26 



THE PHYSIOLOGY OF STOMATA. 



Having satisfied myself that the mechanical disturbances incidental to 

 removing the epidermis do not affect the form of the stomata, at least in the 

 plants with which I was concerned, it remained to find some method of 

 fixing these organs in the form assumed at any particular time, so that they 

 might be studied later at any length desired. Only in this way, if we can be 

 certain of the value of the method employed, can we be informed of the actual 

 condition of the stomatal openings scattered over relatively large areas of the 

 plant surface, unless, indeed, direct observation of the living plant is possible. 

 Such a method I have been able to perfect in its application to the plants 

 ocotillo and Verbena (plates 4 and 6), and I have tested it also on several 

 other species of common cultivated plants with entire success. Since all our 

 ordinary experience leads away from accepting a method of this kind, I 

 shall discuss it in some detail. 



Table 15- — Transverse measurements, ocotillo. 



p. . (In water 



"( In absolute alcohol 



c , < In water 



becona ( In . absolute alcohol 



Outside. 



Inside. 



The method is based upon the fact that if the cell- wall is robbed of its water 

 it becomes inelastic, and it will therefore remain in any position it happens to 

 be in when enough water has been extracted to produce inelasticity.* If, 

 then, it were possible to extract the water fast enough before any appreciable 

 change in the form of the walls of the guard-cells intervenes, this is all that is 

 needed. I have determined to my satisfaction that if the epidermis of the 

 leaf be removed and plunged at once into absolute alcohol, this agent removes 

 the water rapidly enough so that no measurable change in the dimensions of 

 the stomata takes place. It is, then, quite easy to preserve in permanent 

 form for later study the material thus gained, either in vials without further 

 treatment or by adding to the absolute alcohol a suitable stain, which must 

 be soluble in the absolute (99.8 per cent) alcohol. In this the tissue is allowed 

 to remain till deeply enough stained, which occurs in about 12 hours, when it 

 may be passed through clove oilf into balsam. None of these operations 

 affects the form of the stomata in the least, a fact which I have verified time 

 and again both by eye measurements and scale, which in many cases was 

 applied before and after each step was taken in the process. 



*I was led to see the force of this principle in its possible relation to the fixation of stomata 

 during a conversation with Professor deVries, who recalled that during his student days 

 under Sachs, his preceptor pointed out that if a wilted plant is placed in alcohol it will 

 remain in the wilted form. This is clearly due in large part to the loss of elasticity by the 

 cell-walls. 111 



fThe fully formed though young stomata of Agave are properly fixed by absolute alcohol, 

 but become distorted upon being placed in clove oil. 



