194 METHYLEN BLUE 



with weak methylen blue or salt solution, and brought under 

 the microscope. Then as soon as the stain is sufficiently brought 

 out (from forty to sixty minutes) they may be fixed. 



For staining hy immersion the solutions should, if anything, 

 be still weaker. Dogiel {Arch. mik. Anat., xxxv, 1890, p. 305) 

 places objects in a few drops of aqueous or vitreous humour, 

 to which are added 2 or 3 drops of a ^ to y^ per cent, solution 

 of methylen blue in physiological (0-75 per cent.) salt solution, 

 and exposes them therein to the air. In thin pieces of tissues 

 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 necessary. 

 The reaction is quickened by putting the preparations into a stove 

 kept at 30° to 35° C. Rouget {Compt. Rend.. 1893, p. 802) 

 employed a 0-05 per cent, solution in 0-6 per cent, salt solution 

 (for muscles of Batrachia). Allen {Quart. Jour. 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. zviss. Mik., 

 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 | per cent, in white of egg, or serum. Similarly 

 Young {ibid., xv, 1898, p. 253). Michailow {ibid., xxvii, 1910, 

 p. 10) takes | to 3^ per cent, in Ringer's salt solution (for nerves 

 of mammals). 



Apathy {Zeit. wiss. Mik., ix, 1892, p. 15 ; see also his Mikro- 

 tecknik, 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 tlu-ee 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 § 382. This is done by pouring the liquid over them, and 

 leaving them in it zvithout moving them about in it for at least 



