100 BIOLOGY AND TECHNIQUE 



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 



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 begin- 

 ning 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 1 / 10 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 dis- 

 infectant. 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 con- 

 taining 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 after the last tube has been inoculated 

 a subculture is made from the first tube of the series (i.e., 2y 2 

 minutes after this first tube was inoculated) and from the other 

 tubes in order at 15-second intervals. Fifteen seconds after this 

 first series of subcultures is completed, a second series of subcultures 

 is begun which will give the result of a 5-minute exposure to the 

 antiseptic and the subinoculations continued at 15-second intervals 

 until all dilutions have been tested for fifteen minutes. If the 

 strength of the antiseptic is known approximately, subcultures of 

 the lower dilutions for the longer periods may be omitted. It is 

 convenient to have an assistant at hand to call time and to label 



