METHYLEN BLUE 195 



an hour and by preference in the dark. The further treatment 

 is described in § 382. 



The object of the annnonia in these hquids 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 Troft. Lab. Invest. Boli. 

 Univ. Madrid, vii, 1909. fasc. 1 — 4, or Zeit. rviss. 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). 



382. 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 : 



Arnstein {Anat. Anz., 1887, p. 551) puts the tissue for half 

 an hour into saturated aqueous solution of picrate of ammonia. 



S. Mayer {Zeit. iviss. Mik., vi, 1889, p. 422) preferred a mixture 

 of equal parts of glycerin and saturated picrate of ammonia 

 solution, which served to fix the colour and mount the prepara- 

 tions in. This was also in principle the method of Retzius 

 (Intern. Monatsschr. Anat. Phys., vii, 1890, p. 328). 



Dogiel [Enzyk. Mik. Techn., ii, p. 105) puts for from two to 

 twenty-four hours into saturated aqueous picrate of ammonia, 

 and then into equal parts of glycerin and the picrate solution. 

 (Thin membranes, and the like, may be fixed with 1 or 2 per cent, 

 of 2 per cent, osmic acid solution added to the picrate solution 

 and stained with picro-carmine before putting into the glycerin 

 mixture.) 



Other workers have employed saturated solution of iodine in 

 iodide of potassium (so Arnstein) or picro-carmine (so Feist, 

 Arch. Anat. Enticickel., 1890, p. 116 ; cf. Zeit. iviss. Mik., vii, 

 1890, p. 231), the latter having the advantage of preserving the 

 true blue of the stain if it be not allowed to act too long, and the 

 preparation be mounted in pure glycerin. 



Picric acid has been used by Lavdow^sky, but this after care- 

 ful study is rejected by Dogiel. 



Apathy {op. cit., § 381) brings preparations either into a con- 

 centrated aqueous solution of picrate of ammonia free from 

 picric acid, and containing 5 drops of concentrated ammonia for 

 every 100 c.c. ; or, which is generally preferable, into a 1 to 2 

 per cent, freshly prepared solution of neutral carbonate o/ammonia 

 saturated with picrate. They remain in either of these solutions, 

 preferably in the dark, for at least an hour. They are then brought 

 into a small quantity of saturated solution of picrate of ammonia 

 in 50 per cent, glycerin, where they remain until thoroughly 

 saturated. They are then removed into a saturated solution of 

 the picrate in a mixture of 2 parts 50 per cent, glycerin, 1 part 

 cold saturated sugar solution, and 1 part similarly prepared 



7—2 



