100 Iron-haematein Stain 
referred to the original publications, more especially to the first of the 
two referred to. 
Fixation. Tissues must be fixed in sublimate-alcohol, as recom¬ 
mended above. No other method gives, as far as I have tried, useful 
results, and it must be especially mentioned, that the addition of acetic 
acid to the sublimate-alcohol detracts from the value of the stain. 
When films are used, a preliminary fixation may take place in osmic 
acid vapours, as recommended by Minchin (1909). The following 
procedure is the same as that described for the iron-haematein method. 
It is important not to use glycerin albumen for fixing the sections on 
the slides. 
Staining. After taking the sections through the different alcohols 
I have found it preferable, instead of passing them first to distilled 
water, to leave them in tap water until transferred to the staining 
solution. Sometimes it is not necessary to use the somewhat complicated 
process of dissolving any traces of sublimate that may remain, which 
Giemsa recommends. If very thin fragments of tissue are fixed the 
sublimate is completely taken away by the iodine-alcohol which is 
employed during the hardening procedures ; but if only few sections 
are at one’s disposal it may be the wiser course not to omit this 
precaution. A prolonged staining gives the best results. I generally 
use a dilution of 1 to 20 {i.e., 1 ch'op of Giemsa’s solution to each c.c. of 
water) for 1 hour, and then change to a dilution of 1 to 40 (1 drop to 
each 2 c.c.’s), in which the sections are left for about 20 hours, but 
sometimes I have left them for 2 or 3 days in the weaker solution and 
still obtained very good results. The water employed for the dilution 
must be distilled and have been exactly neutralized, or slightly alkalinized, 
after titration, with a haematoxylin solution as indicator, as Giemsa 
describes it. (The water must turn a faint blue in the course of from 
one to five minutes after the addition of one to two drops of an alcoholic 
haematoxylin solution to ten c.c.’s, or potassium carbonate solution must 
be added till that point is reached.) The water, which I have been 
using, has generally needed the addition to each ten c.c.’s of about two 
drops of a cold saturated potassium carbonate solution, but the actual 
proportion undoubtedly varies very much, so that a frequent titration 
is inevitable. An omission of this step in the technique invariably 
spoils the results. The slides are preferably stained with the sections 
downwards, using for that purpose the glass dishes mentioned above. 
It is also important, that both the stronger and the weaker staining 
solutions should be prepared immediately before being used; when left 
