Book - Oral Histology and Embryology (1944) 15
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Orban B. Oral Histology and Embryology (1944) The C.V. Mosby Company, St. Louis.
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Chapter XV - Technical Remarks
This chapter is intended to give the student a general idea of the preparation of microscopic slides, rather than to cover fully the subject of microscopic technique. For detailed information specialized textbooks should be consulted. The various processes to which a tissue is subjected from the time it is taken from the body until it is ready to be examined under the microscope, are termed microtechnique. Its object is to prepare the specimen for examination of its microscopic structure.
2. Preparation of Histologic Specimens
Dissection is the ﬁrst step in the preparation of a specimen; the material may be secured by a biopsy (excision during life) or at an autopsy (postmortem examination). Pieces of tissue are cut as small as possible to insure satisfactory ﬁxation and impregnation. A very sharp knife should be used to prevent tissue structures from being distorted and squeezed.
Immediately after the specimen is removed and the surface washed free of blood, it is placed in ﬁxing solution. The object of ﬁxing is to preserve the tissue elements in the same condition in which they are at the moment the reagent acts upon them, and harden or so affect them that they will not be altered by the processes of dehydration, embedding, staining, clearing and mounting. The amount of the ﬁxing solution should be at least 20 times the volume of the tissue. The ﬁxing tissue coagulates the protein content of the cells, thus preventing decomposition.
First draft submitted by Joan Launspach, research technician or the Foundation for Dental Research, Chicago College of Dental Surgery.
There are several ﬁxing agents in general use, the most common of which are formalin, formalin-alcohol, Zenker-formol solution, and Bouin’s ﬂuid. A good and rapidly penetrating ﬁxative for small specimens is Zenker-formol solution, a mixture of 9 parts of potassium bichromate and bichloride of mercury with 1 part of neutral formalin. Formalin (5 to 10 per cent) is used for large pieces of tissue, e.g., jaws. It does not deteriorate and it penetrates very rapidly. Formalin-alcohol ﬁxes and dehydrates simultaneously, and is used mostly for surgical specimens. Bouin’s ﬂuid is a solution of picric acid and formalin, and is especially applicable in studying cell outlines, but is rather slow to penetrate.
The length of time necessary for a ﬁxing agent to act upon a tissue varies according to the size of the specimen and penetrating power of the ﬁxative. Generally, it should be just long enough for the agent to saturate the piece thoroughly without allowing it to become brittle. Small pieces of tissue, e.g., gingiva, are left in Zenker-formol solution only 4 to 8 hours, while larger pieces such as jaws may be left in formalin for days. In order to obtain good ﬁxation of pulp in an intact tooth, the surface of enamel and dentin is ground away to a thin layer of dentin around the pulp. This process not only insures rapid and thorough penetration of the ﬁxing agent but also reduces the time of decalciﬁcation and permits a thorough impregnation with cellodin. When ﬁxing biopsy specimens, the solution should be at approximately body temperature.
After the specimens are thoroughly ﬁxed, they are washed in running water for twenty-four to forty-eight hours to remove all acids and reagents. Occasionally, however, special treatment is required to remove the precipitates caused by certain agents. Example: specimens ﬁxed in Zenker-formol solution are treated with Lugol’s (iodine) solution and sodium thiosulfate, and specimens ﬁxed in formalin are placed in a mixture of potassium hydroxide before staining. However, there is no need of this if neutral formol is used.
Animal tissues may be classiﬁed as hard and soft, or calciﬁed and noncalciﬁed. The dental histologist is particularly interested in the hard tissues, namely, the enamel, dentin, cementum and bone. These are impregnated with a variable quantity of calcium salts and cannot be seetioned on the microtome unless decalciﬁed.
Decalciﬁcation of a tissue is the removal of its mineral content by an acid such as nitric, hydrochloric, trichloracetic, formic, or sulfosalycilic acid. The length of time a specimen remains in the decalcifying agent is inﬂuenced by the choice and concentration of the acid, and the size of the specimen; however, the shorter the time the better is the staining. A 5 per cent solution of nitric acid seems to be most satisfactory and is, therefore, widely used. It acts quickly without causing swelling of the tissue or any other undue changes in its elements; it does not interfere with the staining process to any marked degree.
