CONTROL OF CULTURES 455 



the majority of ciliates in 3 minutes. It was found that the most satisfactory 

 speed for this purpose was 2000 revolutions per minute. As soon as the 

 centrifuge stopped the supernatant fluid (9 ml.) was immediately withdrawn 

 with a sterile 10 ml. pipette and the tube was allowed to stand for about two 

 minutes in order that the ciliates might swim to the top of the remaining 

 milliliter of water. With a sterile 1 ml. pipette, 0.5 ml. of ciliate suspension 

 was withdrawn and placed in an empty sterile centrifuge tube. This suspen- 

 sion was again diluted, and the process repeated until the ciliates had gone 

 through an average of fifteen such transfers with the accompanying dilutions 

 (a dilution factor of approximately 10^*). This method entails a great loss 

 of ciliates but was found necessary inasmuch as, without removal to fresh 

 tubes with the consequent discarding of the residue, contaminations were 

 invariable. It was demonstrated that the contaminations resulted from the 

 fact that ciliates died or became immobilized during centrifugation and were 

 carried passively to the bottom of the tube with their adhering bacteria. How- 

 ever, by discarding the dead forms we were able to completely sterilize several 

 hundred ciliates at each attempt and these gave us the necessary organisms 

 with which to work [Kidder and Stuart, 1939, p. 332}. 



The washing fluid which was used in this case was sterile Pyrex- 

 distilled water. All pipettes were paper-wrapped and autoclaved. The 

 centrifuge tubes were closed with large cotton plugs and autoclaved. 

 During centrifugation the cotton was folded over and fastened with a 

 rubber band, to prevent the plug from being drawn into the tube. 



B. Migration. — Probably the first report of a technique for obtaining 

 Proto2oa free from bacteria by the utilization of migration was that of 

 Ogata (1893). He reports that he was able to recover as many as fifty- 

 two sterile phytomonads {Polytoma uvella) within five to thirty minutes 

 after the start of the migration. The apparatus he employed was a capil- 

 lary tube 10 to 20 cm. in length, with a 0.3 to 0.5 mm. bore. This tube 

 was filled to within one to 2 cm. from the end with a sterile fluid, and 

 then inserted into a culture of the flagellates and allowed to fill com- 

 pletely. Care was taken to avoid aid bubbles between the layers. Both 

 ends of the capillary were sealed by heat and the whole allowed to stand 

 for from five to thirty minutes. Eventually some of the flagellates were 

 found to have migrated away from their associated bacteria, and when a 

 number had collected in the upper end of the tube, this end was broken 

 off and the flagellates inoculated into nutrient media. 



A refinement of the same technique is reported by Stone and Rey- 

 nolds (1939) for the sterilization of the parasitic flagellate Trichomonas 



