23 



in equal parts of toluene and absolute alcohol for 1 hour to 1 day; (2) 

 this is replaced by pure toluene for an equal period when the tissue 

 will be clear and translucent, except, of course, such as is dark in 

 color. 



Xylene may be used in place of toluene in nearly all cases. Steps 1 and 3 may 

 often be omitted with very small objects. In some cases it is well to clear with 

 cedarwood oil first ( 54) and then transfer to toluene (or xylene), toluene paraffin, 

 etc. Familiarity with the tissue and the reagents will permit considerable depart- 

 ture from a fixed line of procedure. 



53. Infiltration. After the tissue is completely cleared in 

 the toluene, remove it to a dish of (3) melted infiltration paraffin 

 1 part, toluene 1 part, and set it in a warm place (about 38 C.), so 

 that the paraffin may remain melted and the toluene slowly evaporate. 

 After several hours or even days with very large pieces place the tissue 

 in (4) pure paraffin in the paraffin oven for 2 to 24 hours, depending on 

 the size of the piece. Quite large pieces may be left longer ; with them 

 one or two changes to fresh infiltration paraffin may be necessary. 

 The melted paraffin replaces the toluene, filling in the interstices of the 

 tissue. Paraffin melting at about 52-54 C. is used. It is best not to 

 expose to a higher temperature than is necessary, or for a long period 

 of time as the heat tends to shrink and toughen, especially if the dehy- 

 dration (and consequently the clearing) have been incomplete; this 

 is particularly true of organs rich in connective tissue. The paraffin 

 oven w T ill be maintained at a temperature of 54-5 C. 



54. Other Clearers. Cedarwood oil is a good clearer. It will clear from 

 95% alcohol if the dehydration has been carefully done. The tissue should be left 

 in the cedarwood oil until it sinks and the alcohol currents have ceased to rise from 

 it. The steps are given below. Thickened cedarwood oil such as is used for 

 immersion objectives has been found to give excellent results with small objects 

 and is to be recommended for some cell work. Chloroform gives excellent results 

 but it penetrates (clears) slowly, so that it should be used only with small pieces 

 of tissue. Chloroform or thickened cedarwood oil is to be employed with objects 

 in which it is desired to preserve fat which has been blackened by osmic acid 

 ( 227). 



CHLOROFORM. CEDARWOOD OIL. 



Alcohol (97-99%) . 95% or absolute alcohol. 



Chloroform, until it sinks below the sur- 

 face. 



Chloroform and paraffin, equal parts, 

 for 4-48 hours, at incubator tempera- 

 ture (38 C.) . cedarwood oil. 



Pure paraffin, in the paraffin oven. Pure paraffin, in paraffin oven. 



