 5) is known as the "Precision Microtome." It consists of a square frame made of cast-iron, to which the knife is fastened. .3
Fig. 5. Minot automatic precision microtome.
Fig. 6. Minot automatic rotary microtome. 34
Beneath the frame which supports the knife are two horizontal ways, upon which runs the sliding carriage supporting an adjustable object-carrier. The object is raised by a micrometer screw, fed automatically by a largetoothed wheel attached to the bottom of the screw. Both paraffin and celloidin sections may be cut with this instrument. The other type of microtome is known as the "New Rotary Microtome." In this instrument (see Fig. 6) the knife is carried by two upright standards which can be adjusted as to their distance from the object. The object, which needs to be imbedded in paraffin, is fixed to an object -carrier, which may be adjusted to any plane, and which is fixed to a vertical carriage, held by adjustable gibs against the vertical ways, and which is raised or lowered by a crank, working in a slide, and attached to an axle turned by the wheel. The vertical carriage also carries the micrometer screw, to which is attached a toothed wheel ; this is turned by a pawl which acts upon it. This instrument may be most highly recommended for the cutting of serial sections.
In cutting paraffin sections with the sliding microtome the knife is placed at an angle of about 35 to 40 to the horizontal plate of the microtome. Sections are cut more easily with the knife in this position than when the knife is placed at right angles to the microtome, as is often recommended, and it does not seem that the tissues suffer materially from distortion when they are cut with the knife at an angle, as is sometimes claimed.
Before fastening the paraffin blocks into the clamp on the microtome, preparatory to cutting sections, the paraffin is trimmed with a sharp knife from the end of the paraffin block until the tissue is nearly exposed, care being taken, however, to leave a flat surface. The top of the paraffin block is then beveled off on three sides to within a very short distance of the tissue. The fourth side, that which faces the knife when the block is clamped in the microtome, should be trimmed only to within about y& of an inch of the tissue. This edge of paraffin is made use of, as will be seen in a moment, for preventing the sections from curling while they are being cut. The paraffin block is now ready to be clamped in the microtome. This is done in such a way that the paraffin block just escapes the knife when drawn over it. A number of rather thick sections (20 to 40 microns) are cut by moving the micrometer screw from right to left 4 to 8 notches every time the knife has been drawn over the paraffin block and has been brought back again, until it is noticed that the knife touches all parts of the top of the paraffin block, or until the tissue is fairly exposed. (In this description reference is made to the simple laboratory microtome shown in Fig. 4. ) The succeeding sections may now be kept. It may perhaps be well to state that it is better not to try to cut very thin sections at the beginning; sections 15 to 20 microns in thickness will answer very well. To begin with, then, the milled head of the micrometer screw is turned 4 notches from left to right, and the knife is drawn over the block with a steady, even pull, and without using undue pressure. Usually the sections will curl up as they are being severed from the paraffin block. This may very readily be prevented by holding the tip of a camel's-hair brush, which has been pointed by drawing it between the lips, against the edge of the section as soon as it begins to curl. A little practice will enable one to do this almost automatically. The sections are transferred to paper by means of the camel's-hair brush, which process is facilitated if the brush has been slightly moistened with saliva, as the section will then adhere lightly to the brush.
If the tissues are well imbedded and not too hard, and if the knife is sharp and properly adjusted, paraffin sections may be cut in such a way that each succeeding section adheres to the preceding one, so that actual ribbons of paraffin sections may be made. In order to do this, the knife should be at right angles to the microtome. The paraffin block should be trimmed in such a way that when clamped in the microtome ready for cutting sections, the surface of the paraffin block facing the knife should be exactly parallel to its edge, also to the opposite side of the block. In other words, 2 sides of the paraffin block should be parallel to each other and to the knife ; then if the paraffin is of the right consistency, which' must be ascertained by trying, the sections as they are cut will adhere to each other and form a ribbon. If the sections do not adhere to each other it is quite probable that the paraffin is a little too hard. This may often be remedied by holding an old knife or other metallic instrument which has been heated in a flame near the two parallel surfaces for a few moments. Care should be taken not to allow this instrument to touch the paraffin. This is a very convenient and rapid way of cutting paraffin sections. To facilitate the cutting of a paraffin possessing a relatively low melting point in a room with a high temperature, the cooled knife of Stoss may be used. This is so made that a stream of ice-water may be passed through a tube running through the entire length of the back of the blade. Paraffin sections may be cut in ribbons serial sections on an ordinary sliding microtome; for this purpose, however, the "automatic rotary microtome" of Minot is especially recommended.
Celloidin Sections. Before fastening the block of wood or vulcanized fiber to which the celloidin blocks have been fixed in the clamp on the microtome, the celloidin should be trimmed with a sharp knife from the top of the block until the tissue is nearly exposed, care being taken to leave a flat surface. The sides of the celloidin block are then trimmed down, if necessary, to within about Jg- of an inch of the tissue. The block is now clamped in the microtome at such a level that it just escapes the knife when drawn over it. The knife is placed at an angle of about 45, or at even a greater angle. During the process of cutting, the knife, as also the tissue, must be kept constantly moistened with 80 % alcohol. This is perhaps most easily accomplished by taking up the 80% alcohol with a rather large camel' s-hair brush and dipping this on the celloidin block and on the knife. A number of rather thick sections are cut until the knife touches the entire surface of the block or until the tissue is well exposed. The sections may now be kept. The block is raised 20 to 15 microns, and the knife, which should be well moistened with 80% alcohol, is drawn over the block with a steady pull, not with a jerk. The sections are transferred from the knife to distilled water. This is perhaps most conveniently done by placing the ball of one of the fingers of the left hand under the edge of the knife, in front of the section, and drawing the section down onto the finger with the camel's-hair brush. The finger is then dipped into the distilled water when the section floats off. If the sections can not be stained within a few hours after they are cut, they are best transferred to a dish containing 8o c /c alcohol, in which they may be left until it is desired to stain them.
The sliding microtomes may be provided with an arrangement for freezing tissues a so-called freezing apparatus. This consists of a metal plate on which the tissue is laid; an ether or rigolene atomizer plays upon its lower surface, cooling and finally freezing the object, which is then cut.
A drop of fluid (physiologic saline solution, water, etc.) is placed upon the knife, in which the section thaws out and spreads. A better and more rapid method of freezing tissues consists in the use of compressed carbon dioxid, as recommended by Mixter. Cylinders containing about twenty pounds of the liquid gas may be obtained from Bausch & Lomb, who also make a small microtome designed for this purpose. In figure 7 is shown the lower third of a cylinder for compressed carbon dioxid firmly fastened to a thick board, and connected by means of a short piece of strong rubber tubing with the freezing box of the microtome. The handle of the escape valve is from 8 to 10 inches long, so that the quantity of escaping gas may be readily controlled. The pieces of tissue are placed on the freezing box of the microtome and the escape valve slowly opened until a small quantity of the gas escapes. Small pieces of tissue are frozen in about thirty seconds to a minute ; tissues taken from alcohol should be washed for a short time in running water before freezing. A strong razor may be used for cutting sections ; or better, a wellsharpened blade of a carpenter's plane, as suggested by Mallory and Wright. Sections are transferred to distilled water or normal salt solution, and if fixed may be stained at once. Sections of fresh tissue should be taken from the normal salt solution and transferred to a fixing fluid.
Bardeen has devised a microtome to be used with compressed carbon dioxid, which presents many advantages. It admits of better control of the temperature of the freezing stage and there is less carbon dioxid wasted than with other instruments of this type. It freezes almost instantaneously, since the expanding carbon dioxid is caused to pass through a spiral passage contained in the freezing chamber. In this apparatus the microtome is attached to the steel cylinder containing the carbon dioxid. It is impossible to cut thin sections with a knife that is not sharp, or with one that is nicked. A few directions as to sharpening a micro= tome knife may therefore not be out of place. For this purpose a good
Fig. 7- Apparatus for cutting tissues frozen by carbon dioxid.