While tissues are being decalciﬁed they should be suspended in a large quantity of the ﬂuid in order that the salts dissolved may sink to the bottom of the jar. Occasional stirring or gentle agitation of the specimen and heating of the acid may hasten the process of decalciﬁcation, but great care should be taken not to injure the tissues.
To ascertain whether the inorganic salts have been completely removed, the specimen can be pierced with a sharp needle or pin: when no gritty substance is detected, the decalciﬁcation is sufficient. Roentgenographic check-up can also be employed. After decalciﬁcation a tooth should be as pliable as a piece of cartilage. The enamel disappears almost entirely owing to its low percentage of organic matter. The decalcifying agent may also be tested for calcium; the acid is changed periodically until the test is negative.
Following decalciﬁcation the specimen is washed thoroughly in running water for at least 24 hours. From this point it is treated as a soft tissue and is ready for the embedding process. It is possible, however, to embed hard tissues ﬁrst and decalcify them later: the specimen is run through the solutions in routine fashion and, after it is blocked, the excess celloidin is cut away and the tissue is suspended in acid until decalciﬁed. This takes much longer than the usual procedure and the results are often uncertain.
In order that a tissue may be sectioned on the microtome it has to have a certain rigidity to offer sufﬁcient resistance to the cutting edge of the knife. This may be accomplished by freezing the tissue or, as is more commonly done, by using an embedding medium which ﬁlls the interstices of the tissue. The freezing technique is employed where immediate investigation of the specimen is required, as in the course of a surgical operation. Some substances (fat, lipoids, etc.) are dissolved during embedding: tests for such substances can be made only in frozen sections.
Embedding is a much more lengthy process but results are more satisfactory. Before the specimen is embedded, i.e, impregnated with a suitable substance such as parafﬁn or celloidin, the water has to be removed from the tissues. Paraffin embedding is more rapid and is used for small pieces, usually soft tissue, as decalciﬁed pieces become brittle during the heating which is necessary in using this method. Celloidin embedding takes longer but causes less shrinkage. This technique is more commonly used in dental histology when large blocks of decalciﬁed material have to be sectioned.
Dehydration is accomplished by placing the specimens in ascending alcohols (50, 70, 95, 100 per cent) for approximately one day each; the length of time depends on the size and permeability of the specimen. Two consecutive changes of absolute alcohol are used. As, however, paraiﬁn or celloidin is not soluble in alcohol it has to be replaced by a ﬂuid which is a solvent for the embedding medium.
When parafﬁn is selected as the embedding medium, the absolute alcohol is replaced by xylol or oil of cedarwood. The specimens are placed in the solvent 12 to 24 hours, and are then placed in liquid paraﬁin in the incubator (56 C.) for several hours. Finally the specimen is placed in a form ﬁlled with molten paraﬁin and quickly cooled. It is then ready to be sectioned.
If celloidin, a solution of nitrocellulose in absolute alcohol and ether, is selected as the embedding medium, a mixture of equal parts of ether and alcohol is used as a solvent in which the tissue remains for 12 hours. It is then carried through a thin (6 per cent) and medium (121/; per cent) into thick (25 per cent) solution of celloidin. The length of time which is necessary for each of the solutions to penetrate the specimen depends upon size and permeability of the tissue. Soft tissue is well inﬁltrated with celloidin after three weeks while decalciﬁed specimens require at least six weeks; to insure thorough impregnation, it is wise to leave teeth longer in the lower concentration of celloidin and a somewhat shorter time in the stronger concentration. When it is necessary to “rush” a specimen the tissue in thin celloidin may be placed in a 50° C. oven in a tightly stoppered container; the embedding period is thus shortened to two or three days. This method causes considerable shrinkage. Small pieces of tissue may be placed directly on a ﬁber block and left to harden in a desiccator filled with chloroform vapor. Large pieces of tissue, e.g., jaws, are placed in an evaporating dish ﬁlled with celloidin which is allowed to harden down slowly. When the celloidin has reached the desired degree of hardness, blocks are cut out and, after being softened in thin celloidin a few minutes, are placed on ﬁber blocks, allowed to air dry, and then placed in 70 per cent alcohol for storage or sectioning.
