Preparation and Care of Microscope Slides

CARL W. HAGQUIST aqueous solution, formalin, and glacial acetic acid. Specimens may remain unharmed in either fixative for considerable periods of time; but it is not ad- visable to leave them in the fixative indefinitely, because staining qualities of the material may be affected. This is especially true of materials pre- served in Bouin's fluid. Twenty-four hours is usually ONE OF THE MOST COMMONLY USED teaching aids in sufficient for most tissues. They should then be run biology is the prepared microscope slide. It is also through a rather closely graduated series of baths in Downloaded from http://online.ucpress.edu/abt/article-pdf/36/7/414/364621/4444888.pdf by guest on 27 September 2021 one of the most misused, both from the point of a dehydrant, such as ethyl alcohol, until all the water view of technical ability to study it under the has been removed. microscope and of protecting the slide from ordinary wear and tear. Most biology teachers, but especially Clearing and Embedding those in high schools and those teaching introductory courses in colleges, will use prepared microscope When the specimen is completely dehydrated, the slides some time or other. However, a considerable alcohol is removed with xylene or some other clear- number of biology teachers fail to recognize the ing agent, and eventually the tissue is infiltrated qualities of a good slide, and even more of them with melted paraffin. are not aware of what is involved in making a good Because most animal and plant tissues are too preparation. soft to be cut into thin sections, it is necessary to No one should make the mistake of thinking that support them in a matrix of some kind. This may be the microscope slide is the end-all in studying an accomplished by soaking the cleared material in a organism. Biology is the science of the living, and bath of melted paraffin at a temperature just above the prepared microscope slide is nothing more than the melting point of the paraffin. When completely a valuable tool in helping the student to relate penetrated by the paraffin, the tissue and a quantity structure and function. of the paraffin are cooled quickly, to obtain a block The techniques used in preparing slides are so containing the specimen. numerous most of them cannot even be mentioned There are other media that can be used instead of here; but some of them, together with certain prin- paraffin. One of them is celloidin, a solution of ciples that can be applied universally, should be pyroxylin in ether and alcohol. One advantage of familiar to any biology teacher. celloidin is that the tissue can be embedded without using an embedding oven at a relatively high tem- Collecting Material perature. However, there are disadvantages: it takes longer to embed in celloidin; and succeeding Specimens should be fresh and undamaged. They should also be normal-unless, of course, some ab- normal condition is to be shown. The author is vice president of Triarch, Inc., Box 98, Ripon, Wis. 54971. After com- pleting undergraduate work in biology at Fixation Brown University, he did graduate research at Yale School of Medicine and received his The purpose of fixation is to preserve the speci- Ph.D. in microscopic anatomy and endocri- men in as nearly a lifelike condition as possible. nology from Brown in 1938. From 1938 to 1951, Hagquist taught biology, at the Uni- Fixation is, obviously, a drastic procedure, and the versity of Richmond, and at Lewis and Clark student must recognize that extreme changes take College. He left teaching temporarily to become head of the place in the specimen when it is killed and fixed. zoology slide department at Carolina Biological Supply Co. If possible, the specimen should be fixed as soon as from 1942 to 1945, and he came to Triarch in 1951. Hagquist has published technical articles on the cytology of the mam- collected. Necrotic changes may occur rapidly in malian hypophysis and ovary and on reproductive endocri- cells and tissues, and such deterioration affects the nology and behavior in the female mammal in the American appearance and value of the specimen on the slide. Journal of Anatomy, the Anatomical Record, and the Journal One of the best all-round fixatives for plant tis- of Comparative Physiology. He is active in civic affairs: a sues is formalin acetic alcohol (FAA). A com- former alderman of the Ripon City Council and a recipient of the Distinguished Citizen Award from Ripon's Junior Cham- parably versatile fixative for many animal tissues ber of Commerce. His hobbies are music, woodworking, and is Bouin's fluid, consisting of picric acid in saturated bicycling.

414 THE AMERICANBIOLOGY TEACHER. OCTOBER 1974 operations may be more difficult, because sections must be handled more or less individually.

