24 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



It has been found that dioxan is an excellent substitute for alcoholic dehydration. Follow- 

 ing the washing (or even without washing) the tissues are put directly into dioxan for 2 

 two-hour changes. Larger tissues may be left in dioxan for a month without any deleter- 

 ious effects. If the dioxan is kept in covered jars and over copper sulphate, it should 

 last a long time. The dioxan fumes are known to be poisonous to humans. 



Amphibian eggs and yolk-laden embryos should not be completely dehydrated. It is 

 thought that the dioxan method does not give complete dehydration and that this is one of 

 the reasons that dioxan gives better results with amphibian eggs. 



CLEARING 



Clearing follows dehydration and must be accomplished before embedding. In any case, 

 the absolute alcohol should be mixed with the clearing agent so that the transition is 

 gradual. Several steps of 15-30 minutes each are best. The dehydrant dioxan acts as its 

 own clearing agent. 



Xylol - this agent tends to make yolk-laden eggs and embryos rather brittle and sections 

 are apt to crack. This tendency can be somewhat compensated by the addition of 5% 

 lanolin (Sheep fat) to the xylol. Xylol will become cloudy if the embryos were not suf- 

 ficiently dehydrated. 



Benzole - same as xylol. 



Dioxan - this is both a dehydrating and clearing agent, and can be mixed with fixatives. 

 The transfer to paraffin is generally made by way of a paraffin-xylol mash. Used for 

 both cytological and histological preparations. 



Chloroform - tissues are transferred from absolute alcohol to a mixture of equal parts 

 of alcohol and chloroform until they sink to the bottom of the container, thence to pure 

 chloroform for final clearing. Keep the vial covered. 



Wintergreen oil - excellent for yolk masses and glandular tissue, used as chloroform. 

 Tissues become translucent and may thus be photographed. 



Clove oil - used same as wintergreen oil. 



Aniline oil - same as wintergreen oil except that it will mix with lower alcohols down to 

 80%. Stained sections are dehydrated to 95%, then to equal parts of 95% and aniline oil 

 for 10 minutes; finally to 2 ten-minute changes in pure aniline oil. Mount in aniline- 

 balsam. Good for mitotic figures. 



EMBEDDING 



This is accomplished by infiltration with paraffin, wax, rubber, or mixtures of these. 

 Whatever clearing agent is used, the process of embedding should be gradual and at a 

 temperature slightly above the melting point of the mixtures. To the vial containing the 

 clearing fluid and cleared tissue, add shavings of the embedding mixture and bring to 

 about 40°C. for several hours. The paraffin will become gradually more concentrated 

 with the evaporation of the clearing agent. Then transfer to embedding substance for 2 

 half-hour changes. 



If dioxan is used for dehydration and clearing, warm a mixture of 25 cc. dioxan, 5 cc. 

 xylol and 20 cc. of 60°C. paraffin and transfer the tissues to this mixture for 30 minutes. 



