of a Urcdinc, Puccinia dispersa (Eriks.). 455 



To meet this objection I devised operations as follows : 



Clean picked seeds were placed singly, by means of forceps, on filter 

 paper at the bottom of Petri-dishes properly sterilised by heat. When 

 these had germinated, and observation showed that the whole series 

 was free of moulds or other signs of contamination, the seedlings 

 were removed by means of sterile forceps, and transplanted singly into 

 sterilised tubes of various kinds as described below, and the further 

 growth allowed to proceed in the light under conditions varied as will 

 be seen. 



I had already shown that seedlings will continue to grow in such 

 tubes, but, as we have seen, in the cases previously described I had no 

 guarantee that the seedlings introduced into the culture-tubes did not 

 already carry on their leaves wind-borne or otherwise transmitted 

 spores. 



In the case of these seedlings germinated from clean " seed " in 

 sterile dishes and tubes, it is obvious that the only chance of infection 

 depends on spores attached to the " seed " or on mycelium in the 

 seed. 



Experiments with seed gathered even from badly rusted plants and 

 germinated as above, have never given rusted seedlings, although 

 other experiments have shown me that the germ-tubes of attached 

 spores can infect seedlings when the plumule is only 3 5 mm. high. Nor 

 have I ever been able to discover any trace of mycelium in the seeds. 



But if the " seed " of the JJromiis is sterilised before germination as 

 can be done by steeping in various antiseptics, or by heating to 

 60 70 C. it is found that pure cultures of the Brome may be 

 obtained in the tubes, and it is then only necessary to infect such a 

 clean seedling with the spores of the parasite to obtain a pure culture 

 of the latter. 



Preliminary experiments soon showed that the Brome seedlings 

 thus raised from seeds treated antiseptically, and protected from the 

 first by glass, may be grown for weeks and even for a couple of months 

 in such tubes under proper precautions, and I set myself the task of 

 ascertaining how such cultures would behave in infection experiments. 



In the following experiment No. 711 eight upright tubes of the 

 kind known to chemists as " drying towers," were prepared as in the 

 diagram (fig. 1), so that by means of an aspirator attached to the 

 tubing at G, a continuous current of damp air could be slowly drawn 

 through the whole series, aerating the roots of the seedlings F, which 

 burrowed into the cotton-wool B, day and night. The tutes were 

 charged each with one seedling, grown from seeds heated to 65 C., 

 and 48 hours after germination had begun, and the latter allowed to 

 grow in the light on a table outside the laboratory. The tulxjs were 

 charged on June 14, and on June 19, when the first green leaf was well 

 developed, the latter was infected at a definite spot with spores proved 



