250 SELMAN A. WAKSMAN .\ND J. S. JOFFE 



organism. Reactions more acid than 2.0 easily become injurious, 

 although the organisms still continue to live at even as low a 

 reaction as a hydrogen-ion concentration of pH = 0.6, while 

 the medium titrates 0.8 normal acid (with phenolphthalein as 

 indicator, using i^ NaOH: the culture being gro\\Ti on medium 

 1). Reactions ranging in pH from 4.0 to 6.0 are less favorable. 

 Growth is slower to start, but once the reaction, through a 

 slow oxidation of the sulfur, has reached a pH of about 3.0, the 

 growth becomes more rapid. Reactions equivalent to pH 6,0 

 and above are unfavorable for growth. When a culture, at a 

 pH 0.8 to 1.6 (these being the limits tested), is filtered free from 

 any unoxidized sulfur, then stoppered and allowed to stand, 

 the liquid is found to clear up, after a period of time, and the 

 bacteria are agglutinated with the formation of flaky masses at 

 the bottom of the containers. The rapidity of agglutination 

 depends on the reaction of the culture, the more acid cultures 

 agglutinating more rapidly than the less acid ones: at a pH = 

 0.8, agglutination took place in four to five days, while at pH 

 = 1.5, it took more than two weeks for this jahenomenon to 

 appear. It is interesting to note that this phenomenon was 

 never observed in the unfiltered culture, i.e., in the presence 

 of unoxidized sulfur, even if the cultures were kept at ]iH0.6 to 

 0.8 for a long time. 



Neutralizing agents. The acid formed rapidly changes the 

 hydrogen-ion concentration of the medium and growth almost 

 ceases. To obviate a rapid change in reaction by the acid pro- 

 duced from the oxidation of the sulfur, neutralizing agents are 

 to be used. These should be of suth a nature as not to make 

 the medium alkaline or tend to change the reaction rapidly: 

 this eliminates, therefore, the use of carbonates and soluble oxides, 

 like CaO. The best substances are buffers, like phosphates, 

 but to keep the reaction above a very high acidity by means of 

 soluble phosphates, high concentrations have to be used, which 

 will exert an unfavorable physical effect upon the organism. 

 Solid salts, insoluble in water which, on dissolving by the action 

 of the acid, will give a soluble substance and an insoluble residue, 

 are best for this purpose. CaCOa and INIgCOs can be used, but 