Sectioning

Cutting thin sections (thicknesses of 10-15 mi- crons) usually requires the use of a good microtome, a sharp microtome knife, and almost infinite patience. Many variables can affect the quality of the sections. Some soft tissues, such as certain animal glands and 2 chicken embryos, can be sectioned quite easily; others, such as some plant stems, are exasperating in their resistance to being cut uniformly and without damage. In such cases it may be advantageous to try celloidin as the embedding medium (provided a microtome for cutting celloidin sections is available). Downloaded from http://online.ucpress.edu/abt/article-pdf/36/7/414/364621/4444888.pdf by guest on 27 September 2021

Mounting 4~~~~~~~~~~~~~ Affixing the sections to slides involves smearing a thin coat of adhesive, such as Mayer's albumin or Haupt's gelatin, to a scrupulously clean slide and then floating one or two sections on a few drops of liquid (distilled water on the albuminized slides, or 3% formalin on the gelatinized slides) added to the slide. This is done at about 40 'C. The wrinkles and compression resulting from sectioning will usually be flattened at this temperature. The excess liquid is poured off, the sections positioned, and the slides dried thoroughly in a dust-free warming oven at about 35 'C. If the sections are thin enough and flatten readily, they can usually be attached to the slides without an adhesive. The blank slide must be absolutely clean, and the section must be floated on distilled water to which has been added a small quantity of one of the wetting agents; 2 drops of Kodak Photoflow to 60 ml of distilled water is an excellent medium. This technique avoids staining the adhesive-which sometimes occurs, especially with some of the aniline dyes-and thus results in a cleaner preparation.

