6 STUDIES ON RESPIRATION. VIII 



Shearer^ has also demonstrated similar effects of salts upon the 

 viabiUty of meningococcus and Bacillus coli. He found that a com- 

 bination of NaCl and CaCl2 was favorable to growth, while each 

 salt used separately produced decrease in growth. 



It is evident, therefore, that antagonistic effects are to be expected 

 in studying bacteria, and it seemed desirable to investigate these 

 effects in relation to respiration. For this purpose the apparatus 

 described by Osterhout* was employed. 



The organism selected was Bacillus subtilis, the same strain as 

 that used in an investigation previously reported.^ It had originally 

 been isolated from water and grown daily upon agar-agar. In every 

 case an 18 hour culture, inoculated upon agar-agar with a few cc. of 

 0.75 per cent dextrose solution, was used. These inoculations were 

 incubated at 37°C. and the resulting heavy growth of bacteria was 

 washed off with 0.75 per cent dextrose solution and centrifugated 

 to get rid of any foreign substances that may have surrounded the 

 bacteria. The bacteria were then suspended in 0.75 per cent dextrose 

 solution and were ready for experimentation. It is very important 

 to use cultures not older than 24 hours, as the respiration of the older 

 cultures is considerably diminished. 



In making up the solution of dextrose and the salts, distilled water 

 was employed. The salts used were NaCl, CaCl2, and KCl in molec- 

 ular concentrations from 0.05 m to 1.0 m. Experiments on MgCl2, 

 which belong to this series, are in process of completion. 



The temperature varied in the course of the experiments from 

 18-20°C. 



The indicator used was 5 drops of 0.01 per cent phenolsulfoneph- 

 thalein in 10 cc. of water. Tap water was used, as distilled water 

 has a pH value not exceeding 7.1 and this was not alkaline enough 

 for measuring with this indicator. Buffer solutions were made from 

 boric acid and borax, having pH values of 7.78 and 7.60 respectively. 

 These buffers were used as the standard for comparison in determining 

 the color change produced in the indicator as the CO2 was driven over. 



' Shearer, C, Proc. Roy. Soc. London, Series B, 1917, Ixxxix, 440; Proc. Camb. 

 Phil. Soc., 1919, xix, 263. 



* Osterhout, W. J. V., /. Gen. Physiol., 1918-19, i, 17, 171. 

 ' Brooks, M. M., /. Gen. Physiol., 1918-19, i, 193. 



