Fixation For Light Microscopy

Some fixatives are superior for the demonstration of certain histologic details; others are specifically required (or specifically contraindicated) for certain special stains. Special indications are listed with the various fixative recipes. For routine purposes, excellent fixation can be achieved with almost all the mixtures listed below; the choice will depend on availability, costs, technical help, and increasingly, on environmental concerns. Every effort should be made to reduce the use of toxic substances such as mercury.

The smaller the specimen, the sooner the fixation will be completed. The acceptable thickness of tissue is listed with the various fixation mixtures. Larger specimens may remain completely unfixed in the center. The use of small volumes of fixation fluid for large specimens is the most frequent cause of poor tissue preservation. The minimal acceptable volume of fixation fluid is about 15-20 times the volume of the specimen.

No matter what type of fixative is used, the tissues should not touch each other or be pressed against the bottom or walls of the jar. Suspension of larger specimens or use of a cushion of cotton for smaller specimens will permit optimal exposure. If the fixative becomes stained, cloudy, or diluted by blood or other tissue fluids, it must be replaced.

Heating will accelerate the fixation process but, at the same time, will enhance autolytic changes in the unfixed portion of the tissue. Boiling will result in rapid fixation and has been used to prepare rapid-fixed frozen sections. We prefer to use unfixed tissues or formalin-fixed tissues (after routine penetration fixation of small samples) for frozen sections. Decalcification procedures are described in Chapter 8.

FIXATION MIXTURES Many fixatives have been modified by various authors and institutions. However, it seems that the improvements, if any, are minor compared with the results that will be achieved if size and exposure of the specimen and volume and freshness of the fixation fluid are appropriately controlled. Most of the recipes and specifications described here have not been changed since they were listed in the last edition. Several current sources (1,2) provide additional details.

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