Staining and Covering 8 This process is critical for the production of uniformly good slides. Most animal tissues will stain Fi 9 Cleann an lablin side foloigisetin satisfactorily with a combination of Harris's hema- Fig.3*~~~~~~~ICOCP 1. Collecting materialSLDE from41 toxylin and eosin Y. There are, of course, many Cycas cercinalis. other combinations of dyes that can be used, but Fig. 2. Fixing material in formal acetic alcohol. the above-mentioned combination is relatively sim- Fig. 3. Embedding dehydrated ple to use and is fairly foolproof. specimens in paraffin. A stock solution of Harris's hematoxylin consists Fig. 4. Sectioning paraffin-em- of hematoxylin crystals 0.5 g, alum (aluminum bedded specimens. ammonium sulfate) 0.3 g, red mercuric oxide 0.6 g, 4JIFig.5. Selecting and mounting and 100 ml of 50% ethyl alcohol. Dissolve the hema- individual sections. toxylin and alum with the aid of heat. When the Fig. 6. Staining sections already solution begins to boil, add the mercuric oxide and continue boiling for 20 minutes. Cool the solution and add enough 50% alcohol to replace the amount lost by evaporation. Allow the solution to stand several hours (preferably overnight). Filter and 3. Stain in safranin 0 (1% in 50% alcohol) for store in a tightly stoppered bottle. 2-24 hours, depending on the nature of the tissue This stain may be used immediately, but it will being stained. increase in staining capacity for a month or more. 4. Rinse in two changes of water. Tap water If the stain is to be used immediately, add 20 ml of may be used. the stock solution to 80 ml of a saturated solution of 5. Stain in crystal violet (1% aqueous solution) alum in distilled water. If the stain is not to be used for 1 minute. immediately, add 12 ml of the stock solution to 88 6. Rinse and dehydrate in two changes of 100% ml of the alum solution. In either case, the staining alcohol. power of the solution may be modified by adding 7. Bathe for 2 minutes in a bath composed of either a small quantity (1-2 ml) of the stock solu- clove oil saturated with a mixture of 9 parts Orange tion, to increase the staining power, or 10-20 ml of II (gold orange or tropaeolin 000 No. 2) and 1 part the saturated alum solution, if the stain is too dark. 1% fast green FCF in 100% alcohol. The eosin counterstain is made by dissolving 0.5 8. Pass through four more baths of clove oil and g of eosin Y in 100 ml of 90% ethyl alcohol. Orange II, for about 2 minutes each, to remove the When using this method of staining, follow the last traces of water and alcohol and to clear the standard procedure of passing the slides through tissue and the background. two changes of xylene or other paraffin solvent to 9. Clear in three or four changes of xylene, 1 Downloaded from http://online.ucpress.edu/abt/article-pdf/36/7/414/364621/4444888.pdf by guest on 27 September 2021 remove the paraffin, followed by anhydration minute each. through a graded series of alcohol baths, from 100% 10. Cover with balsam or one of the synthetic alcohol to distilled water. Stain in the hematoxylin resins, and apply a #1 or #2 cover slip. for 5-10 minutes, depending on the tissue being 11. Cure the mountant at 60 ?C for 1-4 days, processed and the depth of stain required. Most depending on the thickness of the section and the well-fixed tissues need not be stained longer than diameter of the cover slip. 15 minutes. Wash thoroughly for 15 minutes in A fresh series of stains made up as indicated above gently running tap water. Dehydrate through 50%, will stain the slides too heavily with Orange II and 70%, and 90% solutions of ethyl alcohol in distilled insufficiently with fast green. A small amount of water. If the hematoxylin has not become sufficiently fast-green solution (1-2 drops/50 ml of clove oil) "blued" in the tap-water wash, add 1 drop of am- should therefore be added to the second and third monia per 100 ml of 70% alcohol. Counterstain for clove-oil baths. If a series of slides is run through approximately 30 seconds in the eosin-90% alcohol the schedule, enough green is carried over to each solution. The length of time in the counterstain clove-oil bath from the preceding one to take care of may be varied, depending on the requirements of this problem. Eventually the fifth clove-oil bath will the tissue being stained. Complete the dehydration become too heavily darkened with fast green. The through three changes of 100% alcohol. Do not first clove-oil bath should then be discarded; the leave the slides in the alcohol for more than a few second, third, and fourth baths moved up in the seconds in each change, because the alcohol tends series; and a fresh clove-oil bath placed in the fifth to wash out the stain. Clear through two changes of position. 0.5 ml of fast-green stock (1% fast green xylene. Cover with balsam (or any other suitable in 100% alcohol) should be added to the new first mountant) and apply a #1 or #2 cover slip. bath of clove oil. One of the important advantages of Harris's hema- The chief value of this schedule from the stand- toxylin is that most animal tissues will not over- point of the technician is that it is more or less stain in 15 minutes or less. Thus, the tedious and automatic, particularly if many slides of different sometimes unsatisfactory process of destaining, kinds of plant tissues are being prepared. The sug- which is necessary with the use of Delafield's and gested time schedule may be altered markedly in other hematoxylin stains, can be avoided. The stain- either direction without seriously affecting the re- ing solutions keep well in tightly covered containers, sulting stain. For best results the depth of stain in and the stain in the tissues is about as permanent as the sections should be regulated by varying the any other stain currently in use. thickness of the sections rather than by changing One of the methods of staining plant tissues de- the concentration of the dyes in the different solu- serves a somewhat more detailed description be- tions: the most valuable features of the schedule cause of the uniformly beautiful results that can may be lost by tampering with the concentration be obtained. The late George H. Conant developed of the dyes. Obviously, not all plant structures take a quadruple stain that is essentially a modification a brilliant stain, nor do all tissues exhibit all four and improvement of Flemming's triple stain (a colors. However, if the same schedule is used on standard stain). The dried slides go through the all, an interesting check is given on the varying following steps: chemical composition of the tissues; this is par- 1. Clear in two changes of xylene, to remove the ticularly valuable in a series of stages from a single paraffin. plant-organ. 2. Rinse in two changes of 100% ethyl alcohol, Conant's quadruple stain gives best results, in to remove the xylene. general, after FAA fixation, although brilliant re-

