Preparation and Care of Microscope Slides
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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 with- out damage. In such cases it may be advantageous to try celloidin as the embedding medium (provided a microtome for cutting celloidin sections is avail- able). 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 uni- formly 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.