July IS. 1920 Presoak Method of Seed Treatment 365 



The effect of the various treatments on the blackchaff organism was 

 determined by the following method, devised by Dr. Smith. Wheat 

 seeds in lots of 100 or more were placed in double envelopes of filter 

 paper and sterilized by dry heat at 150° to 160° C. for three hours, in 

 order to kill all internal and surface organisms. After they had cooled, 

 the envelopes were opened with aseptic precautions and the seeds were 

 thoroughly coated with blackchaff bacteria taken from 2- to 4-day-old 

 nutrient agar or potato cultures and used as a heavily clouded bacterial 

 suspension in sterile tubes of tap water. In this way five isolations of 

 the blackchaff organism from as many States were tested. The seeds, 

 after soaking in the bacterial suspension for 20 minutes, were replaced 

 in the envelopes and allowed to dry overnight. By this method each 

 kernel was coated with a dry film of live bacteria such as would occur 

 on badly infected seed under natural conditions. The next day the 

 inoculated and dried seeds were dropped into sterile test tubes containing 

 the disinfectant to be tested. The liquid was drained off after it had 

 acted 10 minutes. The tubes containing the seeds, which now had a 

 thin layer of the solution around each kernel, were placed in moist cham- 

 bers previously rinsed with the same solution. After definite periods 

 the seeds, still moist, were replaced in the sterile envelopes to dry over- 

 night. The next day they were transferred to nutrient agar previously 

 determined to be suitable for the organism, in poured plates, 10 seeds 

 per plate. Each seed was handled with forceps which had been dipped 

 in alcohol and flamed. Control seeds which had been inoculated but not 

 subsequently treated were also planted on this agar. After at least nine 

 days the final records were made. The controls usually developed a 

 typical blackchaff colony around each kernel. Treated seeds, if all the 

 bacteria thereon were killed by the solution used, remained sterile unless 

 contaminated by other organisms or slowly produced blackchaff colo- 

 nies if the disinfectant had not been fully effective. This method is a 

 good index of the effect on the bacteria of the various treatments studied. 

 The seeds are sterilized by dry heat externally and internally without 

 leaving any antiseptic residue such as might be left by chemical sterili- 

 zation. The treatment under consideration is performed upon dried 

 but live bacteria which are found on the seed coat exactly as they would 

 occur in field practice, except that ordinarily they would be less viable 

 and there would be fewer of them. The subsequent exposure on agar 

 plates to optimum conditions for bacterial growth reveals the effect of 

 the treatment, in that the bacteria on the seeds, if uninjured by the 

 treatment, are enabled to develop characteristic colonies, the slowness 

 of their development being a very good index of the proportion killed. 

 Over 5,500 seeds were treated in this manner in the course of the investi- 

 gation. 