416 THE AMERICANBIOLOGY TEACHER, OCTOBER 1974 sults may be obtained after the use of many of the the preparation but do not necessarily mean that the special cytological fixatives, such as Navashin's. Lig- slide should be discarded, particularly if there is nified, suberinized, and cutinized structures stain more than one section on the slide. This would also red, as does chromatin in most instances. Violet ap- hold true for small, localized imperfections, provided pears in the middle lamellae, in pectin structures, the rest of the section is good and the person using and especially in starch grains, for which this stain the slide is aware of the nature of the imperfection. is diagnostic. Orange occurs, more rarely, in tissues Some imperfections, such as lesions caused by a that are being digested and in some fibers and other disease or the imperfect areas found on a "bug-bit- structures not affected by the other stains. The ten" stem or leaf, may actually be valuable, under cytoplasm is uniformly stained with fast green. This certain circumstances. stain also acts as a differentiating agent on the 4. Is the slide esthetically suitable? A poorly safranin in the nuclei. prepared slide is less valuable because of its ap- This combination of dyes may be classed as per- pearance, whereas a clean, properly mounted and manent. The colors do not fade readily if the slides stained slide may well be considered a work of art. are given reasonable care. Slides that have been Sometimes the adhesive used to affix the sections stored for more than 20 years are just as brilliant to the slide takes up some of the stains (especially as when they were first made. the aniline dyes) and will appear as a faintly colored area around the sections. Ordinarily, this is dis- Downloaded from http://online.ucpress.edu/abt/article-pdf/36/7/414/364621/4444888.pdf by guest on 27 September 2021 Inspection tracting unless one knows that the adhesive under the section may also have been stained and thus Probably one of the most important, and often has affected the appearance of the section. Some one of the most neglected, steps in producing a types of preparations-for instance, cross-sections microscope slide of high quality is the final inspec- of Smilax (carrionflower) root, Ranunculus (but- tion and grading of the finished product. The follow- tercup) root, and whole mounts of Clonorchis (a ing criteria should be kept in mind: liver fluke) -are worth examining because of the 1. Does the slide show what was intended? If the symmetry of structure, pleasing combinations of technician wanted slides of mammalian ovary to colors, and other factors, entirely aside from the illustrate a mature follicle with enclosed ovum, it value of the materials as biological specimens. obviously would not do to make slides of juvenile ovary. Or if the technician wished to study lenticels Care of Slides on a plant stem, it would not help to cut sections of stems where these structures are lacking. This is, The treatment given any microscope slide by an of course,- a matter of being knowledgeable in col- instructor or a student will materially affect its lecting material. Even if the organ or tissue is cor- usefulness. It cannot be emphasized too strongly rect, one would want to determine whether or not that a prepared slide is a delicate object and de- it is oriented to show, for example, a cross-section serves careful handling. It would not take very long rather than a longitudinal section. for an excellent preparation to be ruined by leaving 2. Is the slide stained properly to show the the slide exposed to strong light, especially sunlight, structures to be studied? Many organs, tissues, and on a laboratory table or desk. Many dyes are sensi- specific cells or parts of cells may be clearly dif- tive to light and fade readily if left under brilliant ferentiated by a wise choice of stains or combination illumination for periods of time. On the other hand, of stains. Although the staining techniques briefly most preparations will last for many years, with no described above are excellent for fulfilling the re- sign of fading, if they are stored in proper containers quirements of most routine slides, they are by no when not in use. means the only answer to the question of proper It should not be necessary even to mention that staining. For example, are the nuclei stained so slides are often ruined because the student has not that they stand out in contrast to the cytoplasm or been taught by the instructor how to use the micro- cell inclusions? If chromosomes are present in divid- scope properly. Many fine preparations are broken ing cells, are they easily recognizable as such? Is because the student tries to examine the slide under it possible to recognize the stages of mitosis? In the high-power magnification without first orienting quadruple stain, especially, are the features of a himself by using the low-power objective. And plant cell brought out-the type of nucleus present, many students and instructors seem to believe that, the nature of the cell wall with its middle lamella, if a little magnification is good, a lot more mag- the presence or absence of starch granules or other nification is much better; the results are crushed inclusions? sections, cracked cover-glasses, broken slides, and, 3. Is the preparation as nearly perfect mechanical- possibly, damaged objective lenses in the micro- ly as possible? Answers to this question are, per- scope. Fortunately, more of the newer and more haps, less compelling to the teacher as a technician expensive microscopes are being manufactured than to persons who are preparing slides for com- with a spring-loaded feature, which helps prevent mercial purposes. Bits of lint, dust, or other foreign materials detract from the appearance and value of (Concluded on p. 439)

