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
