96 
Iron-haematem Stain 
bichloride and one of absolute alcohol. When this fixation is used, the 
pieces of tissue must be very thin, not more than 2-3 mm., but squares 
of about one cm. may be allowed. The tissues should be left in the 
fixative for 12 hours or more—two hours are sufficient for coverslip- 
films—but both may remain in it for over 24 hours without any 
bad effect. The fragments are then passed into 60 "/o alcohol, and 
subsequently into 70°/o alcohol containing a small quantity of iodine, 
sufficient to give it a pale yellow colour; thereafter to 90"/o alcohol 
and absolute alcohol, allowing one day in each of the four liquids. 
Clear thoroughly in xylol, pass to xylol paraffin then paraffin, and 
finally embed quickly in pure paraffin. The sections should be cut 
as thin as is compatible with the particular purpose in view. For 
trypanosomes it is generally not advisable to use sections of less 
than five yx in thickness. They should be fixed on clean slides after 
simply fioating them on warm distilled water (about 40° C.) and 
without using any fixative such as albumen glycerin. Films are 
treated in the same way, only they are not brought further forward 
tlian 90 "/o alcohol. I have also' stained formalin-fixed sections with 
fairly good results. Osmic vapour fixation of films has so far not pi'oved 
successful, but I have not had the opportunity of trying to bleach these 
preparations with hydrogen peroxide previously to the staining, which 
might well modify the results. On the other hand, the bleaching of 
sections of tissue fixed in Flemming’s liquid has not made it possible to 
obtain a good stain. 
The staining process. After dissolving the paraffin in xylol the 
sections are passed through alcohols of diminishing strength to tap 
water (or distilled water). This is important, as they do not stain well 
when taken directly from alcohol into the stain, in spite of the latter 
being an alcoholic solution. The staining liquid is prepared about 
1.5 minutes before being used, as it does not stain well immediately 
after mixing; it may be kept for about two hours, but will not stain 
after a longer period. It is prepared by thoroughly mixing three parts 
of solution A and two of B. The solution A is prepared by adding one 
gram of pure haematein (Grllbler) to 100 c.cs. of 96 "/o alcohol, and 
shaking repeatedly without heating. The whole quantity is not 
dissolved, but the solution is preserved with the residuum and, if not 
completely transparent, filtered before use. The solution B is com¬ 
posed as follows, after Weigert: perchloride of iron 4, hydrochloric 
acid 1, distilled water 100. The slides are left for about five minutes 
in the mixture, with the sections downwards, and for this pui’pose there 
