334 Journal of Applied Microscopy. 



The transfer from absolute alcohol to the clearing agent should be gradual, 

 like the hardening and dehydrating processes. The following is a good method-: 



3 parts 100 per cent, alcohol, and 1 part xylol, 1 to 10 hours. 

 2 parts 100 per cent, alcohol, and 2 parts xylol, 1 to 10 hours. 

 1 part 100 per cent, alcohol, and 3 parts xylol, 1 to 10 hours. 



Pure xylol until the material becomes transparent. This may require only a 

 moment, but may require hours. Other clearing agents may be used in the 

 same way instead of the xylol. 



5. The Transfer from Clearing Agent to Paraffin. — This should also be a 

 <^ra(inal process. The most convenient method is to place a small block of 

 paraffin in the pure clearing agent with the material. The paraffin dissolves 

 o-radually and produces the same result as if a small shaving of paraffin had 

 been added every few minutes for a day or so. During this process the bottle 

 or dish should be kept lukewarm. Six to ten hours, or over night, should be 

 sufficient for this step, although it would seem that material may be kept here 

 for a much longer time without injury. Excellent preparations of the embryo-sac 

 of Aster have been made from material which had remained in the xylol and 

 paraffin for nearly three years. No more paraffin should be added than will 

 go into perfect solution. The temperature may be gradually increased so that a 

 much greater amount of paraffin will go into solution, but the paraffin must 

 not be allowed to crystallize. 



6. The Paraffin Bath. — This step is usually called infiltration, but when 

 the transfer from the clearing fluid to paraffin is made gradually, as has just 

 been indicated, the process of infiltration is already begun. It is now necessary 

 to get rid of the xylol or other clearing agent. This may be done by simply 

 pouring off the mixture of xylol and paraffin and replacing it with pure melted 

 paraffin. The bath should be kept at a temperature about 1 degree C. higher 

 than the melting point of the paraffin. 53 degrees C. is a good temperature for 

 general purposes, but this may be reduced from 1 degree to 3 degrees C. in winter 

 and must often be raised in summer. For special purposes it is sometimes 

 necessary to use a temperature as high as 70 degrees C. If the xylol or other 

 clearing agent is not thoroughly removed the paraffin will be granular or 

 mealy, and will not cut well. It is well to change the paraffin once or twice 

 to make sure that the clearing agent is all removed. Do not waste this paraffin, 

 for the clearing agent can be driven off by prolonged heating and the paraffin 

 is better than ever. Most people use soft paraffin (about 45 degrees C.) for 

 the first half of the time necessary for infiltration, and a harder grade 

 (4K degrees C. to 54 degrees C.) for the latter part of the process. This is a 

 good plan, for soft paraffin melts at a lower temperature, and it is always well 

 to minimize heat. 



Some think that it is better not to pour ofT the mixture of the clearing agent 

 and paraffin, but rather to evaporate the clearing agent by keeping the tempera- 

 ture just high enough to prevent the crystalization of the paraffin. This method 

 has certainly given good results with small, delicate objects. 



The time required for infiltration varies with the character of the tissue and 

 the size of the piece. Few things can be well infiltrated in less than an hour. 



