28 the; physiology of stomata. 



The stains which have been used are Congo red and Bismarck brown. 

 The former gives very superior results, as it stains much more deeply. No 

 doubt there are others just as good. 



It would seem that this method will enable us to study, with great accuracy, 

 the mechanical aspects of stomata — a matter with which I am at present, 

 however, not concerned. With regard to my own purposes, it enabled me to 

 study the conditions of large numbers of stomata at my leisure and with a 

 high degree of certainty. As a result, however, I found that there are other 

 sources of error inherent in the behavior of the organs themselves. The 

 stomata in a piece of epidermis are seldom of a uniform degree of opening. 

 In the same piece some stomata may be quite shut and others as wide as 6 

 micra, or even more. In one which would be said to have stomata wide open, 

 a glance shows a variation in width of 4 micra, between the extremes of 6 and 

 10 micra. 



In making my observations, therefore, I was compelled to follow a course 

 which should give me the requisite degree of accuracy without too great an 

 expenditure of time. For a while I measured 10 stomata as they came in a 

 line, moving the preparation by means of a mechanical stage. Again I tried 

 to estimate the size of a hundred stomata taken at random, having an eye- 

 piece micrometer scale in position as a standard of comparison. 



I finally decided that I could get as near the truth by the following method 

 as by any way available. This is, to examine the whole of the piece of epider- 

 mis, to see what the range of size is, and to determine whether the majority 

 of the stomata are near the mean size. The extremes are then measured 

 accurately and the measurement recorded. Not infrequently a single stoma 

 may be found shut, or wide open, among a hundred or many more in neither 

 condition. This is disregarded or recorded in parenthesis. In plotting 

 curves the extremes may be given, together with a curve of 'the mean. This 

 method has been found sufficiently accurate for the work, though I can well 

 understand that for further investigation a still more refined method may be 

 found desirable. 



When pieces of epidermis are placed in absolute alcohol it is to be noted 

 that they become more or less twisted. This is due apparently to the cuticle 

 which prevents the synchronous action of the fixing agent on all parts of the 

 tissue. The effect on any one cell is, however, infinitesimal and may be dis- 

 regarded. It should be further pointed out that it does not do to place the 

 whole leaf or even a small part of it in the alcohol and expect the result 

 obtained by using the removed epidermis. It appears that there is quite 

 sufficient water in the tissue of the leaf to dilute the alcohol to the extent 

 that it is made inefficient in rapidity of action, and hence the stomata close 

 more or less. 



I have repeatedly found, especially when large portions of the uninjured 



