558 Waterhouse . — Studies in the Physiology of Parasitism . VII \ 
Methods. 
In April 1920 rusted wheat stubble was collected from a clover field 
at Milton in Pembrokeshire. It comprised sheath and stem, very badly 
rusted with teleutosori of P. graminis , of the crop that had been harvested 
in 1919. During the winter it had of course been fully exposed to the 
weather. The teleutospores proved viable. This was expected, for 
sprouting barberry hedges in the vicinity were at the time showing the 
aecidial stage of the rust. A portion of the straw was kept at ordinary 
laboratory temperature and a portion in cold storage at a temperature a little 
above freezing-point. The former gave excellent germinations up to June, 
and then, as the spores germinated less abundantly, the material that had 
been kept cool was used. Up to the end of July the germination of these 
teleutospores was still very good and the sporidia remained capable of 
infecting the barberry. 
To obtain sporidia for study, small pieces of the washed straw containing 
teleutosori were placed in hanging drops of water on cover-slips with the 
teleutosori facing downwards. These were supported on rings over sterile 
slides on which were placed drops of sterile tap-water, of very dilute turnip- 
juice, or of barberry extract. The latter was prepared by boiling 50 grm. 
of barberry shoots in 1,000 c.c. of water for half an hour, filtering, and 
sterilizing. These slides were kept in a moist chamber at laboratory 
temperature. It was found that better results were obtained by using 
complete sori in this way than by using scrapings of the sori, giving 
separated teleutospores. The sporidia, formed in the moist atmosphere of 
the hanging drop ( 7 ), fell on to the liquid on the slide and germinated 
readily. These cultures were contaminated with bacteria and other 
organisms which spoiled the cultures after about three days. Numerous 
attempts to grow the sporidia on agar media failed for this reason. 
For demonstrating the presence of a mucilaginous sheath, dilute aqueous 
gentian violet gave better results than Indian ink or collargol. This 
investment was best seen in the germinations in dilute turnip-juice. 
Germinating sporidia were also fixed and stained with picro-nigrosin, or 
fixed in Flemming’s fluid and stained in iron-alum-haematoxylin and the 
triple combination, in order to observe the nuclear details. 
For infection work, plants of Berber is vulgaris in pots were obtained 
and placed in a greenhouse. They were pruned back heavily and the 
young developing shoots were used for infections. Cut shoots placed in 
water in sterile glass jars loosely plugged with cotton-wool were tried, but 
abandoned in favour of shoots still attached to the plant. The shoots were 
well watered with a fine spray, and then small pieces of the rusted straw, 
which had been thoroughly washed and then soaked for half an hour in 
sterile water were, placed on the upper surface of the young leaves. 
