434 
AMERICAN JOURNAL OF BOTANY 
[Vol. 8. 
growing in the same habitat were selected whose trunk diameters were 
indicative of youth and of old age respectively. Leaves which had approxi- 
mately equal light exposure were taken from these plants for study. 
Only mature leaves were used, inasmuch as it has been shown by Bene- 
dict (i) and by the writer that the area of vein-islets in immature leaves 
is less than that of vein-islets in mature leaves of the same species. 
From five to fifteen leaves from each plant were selected and taken to 
the laboratory. Portions of each leaf were cleared and stained, and deter- 
minations of the size of vein-islets were made according to a previously 
described method (4). At least four determinations were made from 
different places^ on the same leaf. Thus, from each plant from twenty to 
seventy-five determinations of vein-islet areas were made in order to reduce 
to a minimum the probable error due to variation. 
The method used by Benedict in determining the area of vein-islets is 
as follows: 
The collected leaves were taken immediately to the laboratory, measured as to length, 
breadth, and area, and weighed. The venation was then photographed in the following 
way: a heavy black paper was pasted to a clean glass plate, four by five inches in size. 
Ten openings, approximately four by ten millimeters in size, were then cut in the black 
paper. From the same part of each leaf pieces a little larger than the openings were cut, 
and these were laid over the openings, so that each of the ten leaves was represented. A 
clear glass plate was then laid over all, and the whole was bound together by elastic bands, 
placed in the negative holder of an enlarging camera, and photographed at an enlargement 
of three diameters. Negatives showing the veinlets clearly were obtained after some 
practice, and from these negatives velox prints were made. . . . The counting (of the 
number of vein-islets in the opening) was done under a lens, and a sharp needle was used to 
prick each vein-islet as it was counted on the photograph. 
It occurred to the writer that such a method might be conducive to 
inaccuracy for the following reasons: Leaves growing on the same plant 
and even on the same twig vary greatly in shape and size. There is no 
good reason, therefore, for expecting to find such characters as leaf thickness 
and chlorophyll content constant. In fact, the most casual observation 
soon discloses the fallacy of such a premise. This being the case', the pro- 
portion of vascular bundles visible in the uncleared leaves would vary 
directly with the leaf thickness and the chlorophyll content. 
Only one determination from each leaf appears to be entirely inadequate 
to overcome the probable error. It would also appear that the inaccuracy 
would be exaggerated by each step in the photographing, developing, and 
printing processes, especially when the magnification used was only three 
diameters. 
Experimental Data 
In order to find what percentage of the vascular tissue is hidden by 
chlorophyll, leaves growing under as nearly identical environmental con- 
^ Benedict (i) and the writer (4) have shown that the sizes of vein-islets are quite 
constant in various places in a single leaf. 
