372 APPENDIX 



A saturated corrosive sublimate solution in salt solution with the addition of 5% 

 of glacial acetic acid may be used as a substitute for Zenker's fluid. 



2. Dehydration. After washing for twelve to twenty-four hours in running 

 water, following corrosive sublimate fixation, or simply washing for a few minutes 

 after formalin, the tissues should be placed in 70% alcohol. They may be kept in 

 this indefinitely. If they are to be sent to a laboratory for sectioning, it is advisable 

 to moisten a pledget of cotton in 70% alcohol and fill in the bottom of the bottle with 

 it. Then drop in the tissues and pack in gently over them sufficient 70% alcohol- 

 saturated cotton to fill up the bottle. All the alcohol should be absorbed by the 

 cotton so that if the bottle should break in transit there would be no damage from the 

 alcohol. The stopper of the bottle should be paraffined or sealed with wax. 



Tissues may be left in the 70% alcohol twelve to twenty-four hours and should 

 then be transferred to 95% alcohol for an equal time. They are then transferred to 

 absolute alcohol, where they remain from two to twelve hours and are then placed in 

 xylol. The time in xylol should be as short as possible. So soon as the tissue looks 

 clear it should be removed thirty minutes to two hours. 



3. Imbedding. The tissue is now transferred to melted paraffin. Paraffin melt- 

 ing at 48 C. for winter work, and that melting at 54 C. for summer is to be recom- 

 mended. The time in the paraffin should not be prolonged. Two hours will 

 ordinarily suffice. Some leave in the paraffin for twelve to twenty-four hours. 



Next take a paper box (made of stiff writing-paper folded over a square of wood) 

 and fill with the melted paraffin. As quickly as possible drop in the piece of tissue 

 taken out of the paraffin bath with heated forceps and, so soon as the paraffin begins 

 to solidify on the surface, place the paper box in ice water. When paraffin is rapidly 

 cooled, crystallization is less. 



The Acetone Method. Take the tissues out of the 70% alcohol and place in ace- 

 tone. After remaining in acetone for one to two hours, the tissues should be trans- 

 ferred to fresh acetone for an equal length of time. Dry calcium chloride in the bot- 

 tom of the acetone bottles keeps it dehydrated. They should then be placed in 

 xylol for about one-half hour and then embedded in paraffin as directed above. 



The Chloroform Method. The procedure may be the same as in the method of 

 passing through alcohols to xylol, substituting chloroform for xylol and then trans- 

 ferring to paraffin. 



Where absolute alcohol is not obtainable, very satisfactory results may be 

 obtained by transferring to a mixture of 95% alcohol and chloroform after immer- 

 sion in 95% alcohol. Then going from the alcohol-chloroform mixture to pure 

 chloroform, thence to paraffin. 



Rapid paraffin imbedding methods. 



When a piece of tissue is not more than one-fourth inch square and one-eighth 

 inch thick, it is very easy to run it through in three to six hours. Thus: 



10% Formalin (in 37 C. incubator), i hour. 



70% Alcohol (in 37 C. incubator), i hour. 



95% Alcohol (in 37 C. incubator), i hour. 



Absolute Alcohol (in 37 C. incubator), 1/2 hour. 



Xylol (in 37 C. incubator), 1/2 hour. 



Paraffin (in 55 C. incubator), 1/2 to 2 hours. 



