98 MICROSCOPIC METHODS 



become brittle if overheated, and therefore the best results are 

 obtained by using paraffin melting at a somewhat low tempera- 

 ture. We have used for some years a mixture of one part of 

 paraffin, melting at 48, and two parts of paraffin melting at 

 54 C. This mixture has a melting-point between 52 and 

 53 C., and it serves all ordinary purposes well. An excellent 

 quality of paraffin is that known as the " Cambridge paraffin," 

 but many scientific-instrument makers supply paraffins which, for 

 ordinary purposes, are quite as good, and much cheaper. The 

 successive steps in the process of paraffin embedding are as 

 follows : l 



1. Pieces of tissue, however hardened, are placed in fresh absolute 

 alcohol for twenty-four hours in order to their complete dehydration. 



2. Transfer now to a mixture of equal parts of absolute alcohol and 

 chloroform for twenty- four hours. 



3. Transfer to pure chloroform for twenty-four hours or longer. At 

 the end of this time the tissues should sink or float heavily. 



4. Transfer now to a mixture of equal parts of chloroform and paraffin 

 and place on the top of the oven for from twelve to twenty-four hours. 

 If the temperature there is not sufficient to keep the mixture melted 

 then they must be put inside. 



5. Place in pure melted paraffin in the oven for twenty-four hours. 

 For holding the paraffin containing the tissues, small tin dishes such as are 

 used by pastry-cooks will be found very suitable. There must be a 

 considerable excess of paraffin over the bulk of tissue present, otherwise 

 sufficient chloroform will be present to vitiate the final result and not 

 give the perfectly hard block obtained with pure paraffin. With ex- 

 perience, the persistence of the . slightest trace of chloroform can be 

 recognised by smell. 



In the case of very small pieces of tissue the time given for each stage 

 may be much shortened, and where haste is desirable Nos. 2 and 4 may 

 be omitted. Otherwise it is better to carry out the process as described. 



6. Cast the tissues in blocks of paraffin as follows : Pairs of L-shaped 

 pieces of metal made for the purpose by instrument makers must be at 

 hand. By laying two of these together on a glass plate, a rectangular 

 trough is formed. This is filled with melted paraffin taken from a stock 

 in a separate dish. In it is immersed the piece of tissue, which is lifted 

 out of its pure paraffin bath with heated forceps. The direction in 

 which it is to be cut must be noted before the paraffin becomes opaque. 

 When the paraffin has begun to set, the 'glass plate and trough have 

 cold water run over them. When the block is cold, the metal L's are 

 broken off, and, its edges having been pared, it is stored in a pill-box. 



The Cutting of Paraffin Sections. Sections must be cut as 



1 While the method given is sufficient for ordinary purposes, a more elaborate 

 technique is necessary if it is desired that no changes shall take place in the 

 tissue. Thus after fixation the tissue must be taken up to absolute alcohol 

 through successive dilutions of spirit, but differing from each other by more 

 than 10 per cent. Again, when alcohol has been replaced by chloroform the 

 latter must be saturated with chips of paraffin, first at even temperature, then 

 at 37 C., and must be kept at 55 C. as short a time as possible. 



