BACTERIA IN CULTIVATION OF INTESTINAL PROTOZOA 115 



media that were tried — but the fact was not demonstrated clearly. 

 ... It was impossible to wash the organisms more than four to 

 five times in saline, because they would round up, become non- 

 motile, and die. However, after almost giving up the undertaking 

 entirely, I discovered that the trichomonads could be washed as 

 many times as desired provided sterile serum-saline medium was 

 used instead of saline. It then became necessary to make several 

 gallons of serum-saline medium and to sterilize it by filtration. Two 

 hundred liters of this medium were made, sterilized, and stored 

 away in the cold room. Then the endeavor to free Tritrichomonas 

 from bacteria really began. 



*Tn each attempt to free the trichomonads of bacteria the fol- 

 lowing procedure was carried out : the organism was grown in 

 liter flasks which were examined from time to time to note the 

 abundance of growth. As soon as the trichomonads reached their 

 maximum number in the culture, ten liters were run through 

 filter paper three or four times to remove as many as possible 

 of the particles of debris larger than the trichomonads. Then the 

 Tritrichomonas of the ten liters was concentrated by centrifugaliza- 

 tion. This gave ten to fifteen cubic centimeters of Tritrichomonas 

 having the appearance of heavy cream. Then sixteen sterile fifty 

 cubic centimeters pointed centrifuge tubes were filled with sterile 

 serum-saline medium near the freezing point of the medium and 

 the Tritrichomonas obtained from the ten liters was layered on 

 the surface of the fluid in the centrifuge tubes. The centrifuge 

 (International size 2) was run at step six of the rheostat for 

 fifteen minutes. One-half to one cubic centimeter of Tritrichomonas 

 was packed in the bottom of each tube after centrifugalization. 

 Practically all the fluid was removed from each tube with sterile 

 pipettes. Then the Tritrichomonas was removed from each of 

 the tubes and layered on the surface of the fluid in sixteen other 

 centrifuge tubes. The centrifuge was run as before. Sterile serum- 

 saline medium near the freezing point of the medium, sterile 

 pipettes, and sterile tubes were used in every step of the washing. 

 In the last three or four washings the tubes were plugged with 

 sterile cotton plugs which were held in place by a rubber band 

 around the outside of each tube. After this washing process 

 had been repeated twenty times, the ratio of Tritrichomonas to 

 bacteria was about fifty to one. This was determined by dilu- 

 tion counts of the number of trichomonads in twenty loops (two 



