106 



SCIENCE 



[N. S. Vol. XLIV. No. 1125 



able universal expression of respiratory activity 

 in anaerobic and aerobic tissues in normal con- 

 dition. 



It is extremely important to possess a method 

 of detecting very small quantities of CO„ as it 

 is given off by the organism in the normal en- 

 vironment. The excellent methods devised by 

 Tashiro 2 for the detection of very minute 

 quantities of CO, are unfortunately limited to 

 the study of tissues which are not bathed by 

 solutions. But many of the most important 

 studies on respiration require that the tissues 

 shall be immersed in solutions in order to 

 measure the effect of dissolved substances on 

 respiration. Moreover the methods of Tashiro 

 do not enable us to determine the quantities of 

 CO, produced from moment to moment as the 

 reaction goes on and thus to construct the 

 time curve, which is, in most cases, of primary 

 importance. 



These difficulties are overcome by the method 

 here described. The method consists in adding 

 an indicator to the solution containing the 

 tissue and observing its color changes. 



The indicator should possess the following 

 qualities : (1) it should be non-toxic to the 

 material; (2) it should not rapidly penetrate 

 the tissues; (3) it should be sensitive to very 

 slight increases in the hydrogen ion concentra- 

 tion due to CO,; (4) it should have a suitable 

 working range. 



Phenolsulphone-phthalein with a range of 

 color changes from PIP 6.5 to PIP 8.5 but 

 with extremely sharp differentiations in color 

 between PIP 7.0 and PEP 7.5, has been found 

 to be very satisfactory. 3 Other indicators of 

 various ranges of color change, such as phenol- 

 phthalein, alizarin sodium sulphonate, etc. 

 (sulphonic acid salts being not readily ab- 

 sorbed by cells), are being studied as to their 

 usefulness for such work. 



When salts occur in the solutions used, the 

 salt error for the indicator should be taken into 

 account. Some indicators can not be used with 



2 Tashiro, S., Amer. Jour, of Physiology, 32 : 

 137; Jour. Biolog. Chem., 1914, p. 485. 



3Lubs, H. A., and Clark, W. M., Jour. Wash. 

 Acad. Sci., Vol. V., No. 18, November 4, 1915. 



certain salts on account of being precipitated 

 out of solution, but experimentation alone can 

 tell which, in the large list of accurately de- 

 scribed indicators, 4 are best adapted to a par- 

 ticular need. 



If the material is of the nature of seeds, 

 alga?, or aquatic animals, the whole of which 

 can be submerged, the following procedure is 

 followed : A tube of non-soluble or Pyrex glass 

 of the desired diameter and length (for small 

 seeds, alga?, etc., 16 mm. diameter by about 4 

 to 5 cm. long is very satisfactory; tubes below 

 16 mm. diameter are not recommended) is 

 closed at one end by fusion. A piece of rubber 

 tubing about 7 cm. long is attached at the open 

 end. It is best to boil the rubber tubing re- 

 peatedly previous to using it, in order to insure 

 thorough cleanliness. The rubber tube, while 

 attached to the glass tube, is dipped a few 

 seconds into hot paraffin so as to put a thin 

 coat on both sides of the rubber. The best 

 grade of paraffin (58°-62° C. melting point) 

 is used, and serves to prevent the rubber from 

 possibly giving off substances to the solution 

 and also is advantageous in giving a seal 

 against the CO, of the air. Ordinary soft glass 

 tubing (which gives off alkali) or parawax 

 (which gives off acid) is not suited for accu- 

 rate work. Pyrex tubes, in the absence of Jena 

 glass, can be used to advantage, especially be- 

 cause all sizes can be obtained. 



The material to be studied is placed in the 

 glass tube with a definite number of c.c. of 

 solution containing a definite number of drops 

 of an indicator of known strength. The vol- 

 ume of solution used is always made as small 

 as possible, consistent with the requirements 

 for colorimetric work, but however small the 

 volume of solution used, slightly more than 

 enough to fill the glass tube must be taken. 

 The paraffined rubber tube is then closed with 

 two strong pinchcocks so as to exclude all air 

 from contact with the solution. The paraffin 

 on the rubber tube is prevented from becoming 

 brittle before it is clamped, by working rapidly 

 or if necessary by the use of a lukewarm water 

 bath. In this case the CO, in the solution is 



* Hiiber, ' ' Physik. Chem. der Zelle und der 

 Gewebe," 1914, p. 171. 



