DISINFECTION STUDIES 189 



the numbers of survivors capable of forming colonies on nutrient 

 agar. 



Materials and apparatus 



Close attention was paid to obtaining unifomiity of conditions 

 throughout the work. Constant temperatures (10°, 20°, and 

 30°C.) were secured m an electrically heated and controlled, 

 well-stirred air thermostat, with fluctuations probably not 

 greater than 0.5°C. In the experiments conducted at 0°C. 

 the bottles were maintained at the temperature of melting ice. 



The plating 7natcrials were of the usual kind, consisting of 

 graduated pipettes, petri dishes, dilution bottles and tubes, 

 and nutrient agar. 



The glassware was cleaned in the usual manner and sterilized 

 by dry heat at 160°C. for five hours or longer. The dilution 

 bottles were of about 250 cc. capacity, and were filled from an 

 automatic burette with 100 cc. of distilled water. The dilution 

 tubes were filled with 10 cc. of water. Sterilization in the auto- 

 clave brought the contents down on the average to 99 cc. and 

 9 cc. respectively. 



The procedure in making distilled water the suspension-fluid 

 or the diluting fluid is at variance with the customary practice 

 of using so-called physiological salt solution or mixtures of this 

 solution with nutrient broth. This variation was made advisedly 

 because preliminary tests had shown quite conclusively that 

 for our purposes distilled water was a satisfactory neutral fluid' 

 (cf. Zeug, 1920). 



The nutrient agar for plating was made by a uniform method 

 at various times from a single lot of Difco proteose peptone and 

 Liebig's beef extract. It contained per litre : 



grams 



Peptone 10 



Liebig's beef extract 3 



Shred agar 20 



The reaction of the medium was adjusted colorimetrically to 

 pH 7.0. Occasional tests of the reaction of the nutrient agar 

 at the time it was actually used for plating showed the hydrogen 

 ion concentration to be uniformly between pH 6.7 and 6.9. 



