452 CONTROL OF CULTURES 



by this method is that of Hargitt and Fray (1917), using Paramecium 

 aurelia and P. caudal urn. They experimented with a number of modifi- 

 cations of the washing technique and followed their results by plating 

 on nutrient agar. Their first procedure was dilution by centrifugation, 

 wherein they centrifuged down the paramecia and then quickly with- 

 drew the supernatant fluid with a sterile pipette. The paramecia were 

 then covered again with sterile fluid, and the process repeated five 

 times. At the end of the fifth wash they found that the number of bac- 

 teria "per drop" had decreased from 500 colonies (per plate) in the 

 first wash to 3 colonies in the fifth. These results were not satisfactory, 

 however, and the method was abandoned. The authors offer the follow- 

 ing objections to the method: 



a great deal of time was consumed, the wash waters had to be drawn off^ 

 immediately after the centrifuge stopped or the paramecia rose in a body and 

 prevented the removal of the wash water. . . . Another serious drawback 

 to the centrifuge method is the difficulty of keeping the wash waters free 

 from contamination by bacteria from the air. The air may contain such 

 enormous numbers of bacteria that instruments and media which are sterile 

 to start with will be contaminated unless precautions are taken to prevent 

 the contact of air bacteria. A sterile pipette laid down on the table is no 

 longer sterile, a wash water left unprotected is soon contaminated by air 

 bacteria [p. 435}. 



It will become obvious from later discussions of these points that success 

 of the centrifuge method in the hands of Hargitt and Fray was pre- 

 vented by two principal faults in their manipulations — too few washes, 

 and failure to keep their centrifuge and wash tubes plugged at all times. 



These authors next attempted to reduce the chances of outside con- 

 tamination by the transfer method, using watch crystals enclosed within 

 Petri dishes, and transferring single ciliates through five separate dishes. 

 They again failed to effect sterility, but succeeded in reducing the num- 

 ber of colonies "per drop" from 2,500 in the first wash to one colony 

 in the fifth wash. They blame their lack of success with this procedure 

 upon the fact that the amount of wash fluid was so large that consider- 

 able time was required to locate the ciliate between transfers. 



Successful sterilization was accomplished by transferring individual 

 paramecia through five successive washes of sterile tap water in sterile 

 depression slides. The transfer pipettes were sterilized by dry heat be- 

 fore using, as were the Petri-dish-contained depression slides. The tap 



