212 
Macrostoma mesnili N. Sp. 
Fixing and Staining Methods. 
The fixing and staining of these organisms and indeed of all intes¬ 
tinal Protozoa is best done by the method first advocated by Schaudinn. 
By this method or slight modification thereof it is possible to obtain 
beautiful preparations of the intestinal Protozoa with all the details of 
their anatomy preserved. If one attempts to make preparations by 
spreading faeces on a slide, allowing it to dry and then staining by some 
modification of the Romanowsky method, the results are most unsatis¬ 
factory and indeed the Protozoa are so broken up and disturbed in the 
drying process that they can hardly be recognised. In order to prepare 
specimens by the more rational method a small quantity of the faeces is 
spread out on a cover glass by means of a platinum loop or needle and 
without allowing it to dry it is dropped film side down on to the 
surface of a fixing fluid consisting of two parts of saturated aqueous 
sublimate and one part of absolute alcohol slightly acidified with acetic 
acid. This fixing fluid acts more quickly and better if warmed to the 
temperature of the body. Fixing is complete in a few minutes. The 
films are then carefully washed in weak spirit and finally in weak spirit 
to which a few drops of iodine solution have been added in order to 
completely remove the sublimate. The films are then placed in a 4 °/ 0 
solution of iron alum for several hours. They are then rinsed quickly 
for 2 or 3 seconds in distilled water to remove the excess of iron alum 
and immersed in Heidenhain haematoxylin (Haematoxylin crystals 
1 gram dissolved in 10 c.c. absolute alcohol made up to 100 c.c. with 
distilled water. After ripening for a fortnight add 100 c.c. dis¬ 
tilled water). In this stain the films are allowed to remain about 4—6 
hours or longer. They are then quite black and after washing in distilled 
water are differentiated in a weaker solution (1 °/ 0 ) of iron alum. The 
progress of the differentiation must be watched by taking the films out 
of the solution from time to time, placing them on a slide with film side 
up and examining with the \ and ^ inch objectives. When the nuclear 
structure and other details are clearly seen the film must be removed 
from the iron alum solution and washed well in water. The films are 
taken up through grades of alcohol to absolute alcohol, cleared in xylol 
and mounted in Canada balsam. It is most essential that the films be 
not allowed to dry at any point in this process. Prepared by this method 
or some slight modifications of it nearly all the details of structure of 
the intestinal Protozoa are very clearly brought out. 