Belgian hone is used, which should be moistened or lubricated with filtered kerosene oil or with soap as necessity demands. While sharpening the knife it is grasped with both hands with one by the handle, with the other by the end. The hone is placed on a table with one end directed toward the person sharpening. If the knife is very dull, it is ground for some time on the concave side only (all microtome knives are practically plane on one side and concave on the other), with the knife at right angles to the stone. It is carried from one end of the stone to the other, edge foremost, giving it at the same time a diagonal movement, so that with each sweep the entire edge is touched (see Fig. 8). In drawing back the knife, the edge is slightly raised. The knife is ground on the concave side until a fine thread (feather edge) appears along the entire edge. It is then ground on both sides, care being taken to keep the knife at right angles to the stone, to keep it flat, and to use practically no pressure. It is a good plan to turn the knife on its back when the end of the stone is reached. On the return stroke, the knife is again held at right angles to the stone, the same diagonal sweep is used (see Fig. 8), so that the whole edge of the knife is touched with each sweep. The grinding on both sides is continued until the thread above mentioned has disappeared. The knife should now be carefully cleaned and stropped, with the back of the knife drawn foremost. The strop should be flat and rest on a firm surface.
Fig. 8. Diagram showing direction of the movements in honing.
The Further Treatment Of The Section
1. FIXATION TO THE SLIDE AND REMOVAL OF PARAFFIN.
Sections obtained by means of the microtome undergo further treatment either loose or, better, fixed to a slide or cover -glass, thus making further manipulation much easier.
The simplest, surest, and most convenient method of fixing paraffin sections to the slide is by means of the glycerin-albumen of P. Mayer (83.2). Egg-albumen is filtered and an equal volume of glycerin added. To prevent decomposition of the fluid a little camphor or sodium salicylate is placed in the mixture. A drop of this fluid is smeared on the slide or cover-slip as evenly and thinly as possible. A section or a series of sections arranged in their proper sequence is then placed upon the slide so prepared. Any folds in the section are smoothed out with a brush, and the section or the whole series gently pressed down upon the glass. When the desired number of sections are on the slide or cover-slip, they are warmed over a small spirit or gas flame until the paraffin is melted. At the same time the albumen coagulates. The sections are now fixed, and are loosened from the glass only when agents are used which dissolve albumen, as, for instance, strong acids, alkalies, and certain staining fluids. If it is desired that a given space, say the size of a cover-slip, be filled up with sections as far as possible, an outline of the cover-slip to be used may be drawn upon a piece of paper and placed under the slide in the required position.
A second and in many respects better method is the fixation of the section with distilled water (Gaule). The paraffin sections are spread in proper sequence on a thin layer of water placed on the slide. There should be sufficient water to float the sections. The slide is then dried in a warm oven kept at 30 to 35 C., or gently heated by holding it at some distance from a spirit or gas flame (the paraffin should not melt). By this treatment the sections are entirely flattened out. The superfluous water is either drained off by tilting or drawn off with blotting-paper, the sections are definitely arranged with a brush, and the whole is placed for several hours in a warm oven at 30 to 35 C. The sections thus dried are exposed, over a flame, to a temperature higher than the melting point of the paraffin, and from now on can be subjected to almost any after-treatment. The slide or cover-slip should be thoroughly cleaned (preferably with alcohol and ether), as otherwise the water does not remain in a layer, but gathers in drops.
The advantage of this method lies in the fact that the evaporated water can have no possible influence on the subsequent staining of the sections, while albumen, especially if it be in a thick layer, is sometimes stained, thus diminishing the transparency of the preparation.
This method, although trustworthy for alcohol and sublimate preparations, often fails with objects' that have been treated with osmic acid, chromic acid and its mixtures, nitric acid, and picrosulphuric acid. In such cases advantage may be taken of the so-called Japanese method, which is a combination of the above fixation methods. A little Mayer's albumen is placed on the slide and so spread about that hardly a trace of the substance can be seen. The slide is then put in a warm oven heated to 70 C. This temperature soon coagulates the albumen, after which the sections are fixed to the slide by the water method (Rainke, 95). The procedure can be varied by adding to the distilled water one drop of glycerin-albumen or gum arabic to every 30 c.c. of water (vid. also Nussbaum).
When a large number of paraffin sections are to be fixed to cover-slips, the following method may be recommended : A small porcelain evaporating dish is nearly filled with distilled water and placed on a stand which elevates it 6 to 8 inches from the table. A number of sections are placed on the water, which is then heated by means of a gas flame until the sections become perfectly flat, care being taken not to raise the temperature of the water sufficiently to melt the paraffin. Each section is then taken up on a cover-slip coated with a very thin layer of Mayer's albumen fixative. During this procedure the cover-slips are held by forceps, and the sections are guided by means of a small camel's-hair brush. When all the sections have thus been placed on cover-slips they are placed for four to six hours in a warm oven maintained at 30 to 35 C.
Removal of Paraffin. Before paraffin sections, either fixed or loose, are subjected to further manipulation, the paraffin surrounding the tissues must be removed. This may be done by means of several agents having a solvent action on paraffin, such as xylol, toluol, oil of turpentine, etc. After the paraffin has been dissolved, the sections are transferred to absolute alcohol and by this means prepared for further treatment with aqueous or weak alcoholic solutions.
Dextrin Method of fixing Paraffin Sections. This method is to be recommended for class-room purposes where 30 to 50, or even more sections need to be stained at one time.
The following solutions are kept on hand :
Solution i :
A solution of equal parts of white sugar
and boiling distilled water 300 c.c.
A solution of equal parts of distilled water
and dextrin 100 "
Absolute alcohol 200 "
Mix the sugar and dextrin solutions in a mortar, and add very slowly, while constantly stirring, the absolute alcohol ; filter through fine muslin. Keep in a wide-mouthed bottle through the cork of which there has been placed a broad camel's-hair brush.
Solution 2 :
Photoxylin IO gm.
Absolute alcohol looc.c.
Ether 500 "
The sections to be stained are cut and arranged on a clean piece of paper. A clean glass plate is coated with a thin layer of solution No. i. The sections are arranged on this and pressed against the plate with the finger. The plate is now placed in a warm oven (temperature 40 C.), where it remains for several hours. The plate is then warmed over a flame until the paraffin of the sections begins to melt and is then placed in a tray containing xylol, where it remains until the paraffin is dissolved. It is then transferred to a tray containing 95^ alcohol and the xylol removed. The plate is next taken from the tray and the alcohol drained off. The plate is now covered with a thin layer of solution No. 2, and set aside, at an angle, until the photoxylin dries. The plate is now placed in the staining fluid, in which, or in the water used in washing off the staining fluid, the thin layer of photoxylin, to which the sections adhere, separates from the plate. This thin film may now be treated as one section and carried on in this form through the several stages of staining and clearing until the process is completed. The individual sections are cut from the film with scissors.
Celloidin preparations can not be fixed to the slide with the same degree of certainty, although many sections may be treated at one time. The celloidin sections can be collected in their sequence on strips of paper by gently pressing such a strip, on the blade of a knife, onto the section floating in the alcohol. The sections adhere to the paper, and in this way the entire surface of the strip may be covered by series of sections. To prevent the drying of the sections, a number of such strips are laid in rows on a layer of blotting-paper moistened with 70% alcohol. A glass plate of corresponding size is painted with very fluid celloidin. After the layer of celloidin is dry, the strips of paper are laid, one by one, on the glass plate, with sections downward, and the fingers gently passed over the reverse side. This process is continued until the entire surface of the glass is covered. On carefully raising the strips it is seen that the sections will adhere to the layer of celloidin. (To prevent drying, sections must be kept moistened with 70% alcohol.) After first drying the sections with blotting-paper, a second layer of very thin celloidin is painted on the surface of the glass plate. When this layer is also dry, the plate with its adherent sections is placed in water. Here the double layer of celloidin containing the sections is separated from the glass, and is ready for further manipulation. Before mounting, the sheet of celloidin is cut with scissors into convenient portions.
