434 GEO. T. HARGITT AND WALTER W. FRAY 



mecium were started on depression slides in this sterilized me- 

 dium, and within three days all were dead. Others were started 

 and similar results were obtained. After various tests had been 

 applied to account for this action, it became clear that media 

 such as beef extract and hay infusion were apparently so modified 

 or changed in their composition by the high temperature of the 

 autoclave as to render them unfit for the existence of Paramecium. 

 The bacteria could live in such media but Paramecium invari- 

 ably died in a short time. Another lot of the 0.1 per cent hay 

 infusion was made, placed in tubes and sterilized by heating in 

 the Arnold Steam Sterilizer on three successive days. In this 

 the temperature never gets above 100°C. In hay infusion steri- 

 lized in this manner Paramecium lived satisfactorily and no 

 trouble was experienced after this. 



In order to grow Paramecium in pure cultures of bacteria it 

 was necessary to devise a technic, and to organize a plan of 

 checks and controls over bacterial contamination, chiefly from the 

 air. It was in this preparatory work that the greatest difficulty 

 was experienced. As it worked out finally the precautions are 

 not difficult, but they are efficient. 



One of the first requirements was to get Paramecium free 

 from all bacteria of any sort. If this could not be done it would 

 obviously be of no avail to make use of pure cultures of bac- 

 teria, since they could never be pure after the Paramecium was 

 introduced. Any method which is practical must be expedi- 

 tious, must rid Paramecium of all bacteria, and must leave the 

 animal in the same active, vigorous condition as at the first. 

 Chemicals and heat can not be used, for Paramecium is more 

 sensitive to these than are bacteria in general. The only method, 

 therefore, must be one of washing in sterile liquids till all the con- 

 taminating organisms are removed. Many methods were tried 

 and abandoned as ineffective or impractical. One of the most 

 successful of early attempts was with the centrifuge. A pipette- 

 ful of Paramecium was placed in a sterile centrifuge tube and 

 considerable sterile hay infusion added to dilute the bacteria. 

 The paramecia were thrown to the bottom of the tube and as 

 soon as the tubes came to rest the wash fluid was removed and 



