CONTROL OF CULTURES 461 



dilution part of the technique, and by this part of the procedure alone 

 he was able to obtain many sterile flagellates. Higher percentages of 

 sterile flagellates resulted, however, when he added a final migration to 

 the above washing. After washing, a drop of the packed flagellates was 

 placed in the center of a large Petri dish filled with sterile fluid. The 

 Protozoa migrated in all directions, and after various intervals of time 

 loops of medium, taken two to three inches from the center, were found 

 to contain ten to fifteen trichomonads. These loops were inoculated di- 

 rectly into tubes of media and the majority proved to be sterile. 



Hetherington (1934) described a much simpler technique, wherein 

 he alternated the dilution method with migration. He employed Colum- 

 bia culture dishes in Petri dishes, micropipettes for the transfer of the 

 organisms, and 10 ml. serological pipettes for filling the dishes. The 

 procedure was as follows: A drop of concentrated protozoan suspension 

 was placed in the left margin of the first dish, care being taken not to 

 disturb the one ml. of sterile fluid. By observing the activity of the Proto- 

 zoa, it was seen that numbers of them migrated to the right edge of the 

 fluid. Fifteen to twenty-five Protozoa were picked up in sterile micro- 

 pipettes and transferred to the left side of a second dish. These were 

 allowed to migrate. Those Protozoa which migrated were then placed in 

 a third dish and allowed to remain there for three hours. They were 

 transferred to the left side of the fourth dish and allowed to migrate, 

 then transferred to the fifth dish for a second three-hour period. The 

 sixth and seventh dishes were again used for migration, the Protozoa 

 from the seventh being placed in culture medium. 



By exercising care in the handling of the fluids, the pipettes, and the 

 covers of the Petri dishes, this method gave excellent results. Spores were 

 defecated during the two three-hour periods. Especially adherent bacteria 

 were lost during the migrations and washing, according to Hetherington, 

 owing to the fact that the medium was nutritive (Bacto yeast extract in 

 Peter's medium), resulting in the heightened activity of the bacteria. 



Recently Claff (1940) has described an apparatus designed to sterilize 

 negatively geotropic Protozoa, which employs both the principles of 

 dilution and migration and at the same time reduces the chances of air 

 contamination. This apparatus consists of six flasks in series (Fig. 126). 

 The Protozoa are injected into the bottom of flask 1 through a rubber 

 vaccine cap with a hypodermic needle. From this point on, until they are 



