30 FIXING AND HARDENING AGENTS. 



For the so-called c regeneration ' of reduced solutions, see 

 previous editions. 



Osmic acid is met with in commerce in the solid form in sealed tubes. 

 The assigned weights should be checked, as they may vary greatly. 



Fixation by the Vapours. This is indicated in most of the cases 

 in which it is possible to expose the tissues directly to the action of 

 the vapour. The tissues are treated as described in 695. Very 

 small objects, such as isolated cells, are simply placed on a slide, 

 which is inverted over the mouth of the bottle. They remain there 

 until they begin to turn brown (isolated cells will generally be found 

 to be sufficiently fixed in thirty seconds : whilst in order to fix the 

 deeper layers of relatively thick objects, such as retina, an exposure 

 of several hours may be desirable). It is well to wash the objects 

 with water before staining, but a very slight washing will suffice. 

 For staining, methyl-green may be recommended for objects destined 

 for study in an aqueous medium, and, for permanent preparations, 

 alum-carmine, picro-carmine, or hsematoxylin. 



In researches on nuclei, it may be useful to employ the vapours 

 of a freshly prepared mixture of osmic and formic or acetic acid 

 (Gilson, La Cellule, i, 1885, p. 96). 



The reasons for preferring fixation by the vapour are that osmic acid 

 is more highly penetrating in vapour than in solution ; that the arduous 

 washing out required by the solutions is done away with ; and that all 

 possibility of deformation through osmosis is elminated. See also 

 under. "Cramer's Method," 695. 



Fixation by Solutions. Osmic acid is now very seldom used pure 

 in the shape of solutions. When, however, it is so employed it is 

 used in strengths varying from ^ to 2 per cent. I should say 

 myself that, as a rule, not more than 0-1 per cent., and never 

 more than 2 per cent., should be used. 



On account of its feeble penetrating power the objects to be fixed 

 should be as small as possible. 



The solutions should be kept protected from bright light during 

 the immersion of tissues. (This precaution is not necessary if 

 Flemming's or Hermann's solution be used.) If the immersion is 

 to be a long one the tissues must be placed with the solution in well- 

 closed glass-stoppered vessels. The objects may be deemed to be 

 fixed as soon as they have become brown throughout. But see 

 " Mann-Kopsch Methods," 693. 



After-Treatment. The excess of osmic acid must be well washed 

 out before proceeding to any further steps in preparation ; water 

 should be used for washing. Notwithstanding the greatest care in 