MICROSCOPESLIDES 417 ture extremes (fig. 4 and 5). An additional observa- Mammalian... from p. 413 tion, Bergmann's principle that cooler conditions lead to larger and more stocky animals, can also be made. The box is wiped clean with 70% ethanol after each The laboratory mouse can provide valuable learn- mouse trial. ing experiences for students at all levels by increas- Behavioral studies using only male mice can also ing the depth of observation. The avid interest in be very useful because overcrowding and personality mammalian development which seems to be inherent differences can lead to fighting behavior (Francis in children is capitalized upon by using this gentle, and Peaslee, in press). The presence of an aggressive clean rodent. or dominant male will lead to bites on the tail and external genitalia of the less aggressive cage-mates. REFERENCES If fighting is to be avoided, male mice should be ALLEE,W. C., et al. 1963. Principles of animal ecology. W. B. caged separately or with a female. Saunders Co., Philadelphia. If enough equipment is available, observations can FRANCIS, M. G., and M. H. PEASLEE. Effects of social stress on pituitary melanocyte-stimulating hormone activity in male be expanded to include the differences produced by mice. Neuroendocrinology (in press). extremes in environmental temperature. Control GUNDERSON,D. E., and M. H. PEASLEE.1971. Effect of ethanol mice kept at room temperature can be compared administration on melanocyte-stimulating activity in pitu- with mice raised in warm cabinets, approximately itaries of male rats. Proceedings of the South Dakota Downloaded from http://online.ucpress.edu/abt/article-pdf/36/7/414/364621/4444888.pdf by guest on 27 September 2021 Academy of Science 50:95. 32 0C, or with mice raised in a cold environment, 15- PEASLEE, M. H., and F. A. EINHELLIG. 1973. Tannic acid in- 20 'C. If this type of comparison is made, Allen's rule, duced alterations in mouse growth and pituitary melano- which suggests that tails, ears, and other extremities cyte-stimulating hormone activity. Toxicology and Applied of animals are relatively shorter in the colder parts Pharmacology 25:507. of a species' range than in warmer parts, can be PEASLEE, M. H., M. GOLDMAN, and S. E. MILBURN. 1972. Effects of acute and chronic administration of DDT upon pituitary tested (Allee et al. 1963). This is shown most dra- melanocyte-stimulating hormone activity. Comparative and matically by the tail length in mice kept at tempera- General Pharmacology 3:191.

This method, however, sometimes gives disappoint- MicroscopeSlides. . . from p. 417 ing results, because the macerating fluid often ad- versely affects the staining qualities of the cells. this kind of damage when the student abruptly Well-prepared slides, like good friends, seem to swings from a low-power to high-power objective improve as one gets to know them better. And the without first checking to see if the high-power only way to really understand the qualities of such objective will clear the slide on the microscope tools in biology is to learn what is involved in the stage. making of the slides and then to use them with care and appreciation. Other Preparations REFERENCES There are many methods of using animal and plant materials other than the ones described. CONN, H. J., M. A. DARROW,and V. EMMEL.1960. Staining Stained whole mounts can be made if the specimen procedures, 2nd ed. Williams & Wilkins Co., Baltimore. is small enough to fit on a standard slide and can be EMIG, W. H. 1959. Microtechnique. Privately published by cleared sufficiently for study under a microscope. W. H. Emig, Colorado Springs, Colo. GALIGHER,A. E. and E. N. KoZLOFF.Essenttials of practical Smears of blood, bone marrow, exudates of various microtechnique. Lea & Febiger, Philadelphia. kinds, and soft tissues, such as spinal cord, are GRAY, P. 1952. Handbook of basic microtechnique. Blakiston valuable as a means of treating liquid or semi- Co., Philadelphia. liquid materials. Some tissues and organs, such as HUMASON,G. L. 1962. Animal tissue techniques. W. H. Free- man & Co., San Francisco. developing root-tips and small bits of embryonic JOHANSEN,D. A. 1940. Plant microtechnique. McGraw-Hill, tissue, can be prepared as squashes directly on the New York. slide. This method is particularly useful for deter- JONEs, R. M. 1966. Basic microscopic techniques. University mining the kinds and number of chromosomes in of Chicago Press, Chicago. the cell. Or cellular components of a bit of tissue - -. 1967. McClung's handbook of microscopical techniques, 3rd ed. Hafner Publishing Co., New York. can be dissociated by means of a macerating fluid WILLEY,R. L. 1971. Microtechnique: a laboratory guide. Mac- and by treating the resulting material as a smear. millan Co., New York.

439