USE OF MIXED BUFFER MATERIALS 493 



can be produced and maintained with small experimental errors, 

 as illustrated in curves C and D in figure 1. In another article 

 we are describing a large number of such useful acid and basic 

 mixtures. For example, the mixture of asparaginic acid (K : = 

 1.5 X 10- 4 , K 2 = 10- 10 ) and of orthophosphoric 3 acid (Ki = 

 1.1 X 10" 2 , K 2 = 2 X 10- 7 , K 3 = 3 X 10- 12 ), used by Pieper 

 in his synthetic medium gives nearly a straight line for the hy- 

 drogen ion concentrations when the equimolecular mixture of 

 acids is treated with successive portions of alkali up to 5 mole- 

 cules (fig. 2). This practically straight line is formed because a 

 region of sharp inflection in the phosphoric acid titration curve, 

 for example, is straightened out by adding another acid (or base) 

 which, when it is 65 to 85 (especially when it is 75) per cent neu- 

 tralized, gives a hydrogen ion concentration approximating that 

 of the inflection point (midpoint) of the phosphate inflection 

 curve. The various inflection curves of phosphoric (pyrophos- 

 phoric) acid and asparaginic acid are thus mutually nearly an- 

 nulled. The slight deviation from a straight line will depend also 

 upon the buffer properties of the other materials, if any, present 

 in the medium, such as, for example, Witte's peptone, extracts 



3 Anhydrous sodium glycerophosphate is now sold as a buffer material which 

 we believe will largely replace phosphoric salts for the following reasons. The 

 glycerophosphate is less expensive than the highly purified potassium phosphate. 

 It can be added in sterilized form directly to sterile culture media without pro- 

 ducing the precipitate formed by phosphates. It does not precipitate out the 

 calcium, magnesium and other heavy metals which are necessary for bacteria 

 and can therefore be kept in solution with glycerophosphates and studied accu- 

 rately in different concentrations. The titration curve is very close to that of 

 phosphoric acid and enables workers to duplicate experiments previously buffered 

 with phosphoric acid; the titration curve is furnished by the Grahame Chemical 

 Company, 100 Rockingham Street, Rochester, N. Y., who sell glycerophosphates 

 standardized for pathological and biological work. The glycerophosphates give 

 the organisms a source of carbohydrate food in the glycerol residue which is 

 apparently more readily assimilated than straight glycerol. Avery, Mellon 

 and Acree have grown over 20 organisms, including tubercle bacilli, on solu- 

 tions buffered with glycerophosphates. The glycerophosphates are stable enough 

 to sterilize in accurately standardized tablets or in solution. The salt can be 

 wieghed out and handled accurately as it is not hygroscopic like anhydrous 

 sodium phosphate, for example. Sucrose-phosphate, mannitol-phosphate and 

 other carbohydrate phosphates have the same desirable qualities and will be 

 reported upon later. 



