200 A. B. MACALLUM. 
cent. solutions of osmic acid were found serviceable, the latter 
reagent also having been used in the combination known as 
Flemming’s fluid. 
The corrosive sublimate solution was allowed to act on the 
preparations of tissue for about ten minutes, after which they 
were washed for a few minutes in distilled water, and then in 
50 per cent. alcohol. The hardening was completed with 
alcohol of 70 and 90 per cent. strengths in the usual way. 
When Flemming’s fluid was used the tissue was not allowed to 
lie in it for more than half an hour, while for the osmic acid 
solution not more than ten minutes were given, and the fixa- 
tion was carried on further with alcohol of 50, 70, and 95 per 
cent. strengths. Preparations, whether made with corrosive 
sublimate or with osmic acid solutions, retain, even after 
careful washing, traces of the metallic salt of the reagent used, 
and the black or dark reaction which they give with ammo- 
nium hydrogen sulphide, in consequence of the presence of 
such metals, interferes with the proper demonstration of the 
distribution of iron by that reagent. On this material the acid 
alcohols only were used, and the preparations were subsequently 
treated with the acid ferrocyanide mixture, the Prussian blue 
reaction obtained not having been in the least affected by the 
presence of minute quantities of the metallic salts of the har- 
dening reagents. The latter were free from iron salts, a fact 
of which I convinced myself by qualitative analyses. 
To the use of all hardening reagents other than alcohol there 
are objections. Those which contain an acid may assist in the 
diffusion of iron salts in the tissues, and cause the deposition 
of these in some other parts than those in which they originally 
were held. Further, the acids of some of the reagents (e. g. 
acetic acid in Flemming’s fluid) may liberate the organic iron, 
which cannot in such a case be distinguished from the iron of 
inorganic or albuminate combinations. For these reasons I 
have used acid hardening reagents but occasionally, and then 
the time allowed for their action was short, in order to reduce 
to a minimum the risk of liberating organic iron, and of the 
diffusion of iron salts through the tissues, Against corrosive 
