The effect of keeping Amoeba proteus in dilute basic dye solutions was studied. It was found that Nile blue, neutral red, and neutral violet in particular, and also brilliant cresyl blue, methylene blue, Bismarck brown, thionin, toluidine blue, and azures A and B act as vital dyes, while at comparable molarities crystal violet, dahlia, safranin, methyl green, Janus green, and Victoria blue are lethal, and do not produce any staining until after death. Azure C, basic fuchsin, and particularly pyronine G are relatively harmless, but produce no vital staining.
All the vital dyes stain the food vacuoles, and all produce small, darkly stained granules in colourless vacuoles in the cytoplasm. The latter do not exist in the unstained amoeba. Some of the dyes colour vacuoles around the crystals. These crystal vacuoles also seem to be induced. A few of the dyes colour the spherical refractive bodies, which are at least in part phospholipid.
All the basic dyes used with the possible exception of azure C, methyl green, and pyronine G attach to the external membrane of A. proteus in an orientated manner, as shown by the increase in birefringence of the external membrane induced by these dyes. It is particularly those dyes that act as vital dyes that produce a very pronounced increase in the birefringence of the external membrane.