486 PREPARATION, MOUNTING, AND COLLECTION OF OBJECTS 



same strength. These solutions do not precipitate so readily, and : 

 may be used for fixation by the vapours. 



For it is one of the advantages of osmic acid that it may be 

 employed for fixation in the form of vapour, and its employment in 

 this form is indicated in most of the cases in which it is possible to 

 expose the tissues to be fixed directly to the action of the vapour. 

 For fixation in this way ' the tissues are pinned out on a cork which 

 must fit well into a wide mouthed bottle in which is contained a 

 little solid osmic acid (or a small quantity of 1 per cent, solution will 

 do). 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 wash- 

 ing will suffice. For staining, methyl-green may be recommended 

 for objects destined for study in an aqueous medium, and, for per- 

 manent preparations, alum-carmine, picro-carmine, or hsematoxylin/ 



* The reasons for preferring the process of fixation by vapour of 

 osmium, where practicable, are that osmium is more highly penetra- 

 ting when employed in this shape than when employed in solution, 

 and produces a more equal fixation, and that the arduous washing 

 out required by the solutions is here done away with. In many 

 cases delicate structures are better preserved, all possibility of 

 deformation through osmosis being here eliminated/ (From Mr. Lee's 

 * The Microtomist's Vade-mecum.') 



For fixation by solutions, strengths of from ^ to ^ per cent, may 

 be taken, which may in general with advantage be acidified with 

 about 1 per cent, of acetic acid. Small Crustacea., such as the 

 copepods and the larvae of decapods, may be very well prepared in 

 this way. After fixation, the osmic acid should be very thoroughly 

 washed out with water. 



If it be desired to intensify the grey stain of the osmium, this 

 may be easily done by putting the objects into a weak solution of 

 pyrogallic acid or tannin, which will turn them of a fine black. 



Osmic acid stains most fatty substances of an intense black. 



Osmic acid is now not so much used in the form of a pure 

 aqueous solution as in that of the mixture known as liquid of Flem- 

 ming. This consists of 25 parts of 1 per cent, solution of chromic 

 acid, 10 parts of 1 per cent, osmic acid, 10 parts of 1 per cent, acetic 

 acid, and 55 of water. This mixture blackens tissues much less than 

 the pure aqueous solution. 1 



1 Bleaching. Tissues that have been blackened or browned by osmic or chromic 

 acid or the like may often with advantage be bleached by Mayer's chlorine method, 

 and will then be found to stain much more readily. ' Put into a glass tube a few 

 crystals of chlorate of potash, add two or three drops of hydrochloric acid, and as 

 soon as the green colour of the evolving chlorine has begun to show itself, add a few 

 cubic centimetres of alcohol of 50 to 70 per cent. Now put the objects (which must 

 have previously been soaked in alcohol of 70 to 90 per cent.) into the tube. They 

 float at first, but eventually sink. They will be found bleached in from a 

 quarter of an hour to one or two days, without the tissues having suffered. 

 Only in obstinate cases should the liquid be warmed or more acid taken. 



