1914] Sharp: Diplodinium ecaudatum 55 



avoid the evaporation of the fluid under the cover glass, small dishes 

 of water are placed within the oven, a plan which serves to prolong 

 the period of observation to some degree. Another plan which I use 

 when long continued observations on the same animals are desired is 

 to arrange on the slide a glass cell filled with a drop or two of the 

 stomach fluid and from which a fine cotton thread leads to the fluid 

 under observation. The thread acts as a siphon and serves to keep an 

 ever fresh supply of fluid under the cover glass. 



In brief, it has been possible by means of this apparatus to control 

 the rapidity of the movements of these animals, through temperature 

 regulation, and to keep living Diplodinium under constant observation 

 for from eight to ten hours at a time. In fact the animals have been 

 kept alive for over forty-eight hours after removal from the stomach 

 of the ox and, within this limit, i.e., forty-eight hours, the length of the 

 period during which an individual animal may be kept under observa- 

 tion, barring accidents, depends entirely upon the endurance of the 

 observer. 



Fixation and Staining. — The following fixing fluids have been used 

 with good results ; Schaudinn 's alcoholic sublimate solution, Zenker 's, 

 Flemming's, Worcester's, and Bouin's fluids, formalin (4%), and os- 

 mic acid (1%). Of these Schaudinn 's, used hot, gave uniformly the 

 best results. When it was desired to follow with Mallory's connective 

 tissue stain Zenker's fluid was the best fixing agent. For the study of 

 surface markings it was found necessary to fix in warm four per cent 

 formalin and mount unstained in styrax. This method gives excellent 

 preparations. 



The preparation of fixed material is done on the "killing floor" at 

 the slaughter house. A table is arranged as near to the spot where 

 the cattle are killed as possible. On the table are two pans of hot water, 

 one maintained at 36 '^ C. in which a shallow glass dish is placed and 

 the other which is maintained at near the boiling point serves as a con- 

 tainer for the tubes of fixing fluid. The minute the ox is opened and 

 the stomach removed the assistant obtains the reticulum, turns it inside 

 out in order to get rid of the superfluous food particles, then quickly 

 wrings the fluid from the walls into the glass dish in the water at 36° C. 

 temperature. Instantly the operator dashes the boiling Schaudinn 's 

 fluid into this dish. In this manner the protozoans are obtained with 

 cilia extended and without contractions or contortions of the body. 

 After fixing for five to ten minutes the mixture is shaken up with twice 

 its volume of 50 per cent iodine alcohol. This is repeated until the 



