106 TECHNICAL BULLETIN 4. 



more slowly germinating smut spores. It was found necessary not only 

 to sterilize, bj^ boiling, the slides, Petri dishes and all instruments used, 

 but also to wash the seedlings from which the spores were taken, first, in 

 mercuric chloride, 1 to 1,000, and then in sterile water, before the lesions 

 were opened. In drops of this decoction some of the spores began to 

 germinate within three days at laboratory temperature. The percentage 

 of germination, however, was always very low. In dozens of slide tests 

 made in this way, not over 25 per cent germination has ever been observed ; 

 and in most cases it is lower, averaging 5 to 10 per cent. It is apparent 

 from these tests that there is some substance in the onion which is capable 

 of inducing germination of fresh spores. In the light of other tests de- 

 scribed below, however, one would not be justified in concluding that this 

 substance is peculiar to the onion alone. 



Fresh Spores in Sugar Solutions. — Sterile solutions of |, 1, 2, 3, .5, 7 

 and 10 per cent cane sugar were used just as the onion decoction mentioned 

 above. There was some germination in all of them, but very little in the 

 ^ per cent and the 10 per cent. The highest percentage of germination 

 was in the 2 per cent solution, where 50 per cent of the spores which were 

 on the surface of the drop germinated. When spores are mixed with a water 

 solution of any kind, some of them remain on the surface while others 

 sink to the bottom. Only a very small percentage of those which were 

 immersed germinated. Since the spores on the surface are better located 

 for obtaining air, it is apparent that air is an important factor in germina- 

 tion. It is also apparent that sugar is at least one of the substances which 

 may induce germination. Since onions contain a high percentage of cane 

 sugar, it seems probable that this is also the effective element in the onion 

 decoction which induces germination. 



Fresh Spores on Onion Decoction Agar. — Onion decoction agar was pre- 

 pared by adding 2 per cent of agar to the onion decoction. Sterile plates 

 were poured and permitted to become hard. Spores were mixed with 

 onion decoction or water and floated over the surface of the hard agar. 

 After permitting the spores to settle to the bottom the liquid was poured 

 away and the spores were left distributed over the agar. This insured a 

 sufficient quantity of air, and at the same time access to nutrient substances 

 in the agar. The percentage of germination varied ^vith different experi- 

 ments, but always it was as high as 10 per cent; sometimes 50 per cent. 

 This was found to be the most rehable of all the methods and was largelj' 

 used. Here also it was noticed for the first time that the spores did not 

 all germinate on the same day, but that there was a progressive germina- 

 tion, new ones starting each day for as long as three weeks, after which 

 the plates had dried too much, or possibly the supply of food had become 

 exhausted. 



Fresh Spores on Czapek's Agar, Sugar Potato Agar, etc. — The Czapek's 

 agar contains 3 per cent of cane sugar. Several other agars containing 

 sugar were tried and always with a small percentage of germination, but 

 none higher than on onion decoction agar. 



