304 GOLGI BODIES ETC. 



(see Miss J. C. Hill, Archiv. f. exper. Zellf., 1936, and T. J. 

 Macdougald, ibid.). So far as we are aware, this warning only 

 applies to the delicate fat globules in fibroblasts in vitro. 



The basis of all chrome-osmium techniques consists in a pre- 

 liminary fixation of small pieces of tissue, small embryos or eggs, 

 in such a fluid as Benda, Champy, Flemming- without-acetic acid, 

 or Altmann, for from at least twelve hours to a week. The osmic 

 reaction is then, in some methods (Kull, Benda), " set " or 

 strengthened by the reducing effect of pyroligneous acid ; follow- 

 ing this treatment is a further " chroming " in 3 per cent, bi- 

 chromate of potash, and, finally, a thorough wash out under the 

 tap. Material treated in this way is generally perfectly preserved, 

 and fit for selective staining. Arranged below are chrome-osmium 

 techniques of progressive intensity and difficulty. 



692. Modified ' ' Flemmings ' ' for Cell Inclusions. Benda : 

 15 c.c. chromic acid 1 per cent., 4 c.c. osmic acid 2 per cent., 3 to 6 

 drops of acetic acid. Meves : 15 c.c. of chromic of 0-5 to 1 per 

 cent., containing 1 per cent, sodium chloride, with 3 to 4 c.c. of 

 2 per cent, osmic acid, and 3 to 4 drops of acetic {Enzyk. mik. 

 Techn., 1910). Strong Flemming-without-acetic acid ; and same 

 solution diluted by one-half or one-third (Gatenby, Quart. Journ. 

 Micr. Sci., 1919). The presence of a small quantity of acetic acid 

 is always liable to introduce distortion, but less so among verte- 

 brates than among invertebrates. 



In § 47, a method for making up salt Flemming-without-acetic, and 

 in § 49 salt Champy is given, and should be tried. See also Bensley, 

 § 48, and Baker and Thomas' modification of Altniann's fluid, § 48. 

 Lison (ibid.) attributes Flemming-without-acetic to Heitz (?). Meves 

 sometimes used Flemming-without-acetic, but preferred some acetic 

 acid. The method given below is original so far as times are concerned, 

 and these should be rigidly adhered to for the best results. 



693. Gatenby's Flemming 's Strong Fluid without Acetic Acid, 

 and Iron Haematoxylin. Small organs freshly dissected out in 

 normal saline, or parts of organs cut with safety razor blade, not 

 more than 5 mm. in diameter, are placed in about 15 c.c. of one of 

 the above-mentioned fixing fluids. A glass-covered capsule or 

 vial is the best vessel to use, and the material is left for at least 

 twenty hours, and not longer than one week. We find about 

 twenty-four hours gives a satisfactory fixation of most tissues. 

 After fixation the liquid is poured away, and the material is 

 washed for at least two hours, and not necessarily longer than 

 five, in running tap-water. It is then passed through up-graded 

 alcohols, beginning at 30 per cent., giving the material at least 

 three hours in the strengths 30, 50 and 70 per cent., and overnight 

 in 90 per cent. The pieces of tissue are dehydrated two or three 

 hours in two changes of absolute alcohol, and then transferred to 

 a mixture of half absolute alcohol and half xylol for a quarter of 

 an hour ; then pure xylol, and imbedded in wax. Sections are 



