78 IOWA ACADEMY OF SCIENCE Vol. XXVI, 1919 



ten minutes. Nutrient bouillon is used, being made with Liebig's 

 beef extract and Witte's peptone in the usual manner and giving 

 a reaction of exactly 1.0. 



3. One-fourth minute after the "germicide" tube has had the 

 culture added, a subculture is made from each tube in the series 

 of dilutions by transplanting one loopful to a tube of 10 c. c. 

 sterile broth. (The loops used were of No. 23 U. S. standard 

 gage platinum wire, each loop being 4 mm. in diameter.) 



4. Immediately after the broth tube, inoculated with a loopful 

 of the culture treated with the germicide, has been made, 1 c. 

 c. of this broth culture is plated with 10 c. c. of blood agar. It is 

 mixed in a test tube before being poured into the Petri dish. 



5. At 1-2, 1, 2, 5, 10, 30 and 60 minute intervals cultures are 

 made in the same way as in Direction 3. 



6. The cultures should be incubated at 37° C. for twenty-four 

 to seventy-two hours. "Record" growths usually develop in 

 forty-eight hours. 



CONTROLS 



1. From 0.1 c. c. to 0.5 c. c. (as used in the foregoing) of the 

 same twenty-four-hour broth culture of the micro-organism used 

 in the experiment should be placed in exactly 5 c. c. of plain broth. 



2. One "standard" loopful of the foregoing diluted culture 

 should be transferred to a 10 c. c. broth tube. This will be the 

 "broth control." 



3. One c. c. of the "broth control" should be transferred to 

 a 10 c. c. tube of blood agar. The agar tube should be poured 

 into a Petri dish. This will be the "agar control." 



4. The cultures should be incubated at 37°C. for twenty-four 

 to seventy-two hours. Record growths after the same period of 

 incubation as used for the experiment. 



As a rule, aqueous solutions of the germicide were used. To 

 test the effect of the germicide on the micro-organism in the pre- 

 sence of albuminous material, we also used blood, serum water 

 and dilute serum water. The blood used was defibrinated sheep 

 blood. The serum water was prepared by mixing one part of beef 

 blood serum with three parts of distilled water and sterilizing the 

 mixture on three successive days in the Arnold steam sterilizer. 

 Dilute serum water was prepared in the same way, except that one 

 part of blood serum was used to ten parts of water. 



Blood agar was prepared by the addition of 10 per cent of 

 defibrinated sheep blood to plain agar and by the process of ad- 



