VI Proceedings of the Association of American Anatomists 
A NOTE ON METHYLENE VIOLET AS ONE OF THE NUCLEAR DYES IN 
THE ROMANOWSKY STAIN. By Warp J. MAcNEAL. Department of 
Histology and Embryology, University of Michigan. 
Bernthsen in 1885 showed that decomposition of methylene blue by 
alkalies yields methylene violet, which he obtained pure, methylene azure 
which he was unable to prepare in pure condition, and the leukobases 
of these substances and of methylene blue. 
Michaelis and Giemsa have recognized the nuclear dye of the Roman- 
owsky stain as methylene azure and Giemsa has prepared a pure methylene 
azure by a secret method, and this is on the market. These observers re- 
gard methylene violet as of no value. 
Unna has compared the staining properties of Giemsa’s methylene azure 
with his own polychrome methylene blue solution, and finds the older 
preparation much more active and therefore concludes that azure is not 
the only staining principle of polychrome methylene blue. By adding 
methylene violet and alkali carbonates to azure solutions he approximated 
very closely polychrome methylene blue. 
My first experiment was an attempt to isolate Nocht’s “ roth aus methy- 
lene blau” by extraction with ether. By means of a mechanical ex- 
tractor 0.4 gram of this substance was obtained and identified by its 
chemical reactions as nearly pure methylene violet (Bernthsen). 
Methylene violet was next prepared by Bernthsen’s method and its re- 
actions compared with the ethereal extract of Nocht’s solution. 
The next step was to combine methylene violet into a suitable staining 
mixture. It is insoluble in water, but soluble in aqueous methylene blue 
solution. 
~ 
Methylene violets \ ies. fc rlep iar tes oped a apa 5) g 
Methylene blue (med. pure) ..........«-. 0:5) sa 
Sods scarbomates (CEYSt: )) oll. son ile eede ciate 0.25 g. 
Giivicerane | eater ey wer sre oie 0 Sci Mieka alates 50.00 ce. 
Distilled watermreysts eco ees eee 50.00 ce. ; 
The solid ingredients should be finely divided by trituration. CO, is 
given off for some hours after preparation of this solution. It is used 
by mixing a few drops with dilute aqueous eosin and floating fixed prep- 
aration on the mixture for 2 to 30 minutes. 
A solution of the dye in methylic alcohol is useful for rapid work. 
The following formula has been tried: 
Methivlettesviglegventinecns.tecren sehen est 0.12 g. 
Mlethivdennve "plana ® Si #8 Be aeer Ek Shee ee ee 0.12 g. 
LORI ts Reels ener Cae arte aemees Mad ener eae enege 0.12 g. 
is} 
Methyl: alcohol’ sGpure)s 23 terse neta 100.00 ce. 
