390 
Blackman and Welsford. — Studies in 
nor by any careful microscopic study of the actual phenomena of penetra- 
tion. It is certainly improbable, since no enzyme is known which is able to 
dissolve cuticle. 
A study (Brown, 1 ) in this laboratory of a powerful enzymic extract 
of the germ tubes of Botrytis cinerea had shown that such an extract 
was unable to exert any swelling or dissolving action on the tissues of 
a leaf when placed on the uninjured cuticle, although when injected into 
the leaf it rapidly brought about disorganization of the tissues. It seemed 
then advisable to make a careful study of the early stages of infection by 
Botrytis cinerea , paying particular attention to the phenomena to be 
observed in connexion with the penetration of the cuticle. 
Methods. 
Cut leaves of the broad bean ( Vicia Faba) were used as material for 
infection. The cultures of Botrytis cinerea used were from the strain 
employed by Brown ( 1 ) in his work. It was found that the spores 
germinated in water very slowly, or not at all, and very often failed to 
infect a leaf. For this reason the work has been carried out with spores 
sown in turnip juice on a leaf ; this ensures rapid germination and strong, 
well-nourished hyphae. 
The cultures were grown for ten days at 26° C. on sterilized potato- 
mush-agar. 1 The culture, which by this time has produced a plentiful crop 
of spores, is then flooded with water, and the surface of the medium gently 
scraped with a scalpel to detach the spores and mycelium. The suspension 
of spores and hyphae is passed through fine muslin to remove the mycelium, 
and then centrifuged for a few minutes. The water is then poured off very 
carefully and sterilized turnip juice is added — io c.c. of turnip juice to every 
o-i c.c. of wet spores. The turnip juice is prepared by subjecting peeled 
and chopped turnips to a temperature of i2o° in the autoclave for 
45 minutes, and subsequently extracting the juice by means of a press. 
The suspension of spores in turnip juice is used for infection. 2 Infections 
were also made with a much less concentrated suspension of spores. 
Before infection the leaves are washed with a gentle stream of sterile 
distilled water to remove as far as possible extraneous spores and dust. 
They are then placed on damp filter-paper on a sterile Petri dish, and drops 
of the prepared solution containing spores placed on their upper surfaces. 
Material was fixed at intervals mostly in Flemming’s fluid (the strong 
solution diluted with an equal bulk of water), and some in absolute alcohol 
containing 25 per cent, by volume of glacial acetic acid. It is easy to 
1 See Brown (1), p. 68. 
2 This method was worked out by Brown (1) in this laboratory ; by its means an infecting 
suspension of standard strength is obtained. It has been shown that the spore concentration may 
markedly affect the degree of germination. 
