474 NEUROFIBRILS 



brought into view by means of the methods described in the 

 following paragraphs. These can be used with some slight 

 variations, also for demonstrating neurofibrils in the axis cylinders 

 of nerve-fibres as well as in nerve-endings. 



1008. Apathy's Methods. The gold method (" Nachvergoldimg ") 

 has been given in § 410. The stain is very sharp, but good results 

 are obtained only in certain invertebrates, and even in these with 

 considerable difficulty. 



The hcemateine method {Mitth. Zool. Stat. Neapel, xii, 1897, p. 712) 

 has the same advantages and disadvantages, and has been little used 

 since the discovery of the Cajal and Bielschowsky processes. Material 

 may be fixed with corrosive sublimate, Zenker's fluid, picro-sulphuric 

 acid, or any other mixture which is not inimical to staining with alum 

 haematoxyhn, and should be preserved in 90 per cent, alcohol. Pieces, 

 no more than ^ cm. thick, are stained for at least forty-eight hours in 

 hjemateine I. A. (§ 306), and then washed up to twenty-four hours in 

 absolutely pure distilled water, or preferably suspended therein. Before 

 the stain has become washed out of the neurofibrils entirely, it is fixed 

 by putting the pieces for three to five hours into spring water, after 

 which they are put back for not more than two hours into distilled 

 water, dehydrated as rapidly as possible by hanging them up in absolute 

 alcohol, and imbedded in paraffin or celloidin, after clearing with 

 chloroform, and carefully protecting them from light whilst in chloro- 

 form or celloidin. The sections are mounted either in a resin or in 

 neutral glycerin. 



1009. Bkthe's Molybdenum-Toluidine Blue Method (Ztschr. wiss. 

 Mikr., xvii, 1900, p. 13). Pieces of the central nervous system of 

 vertebrates are fixed for twenty-four hours in 3 to 7-5 per cent, nitric 

 acid, and then brought directly into 96 per cent, alcohol for a day or 

 longer. They are afterwards put for twelve to twenty -four hours in a 

 mixture of 1 part of ammonia (sp. gr. 0-95) with 3 of distilled water and 

 8 of 96 per cent, alcohol ; for six to twelve hours into pure alcohol ; 

 for twenty-four hours into a mixture of 1 part of concentrated hydro- 

 chloric acid, 3 of distilled water, and 8 to 12 of alcohol ; for ten to 

 twelve hours into pure alcohol ; for two to six hours into water. They 

 are now mordanted with 4 per cent, ammonium molybdate, washed 

 again, dehydrated and imbedded in paraffin. The s?ctions, 8 to 10 jj. 

 thick, are seriated on slides by means of egg albumen, but zcithout ivater, 

 then passed through xylol and alcohol and differentiated, viz., covered 

 with water poured on the sections so as to form over them a layer 

 1-5 to 2 mm. deep, and put into an incubator at 55° to 60° C. for ten 

 minites. They are then rinsed with water, covered with a 1 : 3000 

 solution of toluidine blue, stoved for another ten minutes, rinsed with 

 water, and lastly treated with 96 per cent, alcohol till no more colour 

 comes away. After dehydration with absolute alcohol they are mounted 

 in the usual way. 



The method is also applicable to invertebrates for which other fixing 

 agents besides nitric acid are admissible, and the impregnation with 

 ammonium molybdate may be carried out on the sections. 



LuGARo's Modification {Riv. Pat. Nerv. Ment., x, 1905, p. 265) : 



1. Fix in 1 per cent, nitric acid in pure acetone, forty-eight 

 hours. 



2. Acetone twelve to twenty-four hours, changing it three to 

 four times. 



