192 BAKNETT COHEN 



to this procedure were made in the case of the double-distilled 

 water and the filtered tap water experiments. The double- 

 distUled water was freshly distilled directly into sterile paraffined 

 bottles, and the filtered tap water was filtered through a Berke- 

 feld (N) porcelain candle into sterile bottles. The bottles were 

 then placed in the constant temperature box at 0°, 10°, 20° 

 and 30°C., respectively. DupUcate bottles were maintained at 

 each temperature, and kept there at least 12 hours to assume their 

 required temperatures before the beginning of the experiment. 



Cultures of the organisms to be studied were grown on agar 

 slants at 37° for sixteen to eighteen hours. A platinum loopful 

 of the growth was carefully removed and shaken into 9 cc. of 

 sterile distilled water. Precaution was taken to remove only 

 the bacterial growth and to avoid taking up any of the medium. 

 A homogeneous suspension of the organisms was then obtained 

 by thorough shaking. The tube was allowed to stand for several 

 minutes to permit sedimentation of particles and the supernatant 

 fluid was used to inoculate the bottles. 



The bottles, innnediately after being inoculated, were thor- 

 oughly shaken to distribute the organisms evenly throughout 

 the volume, and samjiles were taken to determine the bacterial 

 content by remo\Tng 1 cc. of the suspension and plating it in 

 suitable decimal dilutions. Duplicate plates were made of each 

 dilution and, usually, there were 3 or 4 dilutions and sometimes 

 more, of a single sample. 



After a suitable period of incubation, usually 48 hours at 

 37°C., the bacterial colonies on the plates were counted. For 

 the organisms maintamed at 0°C. it was necessary to incubate 

 for a longer period to permit growth of colonies of adequate 

 size. The plates were counted and the results of dupUcate 

 plates averaged and noted as the bacterial count for the partic- 

 ular moment that the sample was taken. 



Bottles were removed from their respective temperature 

 surroundings only for minimal intervals, not more than a minute 

 or two, usually. Otherwise, they were maintained, protected 

 from light, at their respective temperatures. Successive sam- 

 ples to determine the bacterial content were taken at regular 



