EXPERIMENTAL METHODS. 



The methods employed in this investigation present nothing markedly new. 

 There were, in general, three lines along which the work was carried out. In 

 the first place, there was the determination of the acidity of the tissue both 

 as regards the pure juice and as regards the total amount of acid present. 

 Secondly, determinations of carbon dioxide evolution were made by the well- 

 known Pfeffer-Pettenkofer method, to fix not only the diurnal periodicity in 

 relation to the normal temperature changes, but also to determine the effect 

 of various external agencies upon the rate. The last and most important line 

 of investigation was that of the gas interchange in darkness between the 

 plant and the atmosphere. For this the methods familiar from the work 

 of Bonnier and Magnin and many others of their school were used, and their 

 micro-gas-analysis apparatus was employed in determining the amount of 

 carbon dioxide evolved and the amount of oxygen absorbed, and then the same 

 or parallel material was investigated to determine its acidity. This involved 

 the making of a very considerable number of gas-interchange experiments 

 simultaneously with the acidity measurements. 



DETERMINATION OF ACIDITY. 



At first attention was devoted wholly to the determination of acidity in 

 order to become familiar with the behavior of the plants in this regard, under 

 varying conditions. The acidity may be measured by the expressed juice, 

 and while the writer is very well aware that this juice does not necessarily 

 represent the sap of the cell contents, as it must include also any fluids which 

 are present in the intercellular spaces, it at least gives the amount of free acid 

 present in the tissues, and is the only index of a quantitative sort that we have 

 for such a study. In general, the acid material expressed from the tissue was 

 immediately titrated without further purification or decolorization. In many 

 of the cacti the juice darkens very rapidly on exposure to the air, due perhaps 

 to the formation of oxidases, but Opuntia versicolor presents less difficulty 

 than most in this regard. Only rarely did any marked discoloration of the 

 expressed liquids occur, even after they had stood in contact with the air for 

 as much as two hours. Nevertheless, even when no change of color ensues, 

 there is always danger that oxidation processes may set in and affect the 

 acidity unless titration is made at once. Various methods of extraction and 

 purification were tried, and the method to be described below was adopted, 

 since, in view of the scope of the work which the writer had in mind it was 

 essential to reduce the processes for the determination of acidity to the simp- 

 lest means consistent with reliable results. 



For experimental purposes Opuntia versicolor presents a great advantage 

 over most of the cacti in that the amount of mucilaginous substance, which is 

 squeezed out in the process of pressing the tissue, is minimal. Indeed, there 

 is commonly none whatever except from the very dry, flaccid joints. This is 

 true also of some other species that were tried, such as Mamillaria grahami, 

 Carnegiea gigantea, and Echinocactus wislizeni, but there is not such a wealth 

 of material of these forms within easy reach not, at least, as far as concerns 

 the numbers of individuals. As for the bulk of tissue one giant cactus would, 

 indeed, afford enough for a whole investigation, but for obvious reasons it 

 would hardly be profitable to carry on a research with a single specimen which 

 would necessarily soon become affected with serious traumatic reactions. 



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