208 University of California Publications in Zoology [Vol. 19 



purely vegetative existence. By collecting water from the aquarium 

 and putting in petri dishes I was at times able to get a determinate 

 increase in numbers and low mortality. By killing at such times, 

 material normal as it was possible to get was obtained. Various 

 methods of introducing the material to the killing fluid was used: by 

 pipette, by pouring, or by pouring the killing fluid on concentrated 

 masses of the organisms. The proportion of the killing fluid was never 

 allowed to be less than ten times the amount of the water containing 

 the flagellates. 



The most satisfactory material was obtained by killing either in hot 

 Schaudinn's fluid or strong Plemming. and staining in Heidenhain's 

 aqueous iron haematoxylin. By this method the nuclear differentiation 

 becomes evident as figured in accompanying plates. Counterstaining 

 in eosin, either just after destaining or from 50 per cent alcohol, brings 

 out the flagella. 



Other killing fluids used in addition to Schaudinn's and strong 

 Flemming were: picro-mercuric, Gilson's, Flemming's weak, Carnoy's, 

 Zenker's, Bouin's, and osmic acid. None of these was perfectly satis- 

 factory, possibly through lack of proper proportions or some un- 

 detected flaw in technique. 



Heidenhain's aqueous iron alum haematoxylin I regard as by far 

 the safest, most differential and permanent stain. Alcoholic solutions 

 were about as satisfactory, but required from one to two hours for the 

 mordant and eight to twenty-four for the stain, thus not being much 

 quicker than the aqueous solutions. Acid fuchsin or eosin were 

 excellent counterstains with either of the above. Delafield's haema- 

 toxylin yielded beautiful but not so critical a differentiation. The 

 granular organization of the chromatin, even of the karyosome, was 

 emphasized. Phosphotungstie acid haematoxylin was of assistance in 

 determining the nuclear membrane, but did not show up the spindle 

 fibers as was hoped. Mallory's connective tissue stain as modified for 

 Protozoa yielded only fairly satisfactory results with the fixatives 

 tried. By its use the two basal granules embedded in the blepharoplast 

 could be distinguished. For some reason Collodictyon seems able to 

 withstand chemicals, especially the usually quick stains, far more than 

 other free living Protozoa with which I have dealt, the time required 

 for all stains being longer than that ordinarily scheduled. Flemming's 

 safranin, gentian-violet, orange G was tried, for the purpose of differ- 

 entiating the macrokaryosomes and microkaryosomes, but no variations 

 of color diagnostic of chemical differences were obtained. The 