Tissues are sectioned by means of a microtome, ‘a machine equipped with a knife. There are three different types of microtomes: the freezing, rotary, and sliding, the use of which depends on the kind of tissue and embedding medium used. Each is a heavy specially designed machine precisely constructed, capable of slicing prepared tissues into exceedingly thin sections. The knife is wedge-shaped and made of heavy steel to aﬂord the greatest possible rigidity; it must have a very keen edge as the slightest nick would tear a section. Sharpening a microtome knife is one of the most important as well as the most difficult tasks of a technician. Larger nicks are removed on a coarsely grained stone, and a fine edge is achieved by grinding the knife on a ﬁne hone. The ﬁnal cutting edge is obtained by stropping on a ﬁnishing leather microtome strop. A much more rapid and just as satisfactory a method that has recently been developed is the use of a grinding machine, consisting of an ebony wheel mounted on a rotary motor. A strop, dusted with abrasive powder, is used to put the ﬁnishing edge on the knife. The possibility of making good sections depends upon the type of tissue, its preparation, and the condition of the knife. Sections of 5 to 15 microns (174000 millimeter equals 1 micron) are considered thin.
The importance of the freezing technique in preparing surgical specimens has been mentioned. It is well known that, by exposing tissues to an extreme degree of cold, they become hard and can be easily sectioned with the freezing microtome. The cold is generated by means of carbon dioxide which is sprayed onto the stage holding the specimen: rapid evaporation produces the required temperature.
The rotary microtome is used only for sectioning paraffin blocks; the knife is immovably ﬁxed at a right angle to the block which is carried past the sharp edge of the knife by turning a wheel. With this machine it is possible to out long ribbons of serial sections. The ribbons are placed in lukewarm water where the wrinkles are removed as the paraffin becomes soft. The desired sections are then ﬂoated onto slides smeared with egg albumen and placed in a 37° C. oven for a few minutes. Before staining, the paraffin is dissolved in xylol. the slides rinsed in absolute alcohol, the sections are carried through descending concentrations of alcohols into distilled water; then they can be stained by water-soluble dyes.
A celloidin block is sectioned by a different method. For this purpose the sliding microtome, a heavy sledgetype instrument, is used. The longitudinal angle of the knife is adjusted to each specimen so that the entire cutting edge is used in sectioning. The angle of the cutting edge of the knife should be changed according to the hardness and density of the material. To obtain the most satisfactory results the knife should be in an almost horizontal position for large decalciﬁed pieces, at an acute angle for soft tissue. During sectioning both specimen and knife are continually moistened with 70 per cent alcohol; the sections are placed in distilled water before staining. The celloidin is usually not removed from the section as the stain penetrates the tissues in spite of this embedding medium. However, it has to be removed from the section in the case of speciﬁc stains, i.e., Mallory, azure-eosin, etc. For this procedure the section is mounted on a slide smeared with egg albumen and is ﬂooded with oil of cloves to dissolve the celloidin; the slide is rinsed in 95 per cent alcohol and placed in 70 per cent until ready for staining.“ When serial sections are desired, sections are mounted on glass slides which are then blotted and ﬂooded with a very thin solution of collodion. After a coating is formed, the slides are marked with a diamond pencil or India ink, and stored in 70 per cent alcohol until ready for staining.“
Some special staining methods can be applied only to sections of undecalciﬁed teeth and bone. To obtain such sections mature enamel has to be removed from the teeth. The tissue impregnated With celloidin is placed in a shallow dish and covered with celloidin. The solution is allowed to evaporate slowly until the celloidin is very hard (two to four weeks) . A very hard knife should be used which has been sharpened and stropped ; when checked under the microscope it has deep and even teeth and should be clamped in the microtome at a 13° angle.
