CHAPTER XVI. 191 



p. 125) injected 1 c.c. of saturated (i.e. about 4 per cent.) solution 

 into the vena cutanea magna of frogs, and removed the organ to be 

 investigated after the lapse of an hour. BIEDERMANN (Sitzb. Akad. 

 Wiss. Wien, Math. Nat. Cl, 1888, p. 8) injected 0-5 to 1 c.c. of a 

 nearly saturated solution in 0-6 per cent, salt solution into the 

 thorax of crayfishes, and left the animals for from two to four hours 

 before killing them. S. MAYER (Zeit. wiss. MiJc., vi, 1889, p. 423) 

 took a strength of 1 : 300 or 400 of 0-5 per cent, salt solution. The 

 solutions of EETZIUS are of the same strength. But the tendency 

 of more recent practice is decidedly towards the employment of 

 weaker solutions. APATHY (ibid., ix, 1892, pp. 25, 26 et seq.) finds 

 that it is not only superfluous, but positively disadvantageous, to 

 take solutions stronger than 1 : 1000. DOGIEL (Encycl. Mik. 

 Technik., 1st ed., p. 815) recommends J to J per cent., or at most 

 | per cent. For warm-blooded animals the solution should be 

 warmed to 36 or 37 C., and before sending in the injection the 

 blood-vessels should be well washed out with similarly warmed salt 

 solution. The injected organs may be removed after twenty to 

 thirty minutes. They should be placed on a thin layer of spun 

 glass moistened with weak (J to T ^ per cent.) methylen blue, or 

 simply spread out on a slide, and the whole placed in a Petri dish 

 with a layer of the methylen blue on the bottom. The dish is best 

 placed in a stove at 36 C., and after fifteen to thirty minutes (if the 

 pieces are thin) or one hour to one and a half hours (if they are thick) 

 specimens may be removed for examination or preservation ; or, 

 without using the stove, specimens may be removed ten to twenty 

 minutes after injection, placed on a slide, and moistened with weak 

 methylen blue or salt solution, and brought under the microscope. 

 Then as soon as the stain is sufficiently brought out (forty to sixty 

 minutes) they may be fixed ( 343). 



For staining by 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 Jg to -fg 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, solu- 

 tion in 0-6 per cent, salt solution (for muscles of Batrachia). ALLEN 



