846 The American Naturalist. [September, 
MICROSCOPY .! 
Fixation of the Methylenblue Stain.—In the last number of 
the Zeitschrift fiir Wissenschaftliche Mikroskopie (Vol. VIIL., 1, 1891, 
p: 15), Prof. Dogiel offers some new points on the use of methylen- 
blue in staining nerves.” 
Nerves exposed to the direct action of methylenblue often stain so 
intensely that they appear dark blue, almost black. Such preparations 
should lie in the fixing medium (saturated aqueous solution of picrate 
of ammonium) twenty-four hours or more, otherwise the color fades 
quickly after transfer to glycerine. Long exposure to sunlight causes 
the stain to fade. 
The fixing medium often macerates, loosening the epidermis and 
rendering difficult the investigation of intra-epithelial nerves. The 
macerating effects may be checked by adding osmic acid (1-2 cc. of a I 
per cent. solution to 100 cc. of the fixing fluid). This mixture pre- 
serves the tissue, and at the same time blackens the medullary sheaths 
_of the nerve-fibers. As the axis cylinders stain dark violet, it becomes 
easy to distinguish medullated from non-medullated fibers. The prep- 
arations are mounted in dilute glycerine. 
If the tissues are to be prepared for sectioning, a stronger per cent. 
of osmic is added (1-2 cc. of a 1 per cent. solution to 25-30 cc. of 
picrate of ammonium). The preparation lies in this fluid twenty-four 
hours, and is then cut in liver or pith, or with the freezing microtome. 
Biedermann * recommends as a fixative in the case ‘of invertebrates 
the following mixture : 
Saturated o solution of picrate of ammonium ... .1 Phe 
Glycerin vol. 
Solution of salt, Xu per cent, (sea-water i in case of marine 2 forms) 2 svali 
The nervous system is dissected out for exposure to the staining 
fluid. That the exposure may be as direct as possible, the nerve-cord 
(Hirudo, Lumbricus, etc.) should be freed from its sheath. A` very 
dilute solution of methylenblue is used, and allowed to act from 
two to three hours. If the preparation, after the exposure, be place 
on a slide wet with the staining fluid, and left for one-half to 
one hour in a moist chamber, the color effects will be intensified. This 
t Edited by C. O. Whitman, Clark eas Worcester, Mass. 
2 Cf. this journal, Dec. 1890, p. 
3 Jenaische Zeitschrift fur Mediocre XXV., 3 and 4, 1891, P. 433- 