Dyes used to stain specimens for microscopic examination may be classiﬁed as basic or acid, according to their affinity for different cellular elements. Basic dyes, sometimes called nuclear dyes, primarily stain nuclear chromatin, basic substance of cartilage and mucus; the more commonly used are hematoxylin, methylene blue, safranin, and carmin. Acid dyes color the cytoplasm of the cell, uncalciﬁed bone and dentin matrix, some connective tissue ﬁbers; eosin and phloxin are representative of this group. By using combinations of the two groups, due to their different afﬁnities, a marked diﬁerentiation of the cellular elements of the specimen is possible.
Sections may be stained on the slide or ﬂoating in dishes; in the latter case better differentiation is aﬁorded. Although the steps of the various staining methods differ considerably, they may be arranged in the following order: staining, differentiating, decolorizing, dehydrating, clearing and mounting.
Hematoxylin and eosin is one of the most commonly used combinations of stains because it is the simplest to handle. For the differentiation of more specialized tissues the following are recommended: Mallory stain,“ or Heidenhain’s Azan“ (modiﬁcation of Mallory’s stain) for connective tissue; the latter is more brilliant and has greater capacity for differentiation; Silver Impregnation (modiﬁcation of Foot’s stain by Gomori) for connective tissue ﬁbers and nerve elements ;’ Van Gieson’s stain, a counterstain to hematoxylin, for differentiation of white connective tissue," and Weigert’s stain for elastic tissue.“
After sections of tissues have been stained and differentiated, they are dehydrated and then passed through a medium that will mix with the dehydrating ﬂuid as well as the reagent in which the sections are to be mounted. These intermediary ﬂuids are called clearing agents because they have a high refractive index, thus rendering the sections more or less transparent.
For celloidin sections a variety of clearing agents is used: terpineol (Lilacine), carbol-xylol, oil of cloves, oil of cedar wood, oil of origanum and beechwood creosote; for paraffin sections usually only two are used: xylol or toluol.
After clearing, the sections have to be placed in some medium which will preserve the stain and prevent the tissue from drying. Such solutions are termed mounting agents: among the most common are Gum damar, Canada balsam, and clarite. Loose celloidin sections are ﬂoated onto the slide and straightened out with the aid of a ﬁne camel’s hair brush; they are carefully blotted and covered with a drop of mounting medium and a coverslip. Weights are placed on the coverslips to prevent the formation of air bubbles. When dry, they are carefully cleaned with xylol and labeled with India ink.
The Altmann-Gersh freezing and drying technique for special microchemical studies should also be mentioned. The tissues are frozen instantaneously when placed in a tube of isopentane in a Liquidair container and are dehydrated under vacuum while still frozen, thus avoiding a redistribution of minerals. Fixation, alcohol dehydration, and clearing are omitted as the dehydrated tissue can be immediately inﬁltrated with parafﬁn and sectioned according to the usual methods. This technique has proved valuable in the preparation of tissues for micro-incineration, and for special micro-chemical reactions.
Many submicroscopic structures may be seen with the electron microscope, not visible in sections prepared in the routine manner.
3. Preparation of Ground Sections
Ground sections are prepared by using abrasive stones upon a tooth or bone until the tissue is reduced to translucent thinness. It is the principal method of examining the enamel which has so little organic material that it disappears almost entirely when the teeth are decalciﬁed by ordinary methods. Therefore, this technique should complement the decalciﬁcation method.
To prepare a ground section of a tooth it is ﬁrst ground down on one side on a carborundum stone which rotates at high speed on a laboratory lathe. It is important that the tooth be kept wet constantly with cold water to lessen the heat produced by friction and to prevent the section from drying. If it is allowed to dry its organic constituents will shrink and present a picture untrue to the conditions during life. The tissue is likewise more apt to crack and break up during preparation if it becomes dry. When the desired level is reached and the ground surface is perfectly plane, this surface is polished on wet ground glass and, ﬁnally, on an Arkansas stone. The other side of the specimen is then ground down until the section is sufficiently translucent. This second side is also polished in the above described manner, to remove the gross scratches produced by the carborundum stone. The ﬁnished ground sections should have an average thickness of 25 to 50 microns and, if desired, may be stained before they are dehydrated, cleared and mounted.”
