202 BOTANICAL GAZETTE [SEPTEMBER 
METHODS OF STUDY. 
Microscopic investigations— Material showing various stages 
of the disease was killed by both the chromic acid and absolute 
alcohol methods, dehydrated with alcohol, and infiltrated with 
paraffin in the usual way. Microtome sections were then cut 
and mounted in series, and these were stained with Ziel’s carbol 
fuchsin, and, according to Gram’s method, with anilin water 
gentian violet. The cells of the diseased spot were found to be 
much larger than normal, thin-walled, and oedemic. The chloro- 
plasts were smaller than in healthy cells and were colorless or 
yellowish. Even after the most thorough and careful staining 
no parasitic or saprophytic organism could be detected in the 
tissues of these spots before the epidermal cells collapsed. In 
some cases after they collapsed fungi and bacteria were readily 
distinguished. 
Cultures — After many trials by washing and flaming it was 
found to be impossible to free the surface of a carnation leaf 
from outside germs by any means which might not also destroy 
germs in the tissues. Cultures were therefore made from the 
diseased mesophyll direct by very carefully peeling off the epi- 
dermis and scraping out the inner tissues with a flamed scraper 
which had been allowed to become perfectly cool. From one to 
twenty spots, varying in size from 0.5™™ to 2™™ in diameter, were 
included in each culture. Altogether about 500 cultures were 
made in various media, such as slightly acid, neutral, and 
slightly alkaline beef broth, with and without peptone ; potato 
broth of various strengths; cauliflower broth; potato cylinders; 
agars of various composition; gelatin, acid, neutral, and alkaline 
to litmus, etc. In no case were organisms found in any culture 
made from a spot before the epidermal tissues had collapsed 
In cultures made from spots which had collapsed various fungi 
and bacteria were occasionally obtained, but not constantly or 
always of the same sort. Cultures including the epidermis fre- 
quently contained various organisms, among which was a yellow 
bacterium which occurred frequently on the surface of both dis- 