In the case of celloidin sections, if it be desirable to preserve the surrounding celloidin, care should be taken that the preparations should not come in contact with any agents dissolving celloidin. These latter are alcohols from 95^ upward, ether, several ethereal oils, especially oil of cloves, but not the oils of origanum, cedar wood, lavender, etc.
It is in most cases necessary to stain tissues to bring clearly to view the tissue elements and their relation to each other. The purpose of staining is therefore to differentiate the tissue elements. The differential staining is due to the fact that certain parts of the tissue take up more stain than others. Staining of sections may be looked upon as a microchemic color reaction, and has therefore a value beyond the mere coloring of sections so that they may be seen more clearly.
Broadly speaking, stains used in microscopic work may be divided into basic stains, which show special affinity for the nuclei of cells and are therefore known as nuclear stains, and acid stains, which color more readily the protoplasm protoplasmic stains. Certain stains, which we may know as selective stains (they maybe either basic or acid), color one tissue element more vividly than others, or to the exclusion of others. Since the various tissue elements show affinity for different stains, preparations may be colored with more than one stain. Accordingly we have simple, double, triple, and multiple staining.
Certain stains are also especially adapted for staining in bulk or mass that is, staining a piece of tissue before it is sectioned.
Carmin. Aqueous Borax=carmin Solution. 8 gm. of borax and 2 gm. of carmin are ground together and added to 150 c.c. of water. After twenty-four hours the fluid is poured off and filtered. The sections, previously freed from paraffin and treated with alcohol, are placed in this fluid for several hours (as long as twelve), and then washed out in a solution of 0.5 to i c / c hydrochloric acid in 70% alcohol. They are then transferred to 70% alcohol.
Alcoholic Borax=carmin Solution. 3 gm. of carmin and 4. gm. of borax are placed in 93 c.c, of water, after which 100 c.c. of 70% alcohol is added. The mixture is stirred, then allowed to settle, and later filtered. Sections are treated as in the aqueous borax-carmin solution.
Paracarmin is the carmin stain containing the most alcohol, and is therefore of great value.
Carminic acid I gm.
Aluminium chlorid 0.5 "
Calcium chlorid 4 "
Alcohol, 70% ioo c.c.
Paracarmin stains quickly, is not liable to overstain, and is therefore peculiarly adapted to the staining of large objects. Specimens are washed in 70% alcohol, with the addition of 0.5% aluminium chlorid or 2.5% glacial acetic acid in case of overstating (P. Mayer, 92).
Czocor's Cochineal Solution. 7 gm. of powdered cochineal and 7 gm. of roasted alum are kept suspended in ioo c.c. of water by stirring while the mixture is boiled down to half its volume. After cooling it is filtered and a little carbolic acid added. This fluid stains quite rapidly and does not overstain. Before the sections are placed in alcohol they should be washed with distilled water, as otherwise the alum is precipitated on the section by the alcohol.
Partsch recommends the following solution of cochineal : Finely powdered cochineal is boiled for some time in a 5% aqueous solution of alum, and filtered on cooling, after which a trace of hydrochloric acid is added. It stains sections in two to five minutes.
Alum-carmin (Grenadier). ioo c.c. of a 3% to 5% solution of ordinary alum, or preferably ammonia-alum, are mixed with o. 5 gm. to i gm. of carmin, boiled for one-fourth of an hour, and after cooling filtered and enough distilled water added to replace that lost by evaporation. This fluid stains quickly but does not overstain. Wash the sections in water.
Hematoxy lin. Bohmer's Hematoxylin :
Hematoxylin crystals I gm.
Absolute alcohol 10 c.c.
Potassium alum 10 gm.
Distilled water 200 c.c.
Dissolve the hematoxylin crystals in the alcohol, and the alum in the distilled water. While constantly stirring, add the first solution to the second.
The whole is then left for about fourteen days in an open jar or dish protected from the dust, during which time the color changes from violet to blue. After filtering, the stain is ready for use. Sections, either loose or fixed to the slide or cover-slip, are placed in this solution, and after about half an hour are washed with water. If the nuclei are well stained the further treatment with alcohol may be commenced. Should the sections be overstained, a condition showing itself in the staining of the cell -protoplasm as well as the nuclei, the sections are then washed in an acid alcohol wash (six to ten drops of hydrochloric acid to ioo c.c. of 70% alcohol) until the blue color has changed to a reddish-brown and very little stain comes from the section usually about one to two minutes. They are then washed in tap -water, and passed into distilled water before placing in alcohol.
Delafield's Hematoxylin :
Hematoxylin crystals 4 gm.
Absolute alcohol 25 c.c.
Ammonia alum, saturated aqueous solution 400 "
Alcohol, 95$, ioo "
Glycerin ioo "
Dissolve hematoxylin crystals in absolute alcohol and add to the alum solution, after which place in an open vessel for four days, filter, and add the 95% alcohol and glycerin.
After a few days it is again filtered. This fluid is either used pure or diluted with distilled water. Staining is the same as with Bohmer's hematoxylin.
Friedlander's Glycerin-hematoxylin :
Hematoxylin crystals 2 gm.
Potassium alum 2 "
Absolute alcohol ioo c.c.
Distilled water ioo "
Glycerin ioo "
Dissolve the hematoxylin crystals in the absolute alcohol and the alum in the water ; mix the two solutions and add the glycerin.
The mixture is filtered and exposed for several weeks to the air and light, until the odor of alcohol has disappeared, and then again filtered. It stains very quickly. Sections are afterward washed in water and are placed for a short time in acid alcohol if the nuclei are to be especially brought out.
Ehrlich's Hematoxylin :
Hematoxylin crystals 2 gm.
Absolute alcohol 60 c.c.
Glycerin "1 saturated with .... 60 "
Distilled water J ammonia alum .... 60 "
Glacial acetic acid 3 "
The solution is to be exposed to light for a long time. It is ready for use when it acquires a deep-red color.
Stain as above.
Hemalum (P. Mayer, 91). i gm. of hematein is dissolved by heating in 50 c.c. of absolute alcohol. This is poured into a solution of 50 gm. of alum in i liter of distilled water and the whole well stirred. A thymol crystal is added to prevent the growth of fungus. The advantages of hemalum are as follows : The stain may be used immediately after its preparation, it stains quickly, never overstains, especially when diluted with water, and penetrates deeply, making it useful for staining in bulk. After staining, sections or tissues are washed in distilled water.
Acid Hemalum. To the above hemalum solution is added 2% of glacial acetic acid. Stains even more rapidly than hemalum, and gives excellent nuclear differentiation. Wash sections in tap -water.
Heidenhain's Iron Hematoxylin. Good results, particularly in emphasizing certain structures of the cell (centrosome), are obtained by the use of M. Heidenhain's iron hematoxylin (92. 2). Tissues are fixed in saline sublimate solutions, alcohol, or Carney's fluid. Very thin sections (in case of amniota not over 4, a) are fixed to the slide with water and put into a 2 . 5 ff c aqueous solution of ammonium sulphate of iron for four to eight hours (not longer). After careful rinsing in water, the sections are brought into a solution of hematoxylin prepared as follows : Hematoxylin crystals i gm., absolute alcohol 10 c.c., and distilled water 90 c.c. This solution should remain in an open vessel for about four weeks, and, before using, should be diluted with an equal volume of distilled water. Staining takes place in twelve to twenty-four hours, after which the sections are rinsed in tap-water and again placed in a like solution of ammonium sulphate of iron, until black clouds cease to be given off from the sections. They are rinsed in distilled water, passed through alcohol into xylol, and mounted in balsam. Should a protoplasmic stain be desired, rubin in weak acid solution may be employed.
