234 CHAPTER XVII. 



327. APATHY'S Methods. As a good example of this kind 

 of work, I subjoin a short account of the procedure recom- 

 mended by Apathy (Zeit. f. wins. Mik., ix, 1, 1892, p. 15 ; 

 see also his Mikrotechnilc, p. 172) for Hirudinea. A portion 

 of the ventral cord is exposed, and if it be considered desir- 

 able dissected out, but the sinus and pigmented connective 

 tissue around it had better not be removed till the staining* 

 and fixation are completed. If, however, it be desired to 

 stain as many ganglion ceils as possible, as well as fibres, the 

 lateral nerves, as well as the connectives, should be cut 

 through near a ganglion. The preparation is then treated 

 with the stain. This is, for the demonstration chiefly of 

 fibres in Htrudo and Puntobdeila, either a 1 : 1000 solution 

 in 0'5 to 0*75 per cent, salt solution, allowed to act for ten 

 minutes ; or a 1 : 10,000 solution allowed to act for an hour 

 to an hour and a half ; or a 1 : 100,000 solution allowed to 

 act for three hours (Liunbric-uts requires twice these times ; 

 Antaeus and Unio require three times ; medullated nerves of 

 Vertebrates four times) . For the demonstration of ganglion 

 cells the stain is allowed to act three or four times as long. 



The staining having been accomplished, the preparations 

 from the 1 : 1000 solution are washed in salt solution for an 

 hour ; those from the 1 : 10,000 solution for a quarter of an 

 hour; those from the 1 : 100,000 solution need not be 

 washed at all. They are then treated with one of the am- 

 moniacal fixing and differentiating liquids described below in 

 the next section. This is done by pouring the liquid over 

 them, and leaving them in it without 'moving them about in it 

 for at least an hour, and by preference in the dark. The 

 further treatment is as described in the next section. 



The object of the ammonia in these liquids is to differentiate 

 the stain to produce an artificial " secondary differentiation." 

 It acts by washing out the absorbed colour from certain 

 elements, others resisting its action longer, much as HC1 

 alcohol washes out a borax- carmine stain. In this case the 

 elements that are Avashed out are the protoplasmic parts of 

 nerve-fibres, and their " interfibrillar " and " perifibrillar " 

 substance, the "primitive fibrils" still retaining the colour 

 strongly. It is of theoretical interest to remark that accord- 

 ing to APATHY the coloration thus obtained is a true xtain of 

 the " primitive fibrils/' not an impregnation. The " primitive 



