36 FIXING AND HARDENING AGENTS. 



I therefore recommend that the osmic and chromic acid be kept 

 ready mixed in the proportions given, and 5 per cent, of acetic acid 

 added at the moment of using. 



WEAKER FORMULA. More recently, FLEMMING has been making 

 up the mixture with only 2 parts of the osmic acid instead of 4, and 

 has spoken of this modification as " weaker osmium mixture " 

 (MEVES, in Encycl. Mikr. Techn., p. 476). 



MEVES (loc. cit.) takes for delicate objects 15 parts of chromic acid 

 of only 0-5 per cent., 2 or 4 of osmic acid of 2 per cent., and 1 of 

 acetic acid, and thus gets less shrinkage. 



Under "Cytology" Sections, 678, see BENDA and GATENBY 

 modifications. 



PODWYSSOZKI recommends (for glands especially) the following 

 modification : 



1 per cent. Cr0 3 dissolved in 0-5 per cent, solution 



of corrosive sublimate . . . . . 15 c.c. 



2 per cent, osmic acid solution . . . . 4 c.c. 

 Glacial acetic acid . . . . . 6 to 8 drops. 



The sublimate is said to augment the penetration of the osmium, but 

 is unfavourable to staining (ZIEGLER'S Beitrdge z. path. Anat., i, 1886 ; 

 Zeit. wiss. Mik., iii, 1886, p. 405). 



The first or weak liquid is the better for very small objects, the 

 second or strong one for larger ones, as it has better penetration. 

 These liquids may be allowed to act for many hours or days, or 

 according to some workers even weeks or months ; but this exagge- 

 rated fixation is clearly only justifiable in very special cases, if at 

 all. For chromosome studies some workers fix for only one hour. 

 Others recommend cooling the FLEMMING on ice before using. 

 Wash out very thoroughly in water (running, twenty-lour hours), 

 or treat as directed for chromic acid, 38. Stain with alum 

 hsematoxylin if you wish to stain in toto (staining in this way 

 with other reagents is possible, but difficult). Stain sections 

 with safranin or other basic coal-tar colour, or with iron hsema- 

 toxylin. 



For fixing with the strong mixture you need only take a bulk of 

 liquid of some 4 times the volume of the objects (but with the weak 

 mixture the proportion should be increased). Both of them are 

 first-rate fixatives of cellular structures, both as regards their preser- 

 vation and as regards their optical differentiation. But they must 

 be properly used, and not applied to objects for which they are not 

 fitted. For instance, their power of penetration is extremely bad ; 

 they will not fix properly, even in a loose-celled tissue, through more 

 than a layer of about five cells thick. They are therefore suitable 