Coal-tar or anilin stains. Ehrlich classifies all anilin stains as salts having basic or acid properties. The basic anilin stains, such as safranin, methylene-blue, methyl-green, gentian violet, methyl-violet, Bismarck brown, thionin, and toluidin-blue are nuclear stains, while the acid anilin stains, such as eosin, erythrosin, benzopurpurin, acid fuchsin, lichtgriin, aurantia, orange G, and nigrosin stain diffusely and are used as protoplasmic stains. Safranin :
Safranin , . . I gm.
Absolute alcohol 10 c.c.
Anilin water . . 90 "
Anilin water is prepared by shaking up 5 c.c. to 8 c.c. of anilin oil in 100 c.c. of distilled water and filtering through a wet filter. Dissolve the safranin in the anilin water and add the alcohol. Filter before using.
Stain sections of tissues fixed in Flemming's or Hermann's solutions for twenty-four hours, and decolorize with a weak solution of hydrochloric acid in absolute alcohol (i : 1000). After a varying period of time (usually only a few minutes) all the tissue elements will be found to have become bleached, only the chromatin of the nucleus retaining the color.
Bismarck Brown. A very convenient color to handle is Bismarck brown. Of this, i gm. is boiled in 100 c.c. of water, filtered, and yi of its volume of absolute alcohol added. Bismarck brown stains quickly without overstaining, and is also a purely nuclear stain. Wash in absolute alcohol.
Methyl-green stains very quickly (minutes), i gm. is dissolved in 100 c.c. of distilled water to which 25 c.c. of absolute alcohol is added. Rinse sections in water, then place for a few minutes in 70 f / alcohol, transfer to absolute alcohol for a minute, etc.
Other so-called basic anilin stains can be used in a similar manner. Thionin or toluidin-blue in dilute aqueous solutions are especially useful. Nuclei appear blue and mucus red.
Double Staining. When certain stains are used in mixtures or in succession, all portions of the section are not stained alike, but certain elements take up one stain, others another. This elective affinity of tissues is taken advantage of in plural staining. If two stains are employed, one speaks of double staining.
Picrocarmin of Ranvier. Two solutions are prepared, a saturated aqueous solution of picric acid and a solution of carmin in ammonia. The second is added to the first to the point of saturation. The whole is evaporated to one-fifth of its volume and filtered after cooling. The solution thus obtained is again evaporated until the picrocarmin remains in the form of a powder. A i % solution of the latter in distilled water is the fluid used for staining.
To stain with this solution, one or two drops are placed on the slide over the object and the whole put in a moist chamber for twenty-four hours. A cover- slip is then placed over the preparation, the picrocarmin drained off with a piece of blotting-paper, and a drop of formic-glycerin (i : 100) brought under the cover-slip by irrigation. Proper differentiation takes place only after a few days, and the acid-glycerin may then be replaced by the pure glycerin. In objects fixed with osmic acid, the nuclei appear red, connective tissue pink, elastic fibers canary yellow, muscle tissue straw color, keratohyalin red, etc.
Weigert's Picrocarmin. The preparation of Weigert's picrocarmin is somewhat simpler. 2 gm. of carmin are stirred in 4 c.c. of ammonia and allowed to remain standing in a well-corked bottle for twenty-four hours. This is mixed with 200 c.c. of a concentrated aqueous solution of picric acid to which a few drops of acetic acid are added after another twenty-four hours. The result is a slight precipitate that does not dissolve on stirring. Filter after twenty-four hours. Should the precipitate also pass through the filter, a little ammonia is added to dissolve it. Both picrocarmin solutions dissolve off sections fixed to the slide with albumen.
Carmin=bleu de Lyon (of Rose). Sections or pieces of tissue are first stained with carmin (alum- or borax-carmin). Bleu de Lyon is dissolved in absolute alcohol and diluted with the latter until the solution is of a light bluish color. In this the sections or pieces of tissue are after-stained for twenty-four hours (developing bone stains, for instance, blue).
Picric acid is often used as a secondary stain, either in aqueous (saturated solution diluted i to 3 times in water) or in alcoholic solution (weak solutions in 70^, 80^, and absolute alcohol). Sections previously treated with carmin or hematoxylin are stained for two to five minutes, washed in water or alcohol, and transferred to absolute alcohol, etc. Sections stained in safranin can be exposed to the action of an alcoholic picric acid solution. A solution of picric acid in 70% alcohol may be used to wash sections stained in borax-carmin. This often gives a good double stain. Sections can also be first treated with picric acid and afterward stained with alum -carmin.
Hematoxylin. Van Gieson's Acid fuchsin-picric acid Solution. Stain in any one of the hematoxylin solutions and after rinsing sections in water counter-stain in the following :
Acid fuchsin, \% aqueous solution . ... 5 c.c. Picric acid, saturated aqueous solution . . loo "
Dilute with an equal quantity of distilled water before using. The hematoxylin stained sections remain in the solution for from one to two minutes, are then rinsed in water, dehydrated and cleared.
Hematoxylin=eosin. Sections already stained in hematoxylin are placed for two to five minutes in a i /c to 2 % aqueous solution of eosin or in a \ C J C solution of eosin in 60% alcohol. They are then washed in water until no more stain comes away, after which they remain for only a short time in absolute alcohol. In place of the eosin solution a i c /c aqueous solution of benzopurpurin may be used or the following solution of erythrosin (Held) :
Erythrosin I gm.
Distilled water 150 c.c.
Glacial acetic acid 3 drops.
Hematoxylin-safranin of Rabl (85). Sections of preparations fixed with chromic-formic acid or with a solution of platinum chlorid are stained for a short time with Delafield's hematoxylin, then counterstained for twelve to twenty-four hours with safranin and washed with absolute alcohol until no more color is given off.
Bioridi-Heidenhain Triple Stain. Of the many triple stains in use we mention only the most important, the rubin S orange G methyl-green mixture of Ehrlich and Biondi, employed according to the modification of M. Heidenhain. The best results are obtained with objects fixed in saline sublimate solution. The three stains just mentioned are prepared in concentrated aqueous solutions. (In 100 c.c. of distilled water there are dissolved respectively about 20 gm. of rubin S, and 8 gm. of orange G and methyl-green.) These concentrated solutions are combined in the following proportions: rubin S 4, orange G 7, methylgreen 8. The stock solution thus obtained is diluted with 50 to 100 times its volume of distilled water before using. The sections should be as thin as possible and fixed to the slide by the water method. They remain for twenty-four hours in the stain, and are then rinsed in distilled water or in 90% alcohol or in such with the addition of a little acetic acid (i to 2 drops to 50 c.c.). Before staining it is occasionally of advantage to treat the sections with acetic acid (2 : 1000) for one to two hours.
STAINING IN BULK.
Instead of staining in sections, entire objects can be stained before cutting. This method is in general much slower, and demands, therefore, special staining solutions, as, for instance :
Alcoholic Borax-carmin Solution. Pieces ^ cm. in diameter remain in the stain at least twenty -four hours, are then decolorized for the same length of time in acid alcohol (0.5% to i /o hydrochloric acid in 70% alcohol), and after washing in 70% alcohol are transferred to 90% alcohol. Larger objects require a correspondingly longer time.
Paracarmin. Treatment as in section staining; length of time according to size of object.
Alum-carmin of Grenacher. This never overstains. Time of staining according to size of object. Wash in water, then transfer to 70% and 90% alcohol.
Hemalum, when diluted with water, is very useful for staining in bulk. After staining, objects should be washed with distilled water.
Bohmer's hematoxylin stains small pieces very sharply. Use the same as hemalum.
Hematoxylin staining according to R. Heidenhain 's method is especially recommended for staining in bulk.
