446 BULLETIN 387 



opposition to Pinoy's (1905) statement that there is no development 

 when the spores are not accompanied by a coccus. 



Diseased cabbage roots were disinfected with either mercuric chloride 

 or calcium hypochlorite; if with the former, they were then rinsed three 

 times carefully in different tubes of sterilized distilled water; if with 

 the latter, they were rinsed in muck-soil filtrate, which is acid and tends 

 to neutralize any of the calcium compounds that might adhere to the roots 

 and retard germination of the spores. All the roots were then either 

 transferred to tubes of nutrient agar slants or embedded in agar in petri 

 dishes. If at the end of a week they showed no signs of contamination, 

 those in the petri dishes were placed on agar slants, after which all the 

 roots were minced and left for another week in order to make sure that 

 no bacteria were in the roots and had been liberated by the mincing. 



Seeds of the cabbage were sterilized by the same method as was 

 employed for the roots, but they were not rinsed in sterilized water when 

 calcium hypochlorite was used. The seeds were planted in nutrient 

 agar in petri dishes and the young plants were permitted to develop 

 until they were free of the old seed coats. They were then placed in 

 the tubes with the minced roots that showed no bacterial colonies, and 

 a sufficient amount of the sterilized muck filtrate was added to insure 

 spore germination but not enough to injure the small seedlings. 



This process, tho complicated and long, seems to fulfill all the require- 

 ments that carefulness demands; and in the three series tried, from five 

 to twenty per cent of the cultures were free from bacteria. The chief 

 difficulty lies in the fact that there is such a narrow margin between spore 

 formation and bacterial invasion that it is hard to select swellings which 

 are neither too young to contain mature spores nor yet so old that bacteria 

 have entered. One objection to the experiment is obvious. There is 

 no way of determining contaminations except by the absence of colonies 

 on the agar where the roots have been minced and on which the seedlings 

 grow. Yet it seems hardly possible that bacteria can be present thruout 

 all these operations and not come into contact with the medium. Besides, 

 where no bacterial colonies appear, the plants grow more vigorously, 

 produce larger roots, and show infection sooner than in the contaminated 

 tubes. Swellings apparent to the naked eye were formed at the end 

 of five days the first time the experiment was tried. When the plants 

 were fixed, sectioned, and stained, they showed amoebae in the cortex 

 as well as in a large number of root hairs ; all of which tends to discount 

 Pinoy's (1905) belief that there is no development of the parasites with- 

 out a concomitant bacterium. 



Pinoy based his conclusions in part on the evidence presented by stained 

 sections. Apparently he studied sections of large roots, since the roots 



