METHYLEN BLUE. 83 



for injecting may be employed, but they should, if anything, 

 be still weaker. DOGIEL (Arch. f. mik. Anat., xxxv, 1890, 

 p. 305; Zeit. f. wiss. Mik., vii, 4, 1891, p. 509) places objects 

 in a few drops of aqueous or vitreous humour, to which are 

 added two or three drops of a -^ to -^ per cent, solution of 

 methylen blue in physiological salt solution, and exposes 

 them therein to the air. In thin pieces of tissue the stain 

 begins to take effect in five or ten minutes, and attains its 

 maximum in from fifteen to twenty minutes. For thicker 

 specimens retina, for instance several hours may be neces- 

 sary, the preparation being kept just moist by occasional 

 treatment with a drop or two of indifferent liquid or methylen- 

 blue solution, added by turns. 



118. 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. wiss. Mik., ix., 1, 1892, p. 15) 

 for Hirudinea. A portion of the ventral cord is exposed, 

 and if it be considered desirable, 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-cells 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 Hirudo and Pontobdella, 

 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 (Lumbricus requires 

 twice these times ; Astacus 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 connectives and lateral nerves 

 having been cut as directed above). 



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 ammoniacal 

 fixing and differentiating liquids described below in the 



