EXPERIMENTAL METHODS. 13 



Individual differences in the living shoot, due to a variation in the number of 

 spines and so forth, would amount to a variation larger than the limits thus 

 set. Since we have no idea what their function may be, it seemed unwise to 

 remove the spines, lest a wound stimulus might result; and to obtain, in any 

 case, a strictly quantitative estimate of the amount of tissue actually active in 

 a cactus joint would not be an easy matter. As a rule, it is better not to muti- 

 late tissues used in experimentation more than is absolutely necessary. It 

 will be seen in the later discussion that the possible traumatic reaction, result- 

 ant upon the necessary removal of the joints from the plant, was carefully 

 tested out. 



In most of the work Merck's reagent KOH was employed and as an indicator 

 phenolphthalein was found satisfactory. It takes a little practice to satisfy 

 oneself as to the end-point of the titration, but once familiarity is attained it 

 is entirely possible to get results that are consistent among themselves and 

 which also must closely approximate the absolute quantity of acid present. 



Reference has already been made to the mucilaginous substances so fre- 

 quently found as a result of crushing and pressing tissues of cacti and to the 

 extraordinary freedom of Opuntia versicolor from this disadvantage. The 

 other common cylindrical opuntias (0. mammilata, 0. fulgida, 0. spinosior, 

 and especially 0. leptocaulis) all gave copious amounts of this mucilag- 

 inous material which, showing as it does an acid reaction because of the pre- 

 sumably watery solutions held in it, must be included in the titration. As the 

 alkali penetrates the collodial mass relatively slowly, and as it is not possible 

 to ascertain what reactions may take place between it and the potassic 

 hydroxide, it is obvious that only an approximate determination of the acidity 

 can be made by the method employed. It is not impossible that one might 

 devise a method to obviate these difficulties, but that was not attempted in 

 this work, since the 0. versicolor afforded a sufficient field for study. It should 

 be added that the two flat opuntias common around the laboratory at Tucson, 

 namely, 0. discata and 0, blakeana, aie very mucilaginous. 



DETERMINATION OF RESPIRATION BY THE PETTENKOFER METHOD. 



For the ready and accurate determination of the evolution of carbon diox- 

 ide, there is no apparatus more satisfactory than the Pfeffer-Pettenkofer 

 or some of its modifications. The type employed here was that described by 

 Pfeffer in his paper on intramolecular respiration, with certain modernization 

 in details. The apparatus is so well known that description of it would be 

 superfluous. All of the precautions which are necessary for accurate results 

 were rigidly adhered to. The air passing through the apparatus was carefully 

 washed and the regulation of its flow carefully guarded. In the experiments 

 carried on in New York, the suction used was that produced by an aspirator 

 attached to the faucet, where throughout the building there was maintained 

 by an automatic pump a constant pressure of 60 pounds. In Tucson the 

 exhaustion apparatus consisted of a small electric pump, the suction of which 

 was equalized by a water-column. In both cases an additional safeguard was 

 interpolated between the suction source and the Pettenkofer apparatus by 

 means of a simple mercury safety-valve that has been used by numerous exper- 



"Pfeffer, W. Ueber intramolekulare Athmung. Untersuchungen aua Tubingen, vol. 1, p. 636, 

 1881-85. 