Stain objects fixed in alcohol or picric acid twenty-four hours in a -33% aqueous solution of hematoxylin ; transfer for an equal length of time to a o. 5 cf c aqueous solution of potassium chromate, changing often until the color ceases to run. Wash with water and pass into strong alcohol. This stain also colors the protoplasm, and is so powerful that very thin sections are an absolute condition to the clearness of the preparation.
If the objects have been fixed with picric acid and the latter has not been entirely washed out, staining in bulk by the above methods produces very striking differentiation.
Pieces of tissue stained in bulk may be infiltrated, imbedded, and cut according to the ordinary methods. Under these circumstances, section staining is not necessary unless a still further differentiation be desired.
In general, then, the treatment of the object is somewhat as follows : First, it is fixed in some one of the fixing fluids already described, then carefully washed, and in certain cases stained in bulk before infiltrating with paraffin or celloidin ; or the staining may be postponed until the tissue has been cut. In the latter case, the sections are subjected to the stain either loose or fastened to the slide or cover-slip.
In all cases it is absolutely essential that the paraffin be entirely removed. After the sections have been stained and washed, they are transferred to absolute alcohol in case it be desired to mount them in some resinous medium. They may also be mounted in glycerin or acetate of potash, into which they may be passed directly from distilled water.
The method of staining tissues in sections or in bulk is shown in the following diagrams :
In Bulk, gofc alcohol
In Sections. Celloidin sections Paraffin sections
in 90 tfo alcohol
Wash in water
Wash in acid alcohol
4 Absolute alcohol
70$ alcohol Absolute alcohol
Methods Of Impregnation
The impregnation methods differ from the staining methods in that in the latter the coloration is obtained by reagents in solution, while in the former the tissues are filled with fine particles which enter into combination with certain constituents of the tissue elements and are reduced in them.
Silver Nitrate Method. This method was suggested by Krause ; it was, however, brought to prominence by v. Recklinghausen. It is especially useful for staining the intercellular substances of epithelium, endothelium, and mesothelium and the ground-substance of connective tissues. The method may be used on fresh tissues or on fixed tissues ; the employment of fresh tissue is, however, more satisfactory. The tissues to be impregnated are spread in thin layers, and immersed in a o.$'/c to i'/( solution of silver nitrate for from ten to fifteen minutes; they are then rinsed in distilled water and placed in fresh distilled water or 70% alcohol or a 4% solution of formalin and exposed to direct sunlight, where they remain until they assume a brown color. The sunlight reduces the silver, in the form of fine particles which appear black on being examined with transmitted light. The preparations thus obtained may be examined in glycerin or dehydrated and mounted in balsam. (See methods of injection for staining the endothelial cells of blood and lymph vessels. )
Gold Chlorid Method. In gold chlorid impregnation the cells and fibers of certain tissues are stained while the intercellular substances remain uncolored. The coloration is obtained by a reduction of the gold (either by sunlight or certain reagents formic acid, acetic acid, citric acid, oxalic acid), in the form of very fine particles which impart to the tissues a purplish-red color. This method is especially useful for bringing to view the terminations of nerve-fibers, both motor and sensory ; however, it may also be employed for staining other tissue elements. The method of gold impregnation was introduced by Cohnheim and was used by him in staining the nerve terminations in the cornea. It has received numerous modifications since its introduction. The following may be mentioned :
Cohnheim' s Method. Small pieces of muscle are placed in a i % solution of gold chlorid acidulated by a trace of acetic acid. In this they become yellow (in from a few minutes to half an hour). They are then rinsed in distilled water, placed in water slightly acidulated with acetic acid, and kept in the dark. As a rule, the pieces will change in color, becoming yellowish-gray, grayish- violet, and finally red, from one to three days generally being required for this process. The parts best adapted to examination are those in the transitional stage of violet to red. This procedure has been subjected to innumerable modifications ; of these, the most used are : ( i ) The method of Lowit : Small pieces are placed in a solution of i vol. formic acid and 2 vols. distilled water until they have become transparent (ten minutes). They are then placed in a i % solution of gold chlorid, in which they become yellow (one-quarter hour). They are now again placed in formic acid, in which they pass through the same color changes as above. Finally, they are washed and teased, or subsequently treated with alcohol and cut. (2) Kiihne (86) acidifies with 0.5% solution of acetic acid (especially in the case of muscle), then treats the specimens with a i% solution of gold chlorid, and reduces the gold with 20 to 25% formic acid dissolved in equal parts of water and glycerin. (3) Ranvier (89) acidifies with fresh lemon juice filtered through flannel, then treats with a i % solution of gold chlorid (quarter of an hour or longer), and finally either places the specimen in water acidulated with acetic acid (i drop to 30 c.c. water) and subjects it to light for one or two days, or reduces it in the dark, as in L6 wit's method, in a solution of i vol. formic acid and 2 vols. water. (4) Gerlach uses the double chlorid of gold and potassium, but in weaker concentrations than a i$> solution, otherwise he continues as in the method of Cohnheim. (5) Golgi (94) also uses the same double chlorid, but acidifies with 0.5% arsenious acid, and then reduces in i% arsenious acid in the sunlight.
Golgi's Chromsilver or Chromsublimate Method. This method depends on the formation of a very fine precipitate, which forms in certain tissue elements or in preexisting spaces, when treated first with a solution of bichromate of potassium and secondarily with a solution of silver nitrate or bichlorid of mercury. The nature and precise location of this precipitate is not well understood. It is very probable, however, as Kallius suggests, that an albumin-chromsilver compound, of an unknown constitution, is formed in the cells and processes or in spaces filled with the precipitate. This method is especially useful in bringing to view the cellular elements of the nervous system, both central and peripheral ; further, the end-ramifications of gland ducts, and now and then cell boundaries. Usually only a small percentage of the tissue elements or the spaces of any given tissue are colored. This may, however, be regarded as one of the advantages of the method, since it enables a clearer view of the parts colored. The precipitate appears black in transmitted light. It is necessary to state, however, that this method is very unreliable, and that failures are often met. with, also that an amorphous precipitate is generally formed, both in and about the tissues, which in part at least destroys the usefulness of the preparations obtained.
Golgi's methods will perhaps be better understood if we first give a short historic sketch of their development.
In the year 1875 Golgi applied his method as follows : He fixed (olfactory bulb) in Miiller's fluid, and increased the percentage of bichromate on changing the fluid (up to 4 f ). Fixation lasted five or six weeks in summer and three or four months or more in winter. He then took out pieces of the tissue every four or five days and treated them experimentally with a 0.5^ to ifi> silver nitrate solution. In summer this process took about twenty-four hours, and in winter forty-eight hours, although a longer treatment was not found to be detrimental. This method must be regarded as very uncertain, since the length of time during which the specimens remain in Miiller's fluid must be very closely calculated, as it depends largely upon the temperature of the medium. As soon as the silver reaction was established, the pieces were preserved either in the silver solution itself or in alcohol. The sections were finally washed in absolute alcohol, cleared with creosote, and mounted in Canada balsam. The impregnation disappeared in a short time. In the year 1885 Golgi made a further announcement regarding his method, recommending for fixation the pure bichromate of potassium, as well as Miiller's fluid. Pieces of the brain and spinal cord (from I to 1.5 c.c. in size) from a freshly killed animal were used, and the reaction sometimes took place in from twenty-four to forty-eight hours after death. For fixing, potassium bichromate solution in gradually ascending strengths (i^ to 5^,) was employed, large amounts of the fluid being used and placed in well-sealed receptacles. The fluid was repeatedly changed, and camphor or salicylic acid was added in order to prevent the growth of fungi. Since it is difficult to determine exactly when fixation in potassium bichromate reaches the precise point favorable to subsequent treatment with nitrate of silver, because the process depends entirely upon the temperature and quantity of the fluid, it becomes necessary, after about six weeks' treatment with the bichromate, to experiment every eight days or so to see whether the silver nitrate gives good results. The strength of the latter should be about 0.66^0 and the quantity about 2OO c.c. to a I c.c. object. At first a plentiful precipitate is thrown down, in which case the solution should be changed, and this probably repeated once more after a few hours. After twenty-four hours, at the most forty-eight hours, this process is usually completed, and the tissues may be sectioned. The sections must then be carefully dehydrated with absolute alcohol, cleared in creosote and mounted without a coverglass in Canada balsam (the section is mounted on a cover-glass with Canada balsam, and the cover-slip then fastened over the opening of a perforated slide with the specimen downward).