For surface staining of ground sections the surface is well polished and the section is covered with a 0.25 per cent H01 to decalcify it slightly; then it is stained lightly with hematoxylin.“ By this method only the surface of the ground section is stained and the stained layer can be viewed with high power lenses as it is only a few microns in thickness. This method, however, causes slight decalciﬁcation of the enamel making a marked differentiation of rods from sheaths and cementing substance, If it is necessary to investigate an undecalciﬁed tooth with the surrounding soft tissue, ground sections can be made by using the petriﬁcation method. The specimen is embedded in Kollolith-chloroform solution or in Canada balsam”, 15 where it is left until it is sufficiently hard before it is ground down to a desired thickness. Thin “serial” ground sections of teeth and jaws may be cut in one operation by inﬁltrating the specimen with a plastic material and using a cutting device made up of steel wheels set at various distances."
3. condition not representative of normal enamel. Enamel that has been partially decalciﬁed by caries, or a poorly formed enamel has this appearance.
4. Preparation of Organic Structures in the Enamel
The routine decalciﬁcation of whole teeth in an aqueous solution of acid usually destroys the enamel completely. At most, merely shreds of the organic structures remain near the cervical areas in a tooth of a young person.
The organic structures may be demonstrated by C. F. Bodecker’s celloidin decalcifying method.‘ When dentin is included in the specimen sections are rarely satisfactory because this tissue becomes very brittle as a result of the many media through which it passes. It is necessary only for study of the organic structures in the enamel under high magniﬁcation. In general, this method is erratic and a high percentage of failures must be expected.
The Cape-Kitchin modiﬁcation‘ 5 of Bodecker’s method is quite simple and gives satisfactory results if the structures of the matrix are not magniﬁed more than about 500 diameters.
Frisbie, Nuckolls, and Saunders’ have recently developed a technique for the successful recovery of the enamel matrix. The fresh specimen is immediately ﬁxed in neutral formalin for a long period of time (six months) ; most of the dentin is then removed with a dental bur and the tooth is placed in the ﬁxative again for a shorter period of time, depending on the penetrability of the specimen. The completely ﬁxed enamel is decalciﬁed by placing the specimen on a gauze stretched over a platinum wire frame, and immersing it in a 5 per cent solution of nitric acid in 80 per cent alcohol for 24 to 48 hours. Dehydration is begun with 70 per cent alcohol without preliminary washing. The specimen is inﬁltrated with celloidin at 56 C. for two weeks and then allowed to harden down slowly at room temperature until the block is very hard. Sectioning is done with a sliding microtome at 3 to 4 microns.
An aqueous decalciﬁcation of enamel under a cover-glass is the simplest but the least satisfactory method for its study. It is sufficient to show enamel lamellae, cuticle, tufts, and can be used to demonstrate gross differences in quantity of organic structures of enamel in recently erupted teeth and in teeth of old persons. However, the disadvantages are that only low magniﬁcations up to 100 diameters are possible and that the specimens are not durable.
Another method of differentiating the organic from the inorganic content of the enamel is by incineration. It has been shown that the heating of sections of human adult enamel up to 800° 0. causes a destruction of the organic content but leaves enamel rods intact.
Photomicrographs are photographs of small microscopic objects, made with the aid of a microscope. Most of the illustrations in this book are such pictures. Transmitted light is the most commonly used method of illumination as it permits the sharpest differentiation of details and the highest magniﬁcation of tissue structures in stained decalciﬁed sections.
Reﬁected light is used in oral histology in photographs of ground sections of enamel and dentin. These sections should be ground perfectly smooth. There is no need for extreme thinness because the specimen is viewed only from the surface from which the light is reﬂected.
Polarized light also is useful in the study of the dental tissues. It vibrates in a single known plane and requires special equipment and technique. By this means it is possible to determine details of the submicroscopic structure of tissues, due to the differences in optical properties of various elements. Polarized light is particularly useful in the study of calciﬁed tissues, but is not conﬁned to these, because ﬁbrous and keratinized structures also yield information when studied by this method.
