22O B. R. WEIMER. 



ing as previously described and permit experimentation under 

 more normal, controlled conditions. 



Of the various stains considered, nile blue sulfate seemed the 

 most suitable for the work on Hydra, particularly on Pelmatohydra 

 which is brown in color. The use of nile blue sulfate as a vital 

 stain has been confined for the most part to embryological 

 investigations and applied here for marking the eggs or the 

 embryos. Goodale ('17) used nile blue sulfate in marking the 

 eggs of Spelerpes bilineatus, the dye being applied locally in 

 solid form in the least possible amounts by means of a needle. 

 The dye particle was left for a few moments and then removed 

 by washing. A too prolonged application or the application of 

 the dye in excess amount was found to be toxic. Detweiler 

 ('17) used the dye in aqueous solutions of I : 100,000 up to 

 I : 500,000 but concluded that the ratio of I : 150,000 was 

 optimum. In this case the entire animal was stained. Smith 

 ('14) applied an aqueous solution by means of a fine pipette and 

 was able to get some blue spots which persisted. Vogt ('25) 

 uses nile blue sulfate in vital staining by first staining finely 

 divided agar in aqueous solution of the dye (cone. I : 100 to 

 i : 1,000) and after a few days applying in water one of the 

 colored pieces of agar to the tissue to be stained. The color 

 diffuses over after an interval of several hours to one day. 

 However, the stain is not well localized. To localize the dye 

 by this method, the stained agar must be inclosed in glass, 

 tinfoil or paraffin. 



After various trials, the method which was found most suc- 

 cessful was that of Goodale. The animal, Pelmatohydra oligactis 

 (Schulze) Pallas (Hydra fusca L.), was placed on a glass slide 

 and the excess water removed from around the animal by means 

 of filter paper. The animal was then transferred on the slide 

 to a dissecting microscope and the smallest possible particle of 

 nile blue sulfate applied in the desired region by means of a 

 needle. Almost immediately the animal was washed from the 

 slide into a stender dish containing water and thoroughly washed 

 in currents of water by means of a pipette. A blue spot was 

 found on the animal at the place of application of the dye. The 

 animal was then sectioned in any way desired and in the following 



