100 The Preparation of Microscope Slides 



solvent, there are certain great objections. The first of these is the toxicity 

 of dioxane vapor to humans. Dioxane is a cumulative poison and has been 

 shown to affect seriously the function of both the liver and the kidneys . 

 This does not matter very much where it is used by an individual who 

 knows himself to be free from hepatic or renal disorder, particularly where 

 he is exposed only to low concentrations of the vapor for relatively short 

 periods. However, it militates heavily against the use of this reagent in 

 large classes where the instructor is responsible for the health of individ- 

 uals without knowing their physical idiosyncrasies. Another disadvantage 

 is in the heavy diffusion stresses that are set up when materials are trans- 

 ferred from dioxane to molten paraffin. This is not so important when 

 dealing with tissue blocks for routine histological examination but it is 

 almost impossible to get a good section of a 72-hr chick embryo, for ex- 

 ample, using this shortened dioxane technique. 



As has already been pointed out, dehydrating and clearing plant 

 tissues are altogether different propositions. They may be run through 

 ethyl alcohol, using a very long series of gradually increasing strengths, 

 but it is more usual nowadays to employ mixtures of alcohol and tertiary 

 butyl alcohol. Tertiary butyl alcohol is miscible with molten paraffin so 

 that dehydration and clearing take place at the same time in these graded 

 series. This is described in more detail in Chapter 12, which discusses 

 the process of cutting sections. 



Clearing Agents. Clearing agents, which remove the dehydrating agent 

 from the tissues and leave them in condition either for mounting in 

 balsam or embedding in paraffin, are of two main types. For mounting 

 in balsam, it is customary to use one of the essential oils or their synthetic 

 equivalents. The advantage of these materials is that many of them are 

 readily miscible with 90 per cent to 95 per cent alcohol and, therefore, are 

 capable of removing the last traces of water that may be left in the speci- 

 men after imperfect dehydration. 



In the author's opinion, the best clearing agent for general use before 

 making wholemounts is terpineol (synthetic oil of lilac). This material is 

 readily miscible with 90 per cent alcohol and has the additional advan- 

 tages that it has neither an unpleasant odor nor does it render objects 

 brittle. It is, however, more customary today to recommend clove oil. 

 This has the advantage of being much more fluid than terpineol and the 

 disadvantages of a very pungent odor and the tendency to make small 

 objects brittle. The latter is sometimes an advantage, as when one is en- 

 deavoring to remove appendages from small arthropods, -but on other 

 occasions it is very annoying. Many other oils have been recommended 

 from time to time, but these two between them will be satisfactory for 

 making a wide variety of wholemounts. Under no circumstances what- 

 ever should an essential oil be used to clear objects intended for embed- 