In order to obtain a uniform penetration of the objects by the potassium bichromate, the latter may be first injected into the vessels. Golgi uses potassium bichromate-gelatin (2.5^ of the salt, based on the amount of the softened gelatin ; compare Golgi, 93). After the injection and cooling of the specimen the latter is cut in 4 small pieces and treated in the manner previously described. Instead of Mullet's fluid, that of Erlicki may be used, the time of treatment being then shorter (from five to eight days).
The objects may also be treated with a potassium bichromate-osmic acid solution (2.5^1 solution of potassium bichromate, 8 vols. ; 1% osmic acid, 2 vols.), the sections thus treated being ready for immersion in silver nitrate after two or three days. It is advisable to treat the objects with the potassium bichromate solution first, and then with the potassium bichromate-osmic mixture. By this method the specimens remain under the control of the investigator ; they may be examined either at once, or after an interval varying between three or four and twenty-five to thirty days after immersion. If then one or several pieces of tissue are taken, at intervals of two, three, or four days, from the potassium bichromate solution and placed in the potassium bichromate-osmic acid mixture, and then in the silver nitrate solution, various combinations of the fluids result, and the investigator is usually rewarded with at least some sections giving most excellent results.
Another one of Golgi's methods consists in successive treatment with potassium bichromate and bichlorid of mercury. After remaining in the potassium bichromate for from three to four weeks (a longer period is allowable), the objects are placed in a 0.25 % to I % solution of corrosive sublimate. In the latter the specimens blacken much more slowly than in the silver nitrate solution eight to ten days for smaller pieces ; for larger ones, two months, and in some cases even longer. Before mounting the preparations in glycerin or Canada balsam they must be carefully washed ; otherwise pin-shaped crystals form within the sections and distort the whole view. The metallic white of the preparation may be changed to black by placing the celloidin section in a photographer's toning solution consisting of : (a) sodium hyposulphite 175 gm., alum 20 gm., ammonium sulphocyanid IO gm., sodium chlorid 40 gm., and water looo gm. (the mixture must stand for eight days and then be filtered) ; (b] a I % gold chlorid solution. The specimen is placed for a few minutes in a solution composed of 60 c.c. of a and 7 c.c. of b, washed again in distilled water, dehydrated with alcohol, and mounted in Canada balsam. After toning and washing, the sections may still be stained.
Golgi's methods are extremely inconstant in their results. When successful, however, only a few elements are blackened each time, an advantage not to be underestimated ; for if all nerves should stain equally well, discrimination between the various elements in the preparation would be very difficult. Neither are the same structures always impregnated ; sometimes it is the ganglion cells and fibers, at other times the neurogliar cells, and occasionally only the vessels.
After the foregoing explanation of Golgi's methods as applied by himself, we shall append a description of these methods as modified and employed at the present time (Ramon y Cajal, Kolliker, von Lenhossek and others).
Golgi's methods are classified as the slow, the mixed, and the rapid.
The slow method requires a preliminary treatment. Pieces of tissue from i to 2 cm. in diameter are placed for from three to five weeks in a 2 cfo potassium bichromate solution ; they are then transferred for from twenty-four to forty -eight hours to a 0.75% silver nitrate solution, or for a much longer time to a 0.5% solution of corrosive sublimate.
In the mixed method the specimens are allowed to remain for four or five days in a 2 / aqueous potassium bichromate solution ; then for from twenty -four to thirty hours in a mixture consisting of i% osmic acid i vol., and 2% potassium bichromate solution 4 vols. They are then treated with a 0.75% silver nitrate solution for one or two days
When the rapid method is employed, the specimens are immediately placed in a mixture consisting of i vol. of i % osmic acid and 4 vols. of a Z-S% potassium bichromate solution, and, finally, for one or two days in a 0.75% silver nitrate solution, to every 200 c.c. of which one drop of formic acid has been added.
When employing these methods, and more particularly the one last described (which seems to be the most efficient), the following conditions must be carefully observed : If possible, the material should be absolutely fresh, the specimens must not exceed 3 or 4 mm. in thickness, and for every piece of tissue treated about 10 c.c. of the osmium-potassium bichromate mixture should be employed, the specimens remaining in the latter (in the dark) at a temperature of 25 C. for a length of time varying according to the result desired (two or three days for the neurogliar cells, from three to five days for the ganglion cells, and from five to seven days for the nerve-fibers of the spinal cord). The objects are now dried with blotting-paper or washed quickly in distilled water and then placed for two or three days in a 0.75% silver nitrate solution at room-temperature. In this they may remain for four or five days without damage, but not longer, as otherwise the precipitate becomes markedly granular (vid. v. Lenhossek, 92).
If Golgi's method be unsuccessful (this applies to all its modifications), the preparations may be transferred from the silver nitrate solution back into a potassium bichromate-osmic acid mixture containing less osmic acid, in which they remain several days, and are then again placed in the silver nitrate solution for from twenty-four to forty-eight hours. This procedure may even be repeated.
Cox obtains a precipitate in both cells and fibers by treating small pieces of the central nervous organs with a mixture composed of potassium bichromate 20 parts, 5 % corrosive sublimate 20 parts, distilled water 30 to 40 parts, and 5% potassium chromate of strong alkaline reaction 16 parts. The specimens remain in this mixture from one to three months, according to the temperature, and are then further treated according to Golgi's method.
As the chrome-silver preparations are not permanent, and can not, therefore, be subsequently stained, Kallius has suggested that the chromesilver precipitate be reduced to metallic silver by treatment with the "quintuple hydroquinon developer" (hydroquinon 5 gm., sodium sulphite 40 gm., potassium carbonate 75 gm., and distilled water 250 gm.). For this purpose 20 c.c. of the solution are diluted with 230 c.c. of distilled water ; this mixture may be preserved in the dark for some time if desired. Before using this latter solution, it should be mixed with y$, or at the most y^, of its volume of absolute alcohol. The sections are placed in a watch-crystal containing some of the latter mixture until they turn black (a few minutes). As soon as the silver salt is completely reduced, the sections are placed for from ten to fifteen minutes in 70 % alcohol, then for five minutes in a 2o r / c solution of sodium hyposulphite and, finally, washed for some time in distilled water, after which they may be stained, and even treated with acid alcohol and potassium hydrate.
The following simple method for permanently mounting Golgi preparations under a cover-glass has been recommended by Huber.
After impregnation with chrome-silver the tissues are hastily dehydrated, imbedded in celloidin, and cut in sections varying from 25 ft to 100 fi in thickness. The sections are then dehydrated and placed for from ten to fifteen minutes in creosote, from which they are carried into xylol, where they remain another ten minutes. The sections are then removed to the slide. The xylol is then removed by pressing several layers of filter-paper over the section. On removing the filter-paper the sections are quickly covered by a large drop of xylol balsam and the slide is carefully heated over a flame for from three to five minutes. Before the balsam cools the preparation is covered with a large cover-glass, warmed by passing several times through the flame.
