386 Major W. B. Fry and Captain H. S. Eanken. [Dec. 23, 



VI. Fixed and Stained Specimens. 



The foregoing sections have dealt with living trypanosomes, but we were 

 not able by the ordinary methods to make permanent preparations showing 

 the various stages and forms, and demonstrating the staining reactions of the 

 granule from its origin as a nuclear bud onwards. 



Mr. H. G. Plimmer, F.R.S., has appended a note describing special fixing 

 and staining methods devised by him, and we wish to state that it is only 

 by the use of these methods that we have been able to confirm the 

 appearances we have described in unfixed wet preparations, together with 

 the differentiation between vital and nutritive granules. 



In regard to the granule within the trypanosome, films have been stained 

 showing the granule taking origin from the macro-nucleus itself as a small 

 bud with characteristic chromatin reaction. All the stages of separation 

 have been seen till the granule is a small, independent, dense, chromatin- 

 staining mass in the cytoplasm (Plate 10, figs. 1-6). The granules, as stated, 

 vary in number, and are most frequently seen between the macro- and 

 micron ucleus. They stain a deep red and show a remarkable contrast to the 

 food granules which have taken on the iodine reaction from the fixation, and 

 are visible as bluish-staining bodies or sometimes as a fused mass. This can 

 be better seen in certain bird trypanosomes on account of their large size. 



In a certain number — probably the larger number — of instances the 

 granule at some stage of its development is surrounded by a faint-staining 

 hyaline circular or ovoid area. It is probable that in such cases the granule 

 is really within a vacuole (Plate 10, fig. 8). Sometimes the granule appears 

 to be spherical, but in other cases, even when being budded off from the 

 nucleus, it already shows as an elongated, pear-shaped body; this is well 

 seen in Plate 10, figs. 2-4. 



Granules can be seen actually causing a protuberance on the periplast 

 and evidently on the point of being extruded. Others have been fixed when 

 half-way out, while free granules, which have just effected their escape, have 

 been seen lying close to the parent trypanosome. The early free granule 

 takes on the chromatin stain deeply, and is identical with the body observed 

 by the vital method. 



The observations as to phagocytosis have been confirmed and more 

 advanced forms, showing a macronucleus, micronucleus, and flagellum, have 

 also been seen within polynuclear cells, and rounded forms resembling 

 the Leish man-Donovan body have been demonstrated in large mono- 

 nuclear cells. 



All stages have been seen from the early free granule ; the protoplasm 