Grenz rays are a form of exceedingly soft roentgen rays. When ground sections of teeth are photographed in this way, slight variation in calciﬁcation may be deﬁned thus rendering this method useful in the study of calciﬁed structures." 13
A method has recently been developed by Gurney and Rapp” for studying the ﬁne structural details of tooth surface by adapting the Fax Film technique used for study of metallographic surfaces. Micro-impressions are made of the specimen, using a plastic ﬁlm which may then be mounted on a glass slide for a permanent preparation. Scott and Wyckoff“ obtained similar results by shadowing collodion replicas with vaporized metal in a high vacuum, a more complicated method. The eifect of chemical agents on tooth structure and the changes in tooth surfaces (caries) may be observed using these methods. When examined under the electron microscope, many submicroscopic structures are visible.
Ultraviolet light technique‘ and ﬂuorescence light microscopy, likewise, have been applied in special studies of dental tissues, but have not yet attained wide use.
1. Applebaum, E.. Hollander, F., and Bodecker. C. F.: Normal and Pathological Variations in Calciﬁcation of Teeth as Shown by the Use of Soft X-rays, Dental Cosmos 75: 1097, 1933.
2. Bensley, R. R., and Bensley, S. 8.: Handbook of Histological and Cytological Technique, Chicago, University of Chicago Press.
3. Bodecker, C. F.: Cape-Kitchin Modiﬁcation of Celloidin Deoalcifymg Method for Dental Enamel, J. Dent. Research 16: 143, 1937.
4. Bodecker, C. F.: Enamel of Teeth Decalciﬂed by Celloidin Decalcifying Method and Examined by Ultra Violet Light, Dental Review 20: 317, 1906.
5. Cape, A. T., and Kitchjn, P. 0.: Histologic Phenomenon of Tooth Tissues Observed Under Polarized Light, With a Note on Roentgen Ray Spectra of Enamel and Dentin, J. A. D. A. 17: 193, 1930.
6. Cowdry, E. V.: Microscopic Technique in Biology and Medicine, Baltimore, 1943, Williams & Wilkins Co.
7. Frisbie, H. E., Nuckolls, J., and Saunders, J. B. de G. M.: Distribution of Organic Matrix of Enamel in Human Teeth and Its Relation to Histopathology of Caries, J. Am. Coll. Dent. 11: 243, 1944.
8. Gurney, B. R, and Rapp, G. W.: Technic for Observing Minute Changes on Tooth Surfaces, J. Dent. Research 25: 367, 1946.
9. Guyer, M. F.: Animal Micrology, Chicago, 1943, University of Chicago Press.
10. Hotchkiss, R. D.: Microchernical Reaction Resulting in Staining of Polysaccharide Structures in Fixed Tissue Preparation, Arch. Biochem. 16: 131, 1948.
11. Loosli, C. G.: Outline of Histological Methods, Chicago, University of Chicago Press.
12. Mallory, F. B.: Pathological Technique, Philadelphia, 1938, W. B. Saunders Co.
13. McClung, C. Microscopic Technique, New York, 1937, Paul B. I-Ioeber, Inc., pp. 353-401.
14. McLean, F. 0., and Bloom, W.: Calciﬁcation and Ossiﬁcation. Calciﬁcation in Normal Growing Bone, Anat. Rec. 78: 133, 1940.
15. Meyer, W.: Die Anfertigung histologicher Schliﬁe (Preparation of Histologic Ground Sections), Vrtljschr. f. Zahnheilk. 41: 111, 1925.
16. Scott, D. B., and Wyckoﬁ, R. W. G.: Shadowed Replicas of Tooth Surfaces, Pub. Health Rep. 61: 697, 1946.
17. Sognaecs, R. F.: Preparation of Thin Serial Ground Sections of Whole Teeth and Jaws and Other Highly Calciﬁed and Brittle Structures, Anat. Rec. 99: 133, 1947.
17a. Sognnaes, R. F.: The Organic Elements of the Enamel. I, II, III, IV, J. Dent. Research 27: 609, 1948; 28: 549, and 558, 1949; 29: 260, 1950.
18. Willman, M.: Technique for Preparation of Histological Sections Through Teeth and Jaws for Teaching and Research 16: 183, 1937.
19. Wolf, J.: Plastische Histologie der Zahngewebe (Plastic Histology of Dental Tissues), Deutsche Zahn-, Mund- und Kieferheilkunde 7: 265, 1940.
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