3 o 8 Dey.— Studies in the Physiology of Parasitism . V. 
masses was removed and a uniform suspension was obtained. Ordinary 
tap-water was used instead of a nutrient solution, as used by Blackman and 
Welsford in similar experiments with B.’cinerea , as infection readily occurs 
in this, the normal medium, under natural conditions. The density of the 
suspension was estimated with the naked eye, till a faintly milky appearance 
was arrived at. 
Small drops of this suspension were placed on bean-pods, pre- 
viously washed thoroughly with sterile water to remove dust and foreign 
spores, and kept in a moist chamber. Care was taken that the drops 
spread out in thin films, so that nearly all the spores might be under 
similar condition of oxygen supply. The pods were then incubated at 
25 0 C. On the second day, that is twenty-four hours after sowing, small 
bits of the pod under the ‘ infection drops 1 ( 3 ) were cut out every four 
hours, and fixed, generally in Carnoy’s fluid (absolute alcohol, 6 parts ; 
chloroform, 3 parts; glacial acetic acid, 1 part), but sometimes in 
Flemming’s stronger solution of half strength. The tissue underneath the 
c infection drop’ does not show any sign of infection during early stages, so 
that it is not possible to gauge by the eye the stage of infection reached. 
Now and then small brown spots were visible with the microscope below 
the ‘ infection drop ’ ; Frank noticed those spots about twenty-four hours 
after sowing the spores, and held that they were due to infection. These 
spots, however, appear even when tap-water is substituted for the ‘infection 
drops Thus they are not the result of infection, but may be due to 
osmotic disturbances. 
The fixed material was embedded in paraffin, and in every case 
sections 4 ^ in thickness were cut. Heidenhain’s iron-alum haematoxylin 
and erythrosin were first used for staining, but as erythrosin does not bring 
out the cuticle, a counterstain, Sudan III, was employed instead. In 
making up this stain o-oi grm. of Sudan III (Griibler’s) is first made into a 
paste with absolute alcohol, and then made up to 5 c.c. with 95 % alcohol. It 
is allowed to stand for a few days, and then 5 c.c. of pure glycerine are 
added to it. Sections thus stained were mounted in glycerine jelly (10). 
Observations. The germ-tube produced by the spore soon forms 
a brown, thick- walled, more or less spherical appressorium on the surface 
of the pod (6). The appressorium is pressed closely against the surface, 
the part in actual contact with the pod becoming somewhat flattened. The 
appressorium appears to be held on the surface by means of its mucila- 
ginous sheath. The spore is also fixed in some way to the surface, though 
no mucilaginous envelope has been observed around it ; further growth of 
the germ-tube thus causes the tube to curve up, since it is fixed at both ends 
(Fig. 5). Some pressure is thus exerted on the surface, and, no doubt as 
a result of this, a slight indentation of the wall of the epidermal cell is to 
be observed. This is the first preliminary to penetration. 
