136 THE PLANT 
to the impossibility of distinguishing between the water loss due to light 
and that caused by humidity and other factors. If it were possible to de- 
termine the amount of chlorophyll or glucose produced, these could be used 
as satisfactory measures of response. As it is, they can only be determined 
approximately by counting the chloroplasts or starch grains. The arrange- 
ment of the chloroplasts can not furnish the measure sought, since it does 
not lend itself to quantitative methods, and since the relation to light in- 
tensity is too inconstant. Hesselmann (1. c., 400) has determined the 
amount of carbon dioxide respired, by means of a eudiometer, and has based 
comparisons of sun and shade plants upon the results. As he points out, 
however, light has no direct connection with respiration. Although the 
latter increases necessarily with increased nutrition, the relation between 
them is so obscure, and so far from exact, that the amount of respiration 
can in no wise serve as a measure of the response to light. As a result of 
the foregoing, it is clear that no functional response is able to furnish a 
satisfactory measure of adjustment to light, though one or two have per- 
haps sufficient value to warrant their use. Indeed, structural adaptations 
offer a much better basis for the quantitative determination of the effects 
of light stimuli, as will be shown later. 
In attempting to use the number of chloroplasts or starch grains as a 
measure of response, the study should be confined to the sun and shade 
forms of the same species, or, in some cases, to the forms of closely related. 
species. The margin of error is so great and the connection with light 
sufficiently remote that comparisons between unrelated forms or species: 
are almost wholly without value. It has already been stated that starch is: 
merely the surplus carbohydrate not removed by translocation; the amount: 
of starch, even if accurately determined, can furnish no real clue to the 
amount of glucose manufactured. In like manner, the number of chloro- 
plasts can furnish little more than an approximation of the amount of 
chlorophyll, unless size and color are taken into account. In sun and shade 
ecads of the same species, the general functional relations are essentially: 
the same, and whatever differences appear may properly be ascribed to 
different light intensities for the two habitats. The actual counting of 
chloroplasts and starch grains is a simple task. Pieces of the leaves of the 
two or more forms’ to be compared are killed and imbedded in paraffin in 
the usual way. To save time, the staining is done im toto. Methyl green is 
used for the chloroplasts and a strong solution of iodine for the starch 
grains. When counts are to be made of both, the leaves are first treated 
with iodin and then stained with the methyl green. The thickness of the 
microtome sections should be less than that of the palisade cells in order 
that the chloroplasts may appear in profile, thus facilitating the counting. 
The count is made for a segment 100 m» in width across the entire leaf. 
