1896.] Microscopy. 857 
MICROSCOPY. 
Methylen Blue.—A few points observed in the use of Erlich’s 
methylen blue method by the investigators in the Marine Biological 
Laboratory at Woods Holl, Mass. may be of general interest. 
This method has been successfully applied during the past summer 
to the study of the nervous system in a great variety of forms includ- 
ing vertebrates, crustacea, annelids, echinoderms and tunicates. 
Ehrlich’s intra vitam methylen blue prepared by Griibler was 
used for staining the nerve tissues. The stain was applied by injecting 
a 1-4} per cent solution of the methylen blue made in normal salt solu- 
tion, into the blood vessels, body cavity or lymph spaces or by immer- 
sing small animals or excised pieces of nerve tissue in a weak solution. 
The method of application and strength of the solution were deter- 
mined by experiment for each animal and tissue. During the action 
of the stain, the animal or tissue was kept as nearly as possible in its ` 
normal condition. Everything seems to depend on keeping the tissue 
alive, and in bringing the stain in contact with it in a solution of a 
strength suitable for obtaining the best results. 
The abundant supply of oxygen to the staining tissue was of great 
importance in some cases, while in others it seemed to make little 
difference. 
It was found, as suggested by Dr. C. Huber, that animals which live 
in the dark, stain better in the dark than in the light. 
The relaxation of the tissues by the use of chloroform or chloral 
hydrate seemed to be more favorable for the staining of some elements 
of the nervous system, while others did not stain which stained in the 
unchloroformed animal. 
It was found that recently caught and perfectly normal animals 
stained more satisfactorily than those which had been kept in confine- 
ment for some time, unless under very favorable conditions. 
In the case of the dogfish, active animals were killed by decapitation. 
The stain was applied by injecting a 1—} per cent solution of the me- 
thylen blue into the blood vessels for the central nervous system and 
by immersing small pieces of nerve tissue in a weak solution of the 
stain for the sense organs. 
The length of time required for the intra vitam staining varied 
widely, annelids requiring 4-5 hours, while dogfish only require 1-3 
hours, either by injection or by immersing the tissue in the stain. 
1Edited by C. O. Whitman, University of Chicago. 
