192 METHYLEN BLUE. 



(Quart. Journ. Micr. Sci., 1894, pp. 461, 483) takes for embryos 

 of the lobster a solution of 0-1 per cent, in 0-75 per cent, salt 

 solution, and dilutes it with 15 to 20 volumes of sea- water. 

 SEIDENMANN (Zeit. wiss. MiL, xvii, 1900, p. 239) takes for the 

 choroid a solution of 0-02 per cent, in 0-5 per cent, salt solution. 

 LAVDOWSKY (ibid., xii, 1895, p. 177) takes -^ to J per cent, in white 

 of egg, or serum. Similarly YOUNG (ibid., xv. 1898, p. 253). 

 MICHAILOW (ibid., xxvii, 1910, p. 10) takes ^ to -$ per cent, in 

 Ringer's salt solution (for nerves of Mammals). 



APATHY (Zeit. iviss. Mik., ix, 1892, p. 15 ; see also his Mikro- 

 tecJmik, p. 172) proceeds as follows for Hirudinea and other inverte- 

 brates. A portion of the ventral cord is exposed, or dissected out. 

 If 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 preparations from the 1 : 1000 solution are then 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 in 343. This is done 

 by pouring the liquid over them, and leaving them in it icithout 

 moving them about in it for at least an hour, and by preference in the 

 dark. The further treatment is as described in 343. 



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 longer. 



See also, for Hirudinea, SANCHEZ, in Trab. Lab. Invest. BioL 

 Univ. Madrid, vii, 1909, fasc. 1 4, or Zeit. wiss. Mik., xxvii, 1910, 

 p. 393 (injection of solutions of 0-2, 0-1, or 0-05 per cent., with 

 further treatment as Apathy or Bethe). 



343. Fixation of the Stain. The stain obtained by any of these 

 methods may be fixed, and more or less permanent preparations be 

 made by one or other of the following methods : 



