492 PREPARATION, MOUNTING, AND COLLECTION OF OBJECTS 



The solution known as Kernschwarz may be confidently recom- 

 mended as a powerful, precise, and very safe stain. It is a black 

 liquid imported from Russia by Grubler and Hollborn, and consists, 

 of an iron base united to some gallic acid. Sections may be stained 

 in it, either concentrated or diluted to the required intensity. 

 Overstaining seldom occurs. If it should occur it may be corrected 

 by means of any weak acid (solution of liquor ferri sidfarici oxydati, 

 diluted twentyfold see the iron-haematoxylin of Benda, below is a 

 very fitting decolorant). 



The result is a nuclear stain, sometimes, though by no means 

 always, also taking effect on protoplasm, of a brownish grey or 

 black, powerful and precise, and well adapted for photography. It is 

 permanent in balsam, presumably also in aqueous mounting media. 

 Being a progressive stain, it is possible that it might give good 

 results for staining in bulk. 



The present writer obtains a very similar stain by ' mordanting " 

 for a few hours in Benda's liquor ferri, and then bringing the 

 sections directly for some hours into a 2 per cent, solution of 

 pyrogallol in water. Similar results are also obtained by mordanting 

 in 2 per cent, solution of tincture of perchloride of iron in 70 per 

 cent, alcohol, and then treating with 2 per cent, solution of pyro- 

 gallol in spirit : a process which is applicable to staining in bulk. 



Benda's iron hcematoxylin is a still more powerful and precise 

 stain. Sections of material that has been fixed in any way may be 

 employed. They are ' mordanted ' by soaking for half an hour or 

 for some hours (as much as twenty-four, if a very strong stain be 

 required) in liquor ferri sulfurici oxydati, P.G., diluted with one or 

 two volumes of water. 1 They are then well washed, first with 

 distilled water, then with tap water, and are brought into a 1 per 

 cent, solution of heematoxylin in water, in which they remain till 

 they have become thoroughly black. They are now overstained, 

 and must be ' differentiated.' To this end they are washed and put 

 either into some of the sulphate solution strongly diluted with water 

 (say twenty or thirty fold), or into 30 per cent, acetic acid, the 

 progress of the decoloration being followed in either case under the 

 microscope. They are then mounted in the usual way. 



This gives an extremely powerful blue-black stain, purely nuclear 

 if the differentiation has been pushed far enough, or nuclear and at 

 the same time plasmatic if the differentiation is stopped before the 

 protoplasm has become decoloured. The stain is absolutely 

 permanent in balsam. 



The results obtained by this process are practically identical with 

 those obtained by the iron hcematoxylin process of Heidenhain, with 

 this advantage, that Benda's iron solution is easily made and keeps 

 indefinitely, whereas Heidenhain's process involves the employment 



1 This preparation consists of sulphate of iron 80 parts, water 40, sulphuric acid 

 15, and nitric acid 18. The ingredients should be mixed, and give at first a black 

 liquid which gradually acquires a red colour. The operation should be performed 

 out of doors, or in a chemical laboratory, as during the process of solution voluminous 

 nitrous vapours are given off, which would be hurtful to lenses and delicate instru- 

 ments. 



