ii6 RESEARCH IN PROTOZOOLOGY 



millimeters fused, round, platinum-iridium, which delivered ap- 

 proximately the same amount of fluid each time) of the Tri- 

 trichomonas after washing. The number of bacteria present in 

 each of twenty similar loops was determined by plating on solid 

 media. 



"The ratio of bacteria to TritricJwmonas was so small now that 

 it was an easy matter to inoculate tubes containing from ten to 

 fifteen trichomonads each and no bacteria. This, of course, was 

 done by diluting the trichomonads in sterile serum-saline medium. 

 After the completion of most of the washing experiments, Tri- 

 trichomonas was so abundant that when a two millimeter loop of 

 the organisms was placed in lOO cubic centimeters of serum- 

 saline medium, a two millimeter loop of this dilution would con- 

 tain fifteen to twenty trichomonads. This was determined by 

 counting twenty loops. Inoculations were made then from this 

 dilution of Tritrichomonas to tubes of various sterile media. 

 It was usually found most convenient to make the inoculations 

 when the fluid was in a large Petri plate. From seventy to ninety 

 per cent of the tubes inoculated in this manner showed no growth 

 of bacteria. It was later found that even a higher percentage of 

 bacteria-free tubes could be obtained by securely fastening the 

 bottom of a large Petri plate to a steady table, covering the sur- 

 face of the plate with sterile serum-saline medium, and then 

 placing a loop of the washed trichomonads in the centre of the 

 plate, care being taken to stir or agitate the fluid as little as pos- 

 sible. After various intervals, loops from different distances from 

 the centre were picked up and examined microscopically for tri- 

 chomonads. The trichomonads would swim away from the centre 

 faster than the bacteria. When ten to fifteen trichomonads were 

 found to be present in a loop taken at a certain distance from the 

 centre (usually two to three inches), tubes of sterile serum- 

 saline and other media were inoculated from this place. It was 

 also possible by this method to obtain bacteria-free trichomonads 

 with less washing than when a loop of the washed trichomonads 

 was diluted and thoroughly shaken in serum-saline medium. But 

 the distribution of the trichomonads in the dilution that was not 

 shaken was, of course, not as uniform, and it was impossible to be 

 certain as to the number picked up with a loop. It should also be 

 stated that in the earlier washing experiments, the percentage of 

 bacteria-free tubes was much lower. It was only after the wash- 



