314 GOLGI BODIES ETC. 



for solid tissues like nerve, longer is necessary. These times 

 must be ascertained experimentally. After fixation the pieces 

 are washed in two changes of distilled water for half an hour or 

 less, according to the size of the tissue and its accessibility to 

 the water. The pieces are transferred to a glass-stoppered 

 bottle containing just enough 2 per cent. OSO4 in aq. dest. to 

 cover them. Then they are left in a cupboard at room tempera- 

 ture, for at least ten days, and preferably two or three weeks. 

 Every few days the bottle should be examined to see whether 

 the OSO4 is evaporating, or whether it has completely disinteg- 

 rated. Should either have happened the pieces should be washed 

 quickly in aq. dest., and new OsO^ solution added. It should be 

 noted, however, that the osmic solution nearly always becomes 

 slightly dark, but not until it has gone black or no longer smells 

 of OSO4 should new liquid be added. When the right period has 

 elapsed the objects are taken out of the osmic. 



Ludford's Modification. Experiments have shown that a much 

 better impregnation is obtained if the reduction of the osmic 

 acid is completed by transferring the pieces of tissue to water 

 kept at 38° C. for one or two days before washing in running 

 water preparatory to transference to alcohol (50 per cent.) (see 

 § 714). They are then upgraded and imbedded in hard paraffin. 

 Sections to be cut from 3 to 6 /x. They are stuck on the slide 

 with albumen and water in the usual way and dried overnight. 

 One of the slides is taken, the wax removed in xylol, and it is 

 mounted in xylol balsam. Examination of this slide will enable 

 one to ascertain to what extent the process has acted successfully. 

 In completely successful preparations the Golgi apparatus, yolk 

 and fat alone are blackened, while nuclear organs, mitochondria 

 and cytoplasm are stained in shades of yellow and greenish-brown, 

 Having studied this untreated slide, and noted the extent of the 

 blackening effect of the OSO4, one may then proceed to make 

 experiments. Several alternative methods may be tried : — 



{a) The blackening may be extracted step by step in turpentine, and 

 the appearance of the cell granules studied at intervals. An 

 alcoholic solution of hydrogen peroxide can often be used with 

 advantage for this purpose (20 per cent, of hydrogen peroxide 

 added to 80 per cent, alcohol). 



(6) If the mitochondria are not stained black by the OSO4, one may 

 proceed directly to Altniann's method (see § 694). 



(c) The nuclear structures may be stained in safranin, crystal violet, 

 or acid fuchsin. The sections are brought down to distilled 

 water and transferred to watery solutions of the dye. A few 

 minutes generally suffice to stain the nuclei. 



LuDFORD {Jour. R.M.S., 1925, pp. 31 — 36) recommends the em- 

 ployment of neutral red as a counterstain. Sections which have not 

 been bleached are brought down to water. They are then stained for 

 half a minute, or less, in a dilute solution of neutral red (1 grm. of 

 neutral red in 1000 c.c. of distilled water, with 2 c.c. of a 1 per cent. 



