S08 NINETEENTH REPORT. 



the tissue remains alive during the entire experiment, yet the cells are 

 subject to those serious organic disturbances as a result of the separation 

 from the main plant or organ, as well as oxidation, etc. 



The author has used a means of apph'ing the extract to be tested 

 directly to the leaves of the living plant without detaching them. The 

 method employed was as follows: A cardboard disc about 5 cm. in 

 diameter, with a slit extending from a small hole in the center to the 

 circumference, was slipped around tlie petiole of the clover leaf near its 

 base. This disc was then supported upon a horizontal wire ring bent 

 at right angle to a vertical piece of wire stuck in the ground. As a 

 retainer of the liquid to be tested a Van Tieghem cell was sealed with 

 vaseline upon the upper surface of the leaflet. The weight of the cell 

 served to hold the leaflet in a flat horizontal position upon the cardboard 

 disc. The liquid was then dropped into the cells with a pipette, and 

 cover slips smeared with vaseline pressed down upon the upper edges 

 in order to prevent evaporation. (Fig- 15.) This arrangement retained 

 the liquid for a long time without drying out. In order to examine, 

 macroscopically from time to time, tlie effect upon the leaflet, (where the 

 liquid was dark colored or non-transparent) the liquid was drawn up 

 into a pipette witli a fine point, replaced if necessary and the cell again 

 sealed. That the vaseline has no effect upon the leaf tissue nor upon 

 the liquids tested, has been determined by tlie use of proper controls 

 with eacli experiment. 



Tests of Metabolic By-Products : 



A clover-juice culture 75 days old was used. The fungus growth, 

 which consisted of a heavy mycelial mat was removed by first passing 

 the culture liquid tlirough a filter paper and then through a sterile Berk- 

 feld filter. Transfers were at once made to agar in order to determine 

 later whether or not the filtrate was sterile, although microscopic exam- 

 ination at the time failed to reveal the presence of any fungus or bacterial 

 cells. 



With a sterile pipette, a small quantity of filtrate was added to each 

 cell arranged as above described, (.4-. 5 cc. reaching to within a few mm. 

 of the top of the cell) and sealed with a cover slip. The test was made 

 upon both wounded and unwounded leaves. In wounding, the epidermis 

 was lightly scratched with a fine-pointed, sterile needle, care being taken 

 not to puncture the leaf. Two or three of these scratches, 3 to 4 mm. 

 long, were made in the center of the area enclosed by the cell. Such 

 scratches could barely be seen with the naked eye. In the table below 

 each number corresponds to the three individual leaflets of a single leaf; 

 hence making the test in triplicate for each number. 



