336 University of California Publications in Zoology [Vol. 20 



Micro-injection 



The successful search for a neuromotor apparatus in Paramaecium 

 was the outgrowth of micro-injection studies (Rees, 1922). To 

 accomplish micro-injection, it was found necessary to isolate the 

 organisms in rounded drops, so that they would not be disturbed by 

 the forces of surface tension (Rees, 1922). This led to a search for 

 a method of making stained preparations of undistorted animals, 

 which was accomplished by fixing them in centrifuge tubes instead 

 of applying the fixative after they had been attached to the slide by 

 means of egg albumen. 



TECHNIQUE AND METHODS 

 Culture 



The animals for this work were obtained from wild stock that had 

 been brought in with debris from the creek bed and cultured in 

 covered battery jars. After being injected or otherwise operated 

 upon, they usually thrived if given plenty of food. The importance 

 of food was demonstrated only after the death of many organisms 

 within three or four hours after the operation. The practice had 

 been to place them in small drops of culture fluid from which other 

 indivaduals had been removed by coarse filter paper. Even the 

 controls did not survive twenty-four hours under such conditions. 



A careful search was first made for some toxic substance. It was 

 thought that volatile poisons were present in the vaseline used to seal 

 down the covers or that sufficient of the salts of mercury were formed 

 in the injecting and isolating instruments to prove fatal. After 

 weeks of investigation, a rich autoclaved and fully ripened hay 

 infusion was substituted for the filtered culture fluid. The result was 

 astonishing. The animal survived after the operation and grew 

 rapidly, often dividing twice within twenty-four hours. 



Technique 



Contrary to the statement of Neresheimer (1902), a fibrillar 

 system may be differentiated in Paramaecium with Mallory's triple 

 connective tissue stain, even when the animals are attached to the 

 slide coated with egg albumen. They were fixed in Zenker's fluid 



