34 FIXING AND HARDENING AGENTS 



If this mixture be kept in stock in large quantities, it may go 

 bad, on account of the large proportion of organic acid contained 

 in it. We 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 moinent of using. 



It has been pointed out by J. Z. Young [Nature^ May 18th, 1935) 

 that the addition of 0-75 to 0-95 per cent. NaCl, according as to 

 whether cold-blooded or warm-blooded animal tissues are being 

 fixed, appears to increase the area of cells fixed. In experiments 

 we have made Young's claim appears to be substantiated, and we 

 strongly recommend a trial of this modification. It should be 

 mentioned that Meves sometimes added 1 per cent. NaCl to the 

 weak formula. In our experience the addition of NaCl causes 

 disintegration of the solution in only a few weeks, and the fluid 

 if to be stored shovdd be made up as follows : solution A, 15 parts 

 of 1 per cent, chromic acid in aq. dest. ; solution B, 4 parts of 

 2 per cent. OSO4 in 4 per cent, (actually 4-275 per cent.) NaCl. 

 Dissolve the osmic first and then add the salt. Before fixing 

 material add 15 parts of A to 4 parts of B. This gives a 

 Flemming without acetic acid with 0-9 NaCl. 



Weaker Formula. Later, 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, see Benda, Gatenby, and 

 Guthrie modifications (§ 679). 



PoDWYSSOZKi recommends (for glands especially) the following 

 niodifieation : 



1 per cent. CrOg 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 Beitriige z. path. Anat., i, 1886 ; 

 Zeit. wiss. Mik,, iii, 188G, 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 

 exaggerated 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-four hours), or treat as directed for chromic acid, § 43. 



