342 University of California Publications in Zoology [Vol. 18 



had been treated with a thin coating of albumen fixative. If the 

 animals were not numerous enough for this procedure they were 

 eentrifuged at a low speed to concentrate them. The upper part of 

 the liquid in the tube was poured off and the few remaining drops, con- 

 taining many of the animals, were put on the cover glasses as described 

 above. The cover glasses were allowed to stand until only a thin film 

 of the liquid remained, and then dropped film down on the killing 

 fluid. After a little practice the right degree of dryness can be ascer- 

 tained and the animals killed with little, if any, distortion. 



Several killing agents were employed, depending somewhat on the 

 stain which was to follow. If Mallory's connective-tissue stain was to 

 be used, Zenker's or a picro-mercuric solution made up as follows was 

 used : 



Mercuric bichloride 2 gms. 



Picric acid 1 gm. 



Alcohol 95% 110 cc. 



Ether 20 cc. 



Acetic acid 20 cc. 



Formol 40% 50 ec. 



The latter killing agent seemed to give the best results. If 

 haematoxylin or haematin was to be used, either Carnoy's, Schaudinn's, 

 or the picro-mercuric solution was used with varying results. All of 

 the killing agents except Carnoy's were used hot. The cover glasses 

 to which the animals were attached were allowed to remain in the 

 killing fluid for from three to five minutes, then dehydrated in the 

 usual manner. 



The same processes of staining were employed as were used by 

 Sharp (1913) for staining Diplodinium. The length of time of stain- 

 ing with the modification of Mallory's connective-tissue stain was 

 altered somewhat, for it was found necessary to stain longer in the 

 acid fuchsin and less time in the anilin blue-oxalic acid-orange G solu- 

 tion. This stain was found suitable for both sections and in toto 

 preparations. Sometimes instead of the anilin blue-orange G-oxalic 

 acid mixture, a weak solution of Licht griin in 95 per cent alcohol was 

 used with fairly good results. If this method is used a very few 

 seconds should be allow^ed for the Licht griin, as it takes out the red 

 very rapidly. Balsam was always used as a mounting medium. Some 

 slides stained with the above dyes have been kept for three to four 

 months with very little fading, but experience has proved that the 

 stain is not permanent. 