Kopsch (96) places specimens in a solution composed of 10 c.c. of formalin (40% formaldehyd) and 40 c.c. of a 3.5% solution of potassium bichromate. For objects 2 c.c. in size 50 c.c. of the fluid are employed ; but if the specimens be large, the mixture must be changed in twelve hours. At the end of twenty-four hours this fluid is replaced by a fresh 3.5% potassium bichromate solution, and the specimens are then transferred to a 0.75% solution of silver nitrate (after two days, if the tissue be the liver or stomach ; and after from three to six days, if retina or central nervous system). After this treatment the objects are carried over into 40% alcohol and, finally, into absolute alcohol, imbedded as rapidly as possible, and cut. The sections are mounted in balsam without a cover-glass.
PREPARATION OF PERMANENT SPECIMENS.
The resinous media used in the final mounting of preparations are Canada balsam and damar.
Canada Balsam. Commercial Canada balsam is usually dissolved in turpentine ; it should be slowly evaporated in a casserole and then dissolved in xylol, toluol, or chloroform, etc. The proper concentration of the solution is found with a little experience. A thick solution penetrates the interstices of the section with difficulty, and usually contains air-bubbles which often hide the best areas of the preparation, and can only be removed with difficulty by heating over a flame. Thin solutions, on the other hand, have also their disadvantages ; they evaporate very quickly, and the empty space thus created between the cover-slip and slide must again be filled with Canada balsam. This is best done by dipping a glass rod into the solution and placing one drop at the edge of the cover-slip, whereupon the fluid spreads out between the cover-slip and slide as a result of capillary attraction. Canada balsam dries rather slowly, the rapidity of the process depending upon the temperature of the room. To dry quickly, the slides may be held for a few moments over a gas or alcohol flame, or they may be placed in a warm oven, where the preparations become so dry in twenty-four hours that they can be examined with an oil-immersion lens. The oil used for this purpose should be wiped away from the cover-slip after examination. This can only be done, without moving the cover-slip, when the balsam is thoroughly dry and holds the cover-slip firmly in place.
Damar. Damar is dissolved preferably in equal parts of oil of turpentine and benzin. It has the advantage of not rendering the preparation as translucent as Canada balsam. Otherwise it is used as the latter.
Clearing Fluids. Since alcohol does not mix with Canada balsam or damar, an intermediate or clearing fluid is used in transferring objects from the former into the latter. Xylol, toluol, carbol-xylol (xylol, 3 parts; carbolic acid, i part), oil of bergamot, oil of cloves, and oil of origanum are ordinarily used. The process is somewhat simpler where sections are fixed to the slide. Xylol is dropped onto the surface of the slide, or better, the whole preparation is placed for a few minutes in a vessel containing xylol until the diffusion currents have ceased (which may be seen with the naked eye). The slide is then taken out, tilted to allow the xylol to run off, wiped dry around the object with a cloth, and placed upon the table with the specimen upward. A drop of Canada balsam is now placed on the section (usually on its left side), and a clean coverslip grasped with a small forceps. It is then gently lowered in such a way that the Canada balsam spreads out evenly and no air-bubbles are imprisoned under the glass. When this is done the preparation is finished.
If one is dealing with loose sections, a spatula or section -lifter is very useful in transferring them from absolute alcohol into the clearing fluid carbol-xylol or bergamot oil (xylol evaporates very rapidly) and from this onto the slide. In doing this it is necessary that the section should lie well spread out on the section-lifter, wrinkles being removed with a needle or small camel's-hair brush. In sliding the section off the spatula (with a needle or brush) a small quantity of the clearing fluid is also brought onto the slide. This must be removed as far as possible by tilting or with blotting-paper. The section can now be mounted in Canada balsam as before. For esthetic and practical reasons the student should see that during the spreading of the drop of Canada balsam the section remains under the middle of the cover-slip. Should it float to the edge, it is best to raise the cover-slip and lower it into place again. The cover-slip should never be slid over the specimen.
Glycerin. To mount in glycerin the sections are transferred from water to the slide, covered with a drop of glycerin and the cover-slip applied. This method is employed in mounting sections colored with a stain that would be injured by contact with alcohol, and where clearing is not especially necessary.
Farrant's Gum Glycerin.
In place of pure glycerin the following mixture may be used :
Glycerin 50 c.c.
Water 50 "
Gum-arabic (powder) 50 gin.
Arsenious acid i "
Dissolve the arsenious acid in water. Place the gum-arabic in a glass mortar and mix it with the water; then add the glycerin. Filter through a wet filter-paper or through fine muslin.
To preserve such preparations for any length of time the coverglasses must be so fixed as to shut off the glycerin or acetate of potash from the air. For this purpose cements or varnishes are employed which are painted over the edges of the cover-slip. These masses adhere to the glass, harden, and fasten the cover-slip firmly to the slide, hermetically sealing the object. The best of these is probably Kronig's varnish, prepared as follows : 2 parts of wax are melted and 7 to 9 parts of colophonium stirred in, and the mass filtered hot. Before employing an oil-immersion lens it is advisable to paint the edge with an alcoholic solution of shellac.
METHODS OF INJECTION.
The process of injection consists in filling the blood- and lymph-vessels with colored masses in order to bring out clearly their relation to the neighboring tissue elements. The instruments required are a syringe of suitable size or a constant pressure apparatus and cannulas of various sizes. Serviceable and instructive injections of blood-vessels are readily made ; good injections require skill, experience, and patience. Injection masses may be classed under two heads cold injection masses and warm injection masses. The vehicle of the latter is most generally gelatin. For injecting the blood-vessels either the cold or the warm masses may be employed, although the latter give better results. The cold masses are to be used for injecting the lymphatic vessels. In injecting the blood-vessels it is well to wash out the vessels with warm normal salt solution before the injection mass is forced into the vessels. The following masses may be recommended :
Gelatin=carmin. The first is a gelatin -carmin mass, and is prepared as follows: (i) 4 gm. of carmin are stirred into 8 c.c. of water and thoroughly ground. Into this a sufficient quantity of ammonia is poured to produce a dark cherry color and render the whole transparent. (2) 50 gm. of finest quality gelatin is placed in distilled water for twelve hours until well soaked. It is then pressed out by hand and melted at a temperature of 70 C. in a porcelain evaporating dish. The two solutions are now slowly mixed, the whole being constantly stirred until a complete and homogeneous mixture is obtained. To this mass is added, drop by drop, a 25^ acetic acid solution until the color begins to change to a brick red and the mass becomes slightly opaque. This should be very carefully done, as a single drop too much may spoil the whole. During this procedure the substance should be kept at 70 C. and constantly stirred. The change in color indicates that the reaction of the mass has become neutral or even slightly acid (an ammoniac solution should not be used, since the stain diffuses through the wall of the vessel and colors the surrounding tissues) ; the whole is filtered through flannel while still warm. As this mass hardens on cooling it is injected warm. The instruments used are also warmed before the injection is begun.
Gelatin-Berlin Blue. One part of oxalic acid is powdered in a mortar; to this is added one part of Berlin blue and 12 parts of water. Stir and rub until a solution is obtained. Prepare a gelatin vehicle as directed in the preceding paragraph ; to 12 parts of the gelatin mass add slowly while stirring 1 2 parts of the Berlin blue solution. The whole is filtered through flannel while still warm.
Yellow Gelatin Mass (Hoyer). Prepare a gelatin vehicle consisting of i part of gelatin and 4 parts of distilled water; a cold, saturated solution of bichromate of potassium and a cold, saturated solution of lead acetate. Take equal volumes of each. Add the bichromate of potassium solution to the gelatin and heat almost to boiling; then add slowly, while stirring, the lead acetate solution.
Carmin Mass, Cold (Kollmann). One gm. of carmin is dissolved in a small quantity of ammonium hydrate and 20 c.c. of glycerin added. To another 20 c.c. of glycerin there is added 20 drops of hydrochloric acid and this added to the glycerin-carmin mixture while stirring.
Saturated aqueous solutions of Berlin blue or Prussian blue may also be used for cold injections.
