Appendix I: How to Study a Microscope Slide

Ronald A. Bergman, Ph.D., Adel K. Afifi, M.D., Paul M. Heidger, Jr., Ph.D.
Peer Review Status: Externally Peer Reviewed

In studying a histological preparation, you should acquaint yourself with the following: (a) the name of the organ or tissue; (b) the animal from which it was prepared; (c) the method of fixation (or preservative) employed; (d) the thickness of the tissue slice; and (e) the stain or stain combination used. A sample slide label containing all of the above information is shown below.

It is essential to understand the meaning of each of these notations if you are to gain the maximalamount of information from your subsequent study of the slide.

1. First, familiarize yourself with the overall function of the tissue to be examined. In our example above, one should know that the duodenum is the first segment of the tubular small intestine that receives: (1) partially digested food from the stomach; (2) digestive enzymes from the pancreas; (3) bile from the liver; and (4) its own secretion, the intestinal juices. In the small intestine, the digestion of food, which begins in the mouth and stomach, is completed. The end products of the digestive process (i.e., monosaccharides, amino acids, and fatty acids), as well as vitamins, salts, and water, are absorbed and transported through the columnar epithelium into the capillaries or lymphatic vessels in the wall of the intestine. Any undigested food is carried along through the digestive tube until digestion and assimilation are complete or the undigestible contents are eliminated at the anal orifice.
   
   You should, therefore, examine each of the component parts of this organ. Those parts beginning at the inner or luminal surface include (1) the epithelium, with its four cell types (the simple columnar absorptive cells, goblet cells, Paneth cells, and argentaffin cells); (2) the lamina propria, which contains the capillaries, both blood vascular and lymphatic, arteries and veins, connective tissue fibers and a variety of important, protective migratory cells; strands of smooth muscle, and nodules or collections of lymphocytes; (3) the muscularis mucosa, a thin layer of smooth muscle fibers; (4) the submucosa, in which specific duodenal tubular glands (Brunner's mucous glands) and ducts are found leading to the intestinal lumen, autonomic nerve fibers and cell bodies (Meissner's plexus), as well as loose connective tissue supporting the vascular elements; (5) the muscularis, composed of an inner circular layer and an outer longitudinal layer (between these two smooth muscle layers, autonomic nerve fibers and nerve cell bodies [Auerbach's plexus] may be found); and (6) the outermost loose connective tissue layer, which may or may not be covered with mesothelium.
   
   The basic tissues represented in each slide should be identified and their structure and function reviewed.
   
2. The animal from which the tissue sections are prepared is important. Although the cells and tissues in most mammals bear marked similarity, subtle to gross differences do occur.
   
3. The method of fixation used in the preparation of cells and tissues will determine the type and quality of tissue preservation as well as the subsequent choice of stains that may be used.
   
4. The notation of section thickness on a microscope slide informs the observer of the approximate level of magnification most suitable for examination of the tissue section. The thinner the tissue section is, the higher the magnification and resolution of structural detail possible. In general, cytologists prefer sections 1 to 3 µm in thickness, whereas histologists and neuroanatomists may use sections up to 50 µm or more.*
   
5. The stain notation is most valuable and is an indication of the particular emphasis for which the slide was prepared. This should receive the most careful attention by the student. As will become evident in your reading, a vast array of tissue stains are available to visualize specific cellular and tissue components. Many of the commonly used stains and stain combinations are represented in this atlas. Their potential use extends beyond the educational into the very important realm of biomedical research. The student or researcher in any of the basic or applied biomedical sciences can profit from the use of staining techniques that detail each of the component parts of any tissue under investigation regardless of the specific approach, which may be primarily physiological, chemical, or anatomical.

After a careful reading of the slide label and preparation for study of the microscope slide, examine the slide with the naked eye and/or the microscope ocular and note any gross features of the section that indicate distinctive structural arrangements to be studied with the microscope. These features include size, color, shape, and component parts. If the section is composed of different parts, note their relationship and staining differences, if any.

Examine the section to determine whether the tissue was cut from a larger piece and whether any natural surfaces are present. This will aid your subsequent study at higher magnification. This should be followed by a systematic scanning of the section by the lowest power objective to identify further and locate the components of the tissue. With the low power (10 X objective), continue the study, paying particular attention to smaller and more subtle staining and structural details. The high dry lens (~43 X) will allow the study of most of the details found in routine histologic sections. Switching the lenses frequently from the lowest to the highest magnification will facilitate the study and prevent overlooking important component parts of the section.

The oil immersion lens (100 x) should be used for the study of cytological and other minute tissue components and is best saved for the final stages of study. The oil should be used sparingly; it may interfere with the study of a slide at low magnifications and may come in contact with the high dry objective lens. If contaminated, the lenses should be carefully wiped only with a high-quality, clean lens paper. The use of solvents such as xylol can severely damage or ruin a fine microscope lens. If it is necessary to use a lens cleaner, caution should be exercised.

With practice, most microscopic anatomists find that additional details can be seen by making continuous fine adjustments of the focus at higher magnifications in order to visualize structures more superficial or deep in their histologic sections. This method of continual fine focus adjustment permits the tracing of structures and their relationships through the full thickness of the tissue section. The three-dimensional effect gained adds a richness that is lost if this technique is not used.

Simple sketches of the important features of the tissue section are valuable aids to subsequent review of the major features of the tissue studied. The development of this skill is a valuable technique in the learning process, as it requires attention to detail in translating the visual image to a drawing that will have lasting value in forming a permanent mental image.

When your study of the microscope slide is complete, carefully clean again with lens paper any oil or fingerprints from the section coverglass and slide.

The preparation of microscope slides is very costly and in some instances the sections may be, for other reasons, irreplaceable. Use one slide at a time, replacing it in the slide container before removing another for study.

*1 µm = 1/25,400 inch = 1/1000 mm = 10,000 Å = 1000 nm.

Next Page | Title Page

URL: http://www.anatomyatlases.org/