MICROSCOPY 99 



Preparation of materials 



Some biological materials can be placed on a slide with water, cov- 

 ered by a cover glass, and examined immediately. Many others, however, 

 are too thick to transmit light or there is inadequate contrast between 

 parts. The biological specialty known as microtechnique has evolved as 

 a result of the need to prepare materials for examination. 



Water mounts are satisfactory for some purposes, but the water tends 

 to evaporate. If a specimen must be examined for a longer period of time, 

 mounting in glycerol gives better results. If living materials must be 

 kept alive during examination, either water or glycerol is satisfactory, 

 and certain non-poisonous dyes can be added. Neutral red, methylene 

 blue, and janus green are "nontoxic" and tend to collect mainly in certain 

 parts of cells, increasing the contrast between these parts and the rest 

 of the cell. If the material is too large or too thick, slices can be made 

 freehand with a razor blade. With practice, a steady hand can cut slices 

 only one or two cells thick. 



Prepared slides or permanent mounts are made when more than a 

 preliminary examination is required. The techniques are complex and 

 variable, are frequently long and tedious, but usually involve four major 

 steps: fixation, embedding, sectioning, and staining and mounting. The 

 order of the steps may vary somewhat. Fixation is a method of killing 

 the cells. Ideally, the living material is killed rapidly and in such a way 

 that the various structures do not become disarranged with respect to 

 each other. We should hope that the nucleus of a dead cell on a prepared 

 slide looks about the same as the living nucleus. Obviously changes do 

 occur, but certain fixation procedures apparently bring about a minimum 

 of change. The killing usually is accomplished by chemical fixatives, such 

 as a combination of formaldehyde, acetic acid, and alcohol or a mixture 

 of potassium dichromate, chromic acid, and osmic acid. Some fixatives 

 tend to demineralize bone or to soften hard materials. Often water is 

 withdrawn from the tissue, and another material substituted in its place. 



Before a fixed and softened material can be sliced into thin sections 

 it must be provided with some support. The simplest means of embed- 

 ding is to pour melted paraffin around the object and then allow the 

 paraffin to permeate the material and finally to harden. Paraffin melts at 

 a fairly low temperature, so the biological material ordinarily need not be 

 damaged by a high temperature treatment. More recently several syn- 