Injection masses already prepared are to be had in commerce. Besides those already mentioned, still others colored with China ink, etc., are in general use.
Small animals are injected as a whole by passing the cannula of a syringe into the left ventricle or aorta. In the case of large animals, or where very delicate injections are to be made, the cannula is inserted into one of the vessels of the respective organs. The proper ligation of the remaining vessels should not be omitted.
Organs injected with carmin are fixed in alcohol and should not be brought in contact with acids or alkalies. Such parts as are injected with Berlin blue are less sensitive in their after-treatment. Pieces or sections that have become pale regain their blue color in oil of cloves.
If objects or sections injected with Berlin blue be treated with a solution of palladium chlorid, the bluish color changes to a dark broVn which afterward remains unchanged (Kupffer).
By means of the above injection methods other lumina can be filled, as, for instance, those of the glands. As a rule, these are only partially filled, since they end blindly, and their walls are less resistant and may be damaged by the pressure produced by the injection.
Silver Nitrate. In thin membranes and sections the vessel-walls can be rendered distinct by silver-impregnation, which brings out the outlines of their endothelial cells. This may be done either by injecting the vessel with a i % solution of silver nitrate, or, according to the process of Chrzonszczewsky, with a 0.25^ solution of silver nitrate in gelatin. This method is of advantage, since, after hardening, the capillaries of the injected tissue appear slightly distended. Organs thus treated can be sectioned, but the endothelial mosaic of the vessels does not appear definitely until the sections have been exposed to sunlight.
The injecting of lymph -channels, lymph -vessels, and lymphspaces is usually done by puncture. A pointed cannula is thrust into the tissue and the syringe emptied by a slight but constant pressure. The injected fluid spreads by means of the channels offering the least resistance. For this purpose it is best to employ aqueous solutions of Berlin blue or silver nitrate, as the thicker gelatin solutions cause tearing of the tissues.
Altman's Process. To bring out the blood capillaries and the lymphatic channels, Altman's process (79), in which the vessels are injected with olive oil, is useful. The objects are then treated with osmic acid, sectioned by means of a freezing microtome and finally treated with eau de Javelle (a concentrated solution of hypochlorite of potassium). By this process all the tissues are eaten away, the casts of the blood-vessels remaining as a dark framework (corrosion) . The manipulation of these preparations is extremely difficult on account of the brittleness of the oil casts. For lymph-channels Altman (ibid.) used the so-called oil-impregnation. Fresh pieces of tissues, thin lamellae of organs, cornea, etc., are placed for five to eight days in a mixture containing olive oil i part, absolute alcohol */2 part, sulphuric ether ^ part (or castor oil 2, absolute alcohol i, etc.). The pieces are then laid for several hours in water, where the externally adherent globules of oil are mechanically removed and those in the lymph-canalicular system are precipitated. The objects are now treated with osmic acid, cut by means of a freezing microtome, and corroded. In this case, the corrosive fluid (eau de Javelle) should be diluted two or three times.
Reconstruction By Means Of Wax Plates
It is often impossible to obtain a clear conception of the form of minute anatomic structures, nor of their relations, by means of sections or by the methods of maceration and teasing. To obviate such difficulties methods of reconstruction have been devised, by means of which such structures may be reproduced in an enlarged form without losing their inherent morphologic features. Of these methods, we shall here describe that suggested by Born (1876) and known as Bern's method of reconstruction by wax plates. This method has found wide application in embryologic investigations, and has proved very valuable in ascertaining the form, relation, and metamorphosis of embryonic structures and organs. It has not been so extensively used in the study of the form of fully developed anatomic structures ; it deserves, however, a fuller appreciation of its value as- an aid in microscopic study. Necessary are serial sections, wax plates of desired thickness, and a drawing apparatus.
Serial Sections. One of the requisites of wax plate reconstruction is a perfect series of sections of uniform thickness. The thickness of the sections should depend on the character and size of the object to be reconstructed and on the magnification necessary to give the model obtained such a size as to enable it to be readily manipulated. In the reconstruction of fully developed anatomic structures, such as parts of glands or entire glands, it is generally not possible to make an outline drawing of the parts to be reproduced. When this is possible, it forms the first step of the method.
Wax Plates. Several methods have been suggested for obtaining wax plates of uniform and desired thickness. The instrument devised by
Fig. 9. Apparatus for making wax plates, used in reconstruction by Bern's method.
Huber and figured in Fig. 9 may be recommended for this purpose. It consists of a heavy cast-iron plate, supported by three adjustable legs. On two sides of the plate are found movable side-pieces which may be raised or lowered by micrometer screws to a desired height and then tightly clamped. There is, further, a heavy iron roller which runs on the adjustable side pieces. This roller needs to be heated in boiling water before use, and is kept in boiling water when not in use during the process of making wax plates. The method of making plates is as follows: The side plates are adjusted so that their upper surface projects above the main plate for a distance representing the thickness of the wax plate desired. Melted wax is then poured on the main plate, in an even layer somewhat thicker than the wax plate desired, and then rolled out with the hot roller until the roller runs evenly on the side pieces. The wax plate is now allowed to cool, when it is removed from the apparatus and placed in a pan of cold water, where it remains for a few minutes or until thoroughly cooled.
Drawing of the Portions of the Sections to be Reconstructed. The drawings of the portions of the sections representing the portion to be reconstructed, at the magnification selected, may be made with the aid of a camera lucida, or by means of a projection apparatus. Bardeen has devised a drawing table which is placed horizontally, over which is placed a mirror at an angle of 45 degrees. The table may be made to move by means of a windlass toward or away from the microscope so that any magnification may be quickly obtained. An ordinary microscope with the tube placed horizontally may be used, the illumination being obtained from an arc light. (For further details see Bardeen, "Johns Hopkins Bulletin," vol. xii, p. 148.) Sharp outlines of the parts to be reconstructed should be made and the drawing for each section labelled with reference to the series of drawings and with reference to the number of the section, as it is often necessary to refer to the sections while reconstructing. After the drawings have been completed they are transferred to the wax plates, which is conveniently done by placing the drawing over the wax plate and tracing the outline with a blunt-pointed instrument, using some pressure while doing so. The wax plates are numbered with reference to the drawings. It is necessary to maintain an equal ratio between the diameter of the magnification of the drawing of the sections, the thickness of the plates used and the thickness of the sections. Thus, if it is desired to reconstruct portions of a series of sections 5 /JL in thickness and to use wax plates 2 mm. thick, the drawings need to be made at a magnification of 400 diameters.
Cutting Out the Parts to be Reconstructed and Completing the Model. Those portions of the wax plates representing the parts to be reconstructed as outlined by the tracings are cut out with a sharp knife with narrow blade, the wax plate being placed on a glass plate during this procedure. If the parts of the sections to be reconstructed consist of a number of disjointed pieces, these are retained in their relative positions by means of remaining bridges of wax, which should be firm enough to keep all parts in their proper relation. The parts of each wax plate representing the portions of the section to be reconstructed are piled up in their proper sequence as they are cut out. The completion of the model consists in accurately adjusting the portions obtained from each wax plate to those which precede and follow them. This process is facilitated by building up the model in blocks representing five sections, as has been suggested by Bardeen. Those parts representing the portions of the sections to be reconstructed are united together by pins or small nails ; other parts, such as wax bridges, are removed by means of a hot knife. The successive blocks are then similarly united and the model is completed by smoothing over the surfaces by means of a hot iron.
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Reference: Böhm AA. and M. Von Davidoff. (translated Huber GC.) A textbook of histology, including microscopic technic. (1910) Second Edn. W. B. Saunders Company, Philadelphia and London.
Cite this page: Hill, M.A. (2020, February 23) Embryology Book - A textbook of histology, including microscopic technic (1910) microscopic technic. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_A_textbook_of_histology,_including_microscopic_technic_(1910)_microscopic_technic
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