366 University of California Publications in Zoology [Vol. 20 



staining. Some twenty-five of the more common anilin stains in 

 1 per cent normal salt solution were also used on these cysts, which 

 included those of Endanweha dysenteriae, large and small races; 

 Endamoeha nana, small race; lodamoeba btietschlii; Chilomastix 

 davainei, and Giardia ent erica, in addition to Council mania and Enda- 

 mocba coli. In no cyst, and with no stain, was there any staining 

 of live cysts except with the Congo red on Councilmania and Enda- 

 moeba coli and, as already stated, this stain acted differentially on 

 the whole between these two. 



There were a number of variables to be considered. First of all 

 there was the matter of determining whether the cysts were alive or 

 dead, as the dead cysts stained intensively with any of the dyes used. 

 It was soon found that in Congo red dead cysts stained a deep orange 

 red with nuclei showing very dark ; it also became evident that in 

 live cysts the dye did not penetrate the cyst wall, therefore did not 

 stain either the cytoplasm or the nuclei. 



This fact was therefore established early, that in live cysts the 

 dye did not stain the cytoplasm or nuclei of the cyst, but only the 

 cyst wall. It was also found that there were degrees of intensity of 

 the staining of the cyst wall, and for this reason it was decided to 

 designate three degrees of staining as follows : the intense shade 

 as "stained," the light shade "pinkish," and where no staining was 

 apparent as "unstained." It was found in many of the cysts which 

 were ordinarily regarded as " un,stained, " that in a powerful light a 

 faint pinkish shade might be seen. This might have been due, how- 

 ever, to the film of staining fluid around the cj'st. 



The other variables were the amounts of material used and the 

 dye used. Obviously both of these could not be kept absolutely con- 

 stant, but continuous practice reduced to a minimum error due to 

 these factors. 



After a few trials it was found that if a dye was going to act at 

 all on a cyst it acted immediately, and that the cyst did not stain 

 more intensively if exposed for a long time, even as long as two days 

 in the dye. So there was no error due to the time element. Variations 

 in room temperature did not make any difference in the results. 



It is obvious that in a study like this the personal element enters 

 in a large way, since what would be considered stained by one might 

 be considered pinkish bj'' another, and even with the same person, 

 there would be variations of judgment as to the intensity of the color, 

 especially with different densities of the surrounding field of material 



