Appendix II: The Preparation of Cells and Tissue for Microscopic Study

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

The photographs that comprise this atlas were prepared from tissues obtained either from animals that were sacrificed for the purpose or, in the case of human tissues, from surgical or autopsy specimens.

The tissues were first placed in a solution called a fixative, which ideally preserves and hardens them with minimal distortion of the physical features, chemical characteristics, and staining properties. No matter how successful this fixation process, the death of the cells and tissues will ultimately result in artifacts that must be recognized, understood, and controlled. As a consequence of this, the histologist is always modifying existing methods and experimenting with new fixatives in order to preserve the essential features of living cells for microscopic study.

In general, the stepwise procedures that follow are used to prepare stained tissue sections or slices of fixed tissues for microscopic study. The procedure to , be outlined relates to paraffin (wax)-embedded tissues, the most commonly used method, but, in principle, is similar to those used with other embedding media. Specific details for the wide range of techniques developed by anatomists can be found in the cited references (Appendix V).

The paraffin or wax method includes the following steps:

1. Fixation (as in 10 per cent formalin, Zenker's fluid, etc.).
   
2. Rinsing (excess fixative removed with water).
   
3. Dehydration (in graded ethanol from 70 to 100 per cent).
   
4. Clearing (to replace ethanol with a solvent miscible with both ethanol and paraffin). Cedar wood oil, xylene, or others may be used.
   
5. Embedding (impregnation of tissue in molten paraffin and subsequent hardening by cooling).
   
6. Sectioning (slicing the wax-impregnated tissue on a microtome).
   
7. Affixing sections on glass slides (usually with egg albumin).
   
8. Dewaxing sections in xylene and hydrating the tissue with decreasing grades of ethanol an distilled water.
   
9. Staining or dyeing the tissue.
   
10. Rinsing (to remove excess dye).
    
11. Dehydration in graded ethanol and clearing the tissue sections in xylene.
    
12. Covering the sections with Canada balsam or a synthetic medium and a thin glass coverslip.

These procedures can and do in many instances result in the loss of cellular constituents and inclusions and thereby alter the microscopic appearances of cells and tissues. Because of this, a variety of fixatives are used to preserve and stabilize certain structures and inclusions to withstand the procedures just outlined. In addition, the choice of fixative must be compatible with the dyes or stains to be used. Appendix III provides comments about the fixatives and stains used on tissues photographed for this atlas.

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