METHYLEN BLUE 193 



and in order to prevent this washing-out being excessively rapid, 

 it is desirable to have it go on in presence of as little liquid as 

 possible. 



380. The Modes of Staining. The practice of the earlier workers 

 at this subject was (following Ehrlich) to iy^ject niethylen blue 

 into the vascular system or body-cavity of a living animal, wait 

 a sufficient time, then remove the organ for further preparation 

 and study. And there appears to have been a belief with some 

 workers that it was essential that the stain should have been 

 brought about by injection of the colouring matter into the entire 

 animal. It is now known that the reaction can often be equally 

 well obtained by removing an organ and subjecting it to a hath 

 of the colouring matter in the usual way. But in some cases it 

 seems that injection is preferable, if not necessary. 



381. The Solutions Employed. The solutions used /or injection 

 generally made in salt solution (physiological, or a little weaker) ; 

 those for staining by immersion, either in salt solution or other 

 " indifferent " liquid, or in pure water. The earlier workers 

 generally took concentrated solutions. Thus Arnstein (Anat. 

 Anz., 1887, ]). 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. Wein. Math. Nat. CI., 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 cray-fishes and left the animals for 

 from two to four hours before killing them. S. Mayer {Zeit. 

 wiss. Mik., vi, 1889, p. 423) took a strength of 1 : 300 or 400 of 

 0-5 per cent, salt solution. The solutions of Retzius 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 super- 

 iluous, but positively disadvantageous, to take solutions stronger 

 than 1 : 1000. Dogiel {Enzyk. Mik. Technik., 1st ed., p. 815) 

 recommends | 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 injecting 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 

 ("8 to xV per cent.) methylen blue, or simply spread out on a 

 slide, and the whole placed in a Petri dish with a layer of the 

 niethylen 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) speci- 

 mens may be removed for examination or preservation ; or, 

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

 twenty minutes after injection, placed on a slide, and nioistened 



VADE-MECl'-M. 7 



