380 ANNALS OF THE MISSOURI BOTANICAL GARDEN 



[Vol. 9 



kali. "Replace in the steamer for twenty minutes (to complete 

 the precipitation of the phosphates, etc.)." 



Which phosphates are involved in this precipitate has not 

 been determined. In experimenting with anhydrous disodium 

 glycerophosphate Mellon and his associates (II, '21) decided that 

 this substance acts as a solvent for calcium and magnesium salts. 

 Accordingly, it was incorporated into nutrient agar in the pro- 

 portion they recommend. The medium was divided into sev- 

 eral parts, some being rendered acid and some alkaline by the 

 addition of hydrochloric acid or by sodium hydroxide giving the 

 following reactions: P H 6.0, 6.3, 6.6, 6.8, and 7.4. Growth on this 

 medium as a whole is somewhat better than on ordinary nutrient 

 agar. It makes a clear medium although a dense precipitate 

 settles at the bottom of the tubes after sterilization. No white 

 precipitate was produced on either the acid or alkaline side. It 

 is of course difficult to say how this is brought about, whether 

 the presence of this salt prevents precipitation by its solvent 

 action on calcium and magnesium salts, or because this salt 

 when added to peptone-beef extract agar precipitates out ma- 

 terials which would otherwise enter into the production of white 

 precipitate, or because of its buffer action in preventing a change 

 in the hydrogen-ion concentration. At any rate, it is quite prob- 

 able that the white precipitate is in the nature of a complex 

 phosphate and not a simple calcium or magnesium salt. 



PHYSIOLOGICAL REACTIONS 



Effect of temperature on growth. — The medium used was nu 

 trient broth testing P H 6.8, care being taken to add approximate!; 

 the same amount of inoculum, 0.1 cc. of 48-hour broth culture, t< 

 each 10 cc. of medium. The organism was subjected to the fol 



temperatures 



10, 14, 20, 25, 30, 35, 



40°. With the exception of the 30° incubator which maintained 

 very nearly a constant temperature, there was some variation in 

 all of the others amounting to 1-2 degrees, so that the tempera- 

 tures given are not absolute. In 24 hours there was a heavy 

 clouding at 34-35°, almost as heavy at 30°, noticeably less at 

 25 ° , only a slight growth in the upper fourth of the broth at 20 

 no visible growth at 14°, and none at 10°. In 48 hours a slight 

 growth was visible at 14°. At the end of a week there was a 

 faint clouding at 10,°, more noticeable clouding at 14°, and 

 beginning with 20° growth was very marked up to 35°, with 



