FIXING AND HARDENING REAGENTS. 33 



tissue is to be hardened in a certain fluid, and to be treated with 

 osmic acid afterwards, that tissue had better not be put into alcohol 

 if it contains fat or fatty particles, as the alcohol dissolves the fat. 

 Thus, a tissue hardened in Miiller's fluid may be put into osmic acid, 

 and the fat-cells will still be blackened. 



It has recently been shown that the prolonged action of turpen- 

 tine, toluol, xylol, ether, and creasote, but not clove-oil or chloroform, 

 will decolorise particles of oil (fat) blackened by osmic acid. This 

 is most important in connection with the osmic method for studying 

 the absorption of fat in the small intestine (Lesson XXV.). 



Tissues containing fat maytherefore be embedded in paraffin after 

 being passed through chloroform (p. 43). 



Osmic acid fixes fresh tissues very rapidly, but it does not pene- 

 trate deeply ; therefore the tissues must be cut into very small pieces 

 to get them fixed throughout by the acid. It has the power of 

 differentiating tissues, the nuclei become yellow, fat, and the nervous 

 system black. After twenty-four hours or so, the tissues are 

 thoroughly washed in water and hardened in 90 per cent, alcohol. 

 To avoid the blackening which is apt to occur in tissues still con- 

 taining a trace of osmic acid, it has been proposed to treat the 

 tissues with a weak solution of cyanide of potassium, and then to 

 harden in alcohol. It enters into the composition of several 

 hardening fluids (p. 32), and Hermann's fluid (Lesson XXXV.). 



D. Other Hardening Fluids. 



1. Bichloride of Mercury or Corrosive Sublimate. A saturated 

 watery solution contains about 5 per cent, of the salt; but it is 

 much more soluble in alcohol, especially alcohol of 50 to 60 per 

 cent. Make a saturated watery solution, and also a saturated 

 alcoholic solution. A saturated solution in 0.5 NaCl solution is 

 also used. 



A cold saturated solution is best made as follows : Place 60 

 grams of it in i ooo cc. of water, and dissolve with the aid of heat. 

 Filter the warm solution and allow it to cool. On cooling, long 

 white needle-shaped crystals of the sublimate separate. The 

 supernatant fluid is used, and the tissues, which must not be more 

 than J cm. in diameter, remain in the fluid from 1-3 hours, accord- 

 ing to their size. After fixation they are hardened in 70, 80, and 

 90 per cent, alcohol. Xo metallic instruments are to be used. 

 Use glass or wooden instruments. 



This is a most excellent hardening reagent, and it hardens tissues 

 with great rapidity, so that tissues must not be left in it for too 

 long a time. For small pieces, u quarter of an hour or thereby is 

 5 C 



