THE DESTRUCTION OF BACTERIA 81 



standard (phenol) are first prepared and a series of accurate dilutions 

 made with distilled water using graduated pipettes. (To make 1 : 70 take 

 4 c.c. of stock and 10 c.c. distilled water; 1:80 = 4 c.c. of stock + 12 

 c.c. distilled water; 1:90 = 4 c.c. stock + 14 c.c. distilled water; 1:500 

 = 2 c.c. of stock -f- 48 c.c. of distilled water. Complete dilution tables 

 are given in their original article.) The series should include dilutions 

 strong enough to kill B. typhosus in two and a half minutes and weak 

 enough to fail to do so in fifteen minutes. If dilutions greater than 1- 

 500 are required, a second 1 per cent stock solution is prepared. They 

 adopted the following scale for their tests: Dilutions up to 1:70 should 

 vary from the next in the series by a difference of 5 (i.e., 5 parts of water). 



From 1 : 70 to 1 : 160 by a difference of 10 

 From 1 : 160 to 1 : 200 by a difference of 20 

 From 1 : 200 to 1 : 400 by a difference of 25 

 From 1 : 400 to 1 : 900 by a difference of 50 

 From 1 : 900 to 1 : 1800 by a difference of 100 

 From 1 : 1800 to 1 : 3200 by a difference of 200 



and so on if higher dilutions are necessary. 



Short wide test tubes 1 inch by 3 inches are used in making the test. 

 These are placed in a rack in a water bath at 20 C. Five c.c. of each 

 dilution are measured into a series of these tubes beginning with the 

 strongest specimen and rinsing the pipette once with each dilution 

 before the 5 c.c. are measured out. For inoculation, a 24-hour broth 

 culture of B. typhosus is prepared which has been transferred daily for 

 at least 3 days. Before use it is shaken and filtered through sterile 

 filter paper. The wide test tubes containing diluted disinfectant are 

 inoculated with /i c.c. of this culture with a graduated pipette. The 

 tip of the pipette is held against the side of the tube to insure accurate 

 measurement and the tube immediately shaken to mix the bacteria 

 thoroughly with the disinfectant. Test inoculations are made from 

 this mixture at proper intervals into tubes containing 10 c.c. of standard 

 extract broth of + 1.5 acidity, using loops 4 mm. in diameter. At least 

 four such loops should be at hand, supported on a rack or wooden block 

 so that a fan-tail Bunsen burner may be placed under each wire in turn. 

 Each one is sterilized after a plant is made and allowed to cool while the 

 other three are being used in order. 



The test is conducted as follows: A row of ten wide tubes containing 

 dilutions of the antiseptic is placed in the water bath at 20 C. and time 

 allowed for them to reach the temperature of the bath. They are then 

 inoculated in order at intervals of exactly 15 seconds. Fifteen seconds 



