28 FIXING AND HARDENING AGENTS 



action of the vapour. The tissues are treated as described in 

 § 717. Very small objects, such as isolated cells, are simply 

 placed on a slide, which is inverted over the mouth of the bottle. 



Thev 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 hsematoxylm. 



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

 freshly prepared mixture of osmie 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 possibihty of deformation through osmosis is eliminated. 

 See also under " Cramer's Method," § 717. 



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 ^o to 2 per cent. Never more 

 than 2 per cent, need be used. ^ . n ^ 



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

 should be as small as possible. ■ -.^ r \.i- 



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," § 710. n i j 



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 soaking, it frequently happens that some of the acid remains 

 in the tissues, and causes them to blacken in time, and m any 

 case hinders staining. To obviate this blackening it has been 

 advised to soak them for twenty-four hours in a solution ot 

 bichromate of potash (Miiller's solution or Erlicki's wi 1 do) or in 

 0-5 per cent, solution of cliromic acid, or in Merkel s solution. 

 The treatment with bichromate solutions has the great advantage 

 of highly facilitating staining with carmine or haematoxylm. Max 

 Schultze recommended washing, and mounting permanently m 

 acetate of potash ; Fol, treatment with a weak solution ot car- 

 bonate of ammonia. But the best plan of all is to properly 

 bleach the preparations. See " Bleaching." This may be done 

 by means of peroxide of hydrogen. Overton [Zeit. wiss Mik., 

 vii 1890, p. 10) finds that it is completed in a few minutes m a 



