38 FIXING AND HARDENING AGENTS 



especially was at one time universally employed for hardening all 

 sorts of tissues. 



Flemming {Arch. mik. Anat., xviii, 1880, p. 352) pointed out 

 that though it preserves cytoplasm well it causes chromatin to 

 swell, and therefore should not be employed /or the study of nuclei. 

 But, duly corrected with acetic acid, it affords a correct and fine 

 fixation of nuclei ; whilst preserving secretions, etc., much better 

 than chromic acid. 



For general work Kultschitzky and Zenker or Zenker Formalin are 

 the only bichromate formulae which are recommendable, and probably 

 only for vertebrate material. The chromates are of great importance 

 in neurological work, and as fixing fluids before staining bacteria in 

 tissues. 



None of these solutions approaches Bouin's fluid in general applica- 

 bility, TeUyesniczky and Miiller usually giving atrocious results. 



For an elaborate study of the action of chrome salts on nucleus and 

 cytoplasm, see Burckhardt, La Cellule, xii, 1897, p. 335. 



For the demonstration of the achromatic figure of cell division he 

 recommends — 



Chromic acid, 1 per cent, solution . . .60 vols. 



Bichromate of potassium, 5 per cent, solution . 30 ,, 

 Glacial acetic acid . . . . . 5 „ 



56. Bichromate of Potash.* Perhaps the most important of all 

 known hardening agents, senu stricto. It hardens slowly, much 

 more so than chromic acid, but it gives an incomparably better 

 consistency to the tissues. They may remain almost indefinitely 

 exposed to its action without much hurt. 



The strength of the solutions employed is from 2 to 5 per cent. 

 As with chromic acid, it is extremely important to begin with weak 

 solutions and proceed gradually to stronger ones. About three weeks 

 will be necessary for hardening a sheep's eye in solutions gradually 

 raised from 2 to 4 per cent. Spinal cord requires from three to six 

 weeks, a brain at least as many months. 



After hardening, the objects should be well soaked out in water 

 before being put into alcohol, or be treated as directed for chromic 

 acid, § 43. They had better be kept in the dark when in alcohol. 

 See § 43. (Boiim and Oppel [Taschenbuch, 3 Auf., 1896, p. 22] fix 

 in the dark.) // you wish to have a good stain with carmine you should 

 not ijut the objects into alcohol at all, even for a second, until they have been 

 stained. 



You may stain either with carmine or haematoxylin, as well as with 

 tar colours. 



Bichromate objects have an ugly yellow colour which cannot be 

 removed by mere soaking in water. It is said that it can be removed 

 by washing for a few minutes in a 1 per cent, solution of chloral hydrate. 



Prof. GiLSON wrote that alcoholic solution of sulphurous anhydride 

 (SO2) is very convenient for the rapid decoloration of bichromate 

 objects. A few drops suflice. See also § 43, and " Bleaching." 



To facilitate staining with haematoxylin, Wolff {Zeit. wiss. Mik., xv, 

 3, 1899, p. 311) first stains in Boeluner's haematoxylin for twenty-four 

 hours, and then for a few minutes in the same haematoxylin to which 



* Regaud's fluid, § 699. 



