MEDICAL AND BIOLOGICAL STAINING TECHNIQUES 



Note: Where possible use a series of corked specimen tubes for 

 the above procedures. By occasionally shaking the tube containing 

 the specimen the process of penetration of the alcohol is acceler- 

 ated. Twelve hours in each change of alcohol is sufficient for small 

 pieces, but larger pieces of tissue usually require eighteen or 

 twenty-four hours. Hard tissues may be softened by Lendrum's 

 technique which consists of immersing the tissues in 4% aqueous 

 phenol for one to three days, after washing out the fixative; de- 

 hydration is then carried out in the manner described above. 



Rapid dehydration of small slices of tissue: 



Thin slices not more than 5 mm. in thickness are immersed for 

 half an hour in each of 50%, 70%, 90%, 96% and absolute alcohol. 



{h) Clearing 



Xylol, cedarwood oil, benzene, toluene or chloroform are the 

 reagents most frequently used for this purpose. 



Xylol is the most rapid of these in displacing the absolute 

 alcohol, but it has the disadvantage of rendering tissues brittle; 

 therefore, if xylol is used as the clearing agent tissues must be sub- 

 jected to it only for the minimum time necessary to displace the 

 absolute alcohol. 



Cedarwood oil is slow in its action but it has the advantage of not 

 hardening tissues even after prolonged immersion. 



Benzene is the best clearing agent and may be employed for the 

 most delicate tissues : it causes the minimum shrinkage, penetrates 

 tissues fairly rapidly and subsequently evaporates from them in 

 the paraffin embedding bath. 



Toluene is also a very satisfactory clearing agent in that tissues 

 can be subjected to it for at least twenty-four hours without risk of 

 their undergoing shrinkage. 



Chloroform hardens tissues to a lesser degree than xylol but 

 requires two or three times as long to penetrate and clear the tissue. 

 It rapidly evaporates from the paraffin embedding bath, and it is 

 particularly suitable for large pieces of pathological tissue. 



Technique: 



I. Small pieces of material not more than 5 mm. thick may be 

 cleared by immersing for fifteen to thirty minutes in each of two 



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