1X48-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



medium. To do this, it is necessary first to select a baseline — that 

 is, a pH number which is to be used as an endpoint in the titration 

 and for the selection of an appropriate acid-base indicator. In the 

 absence of special criteria, it is reasonable to choose as a baseline the 

 pH of the uninoculated medium. The selection of pH 7 as a baseline 

 may be acceptable, because many bacteria grow optimally in this 

 region, not necessarily because it represents the pH of theoretical 

 "neutrality". Other baselines may be chosen in accordance with 

 the special requirements for which the titration is to be made. 



The titratable acidity of the culture can be measured by titration 

 of a known volume of the fluid with 0.1 N NaOH to the predeter- 

 mined endpoint as shown by a standardized glass electrode or by the 

 color of a suitable indicator. In the latter case, it is necessary to pre- 

 pare for comparison an appropriate color standard representing the 

 pH of the chosen endpoint (see earlier discussion of the essential re- 

 quirements for adequate color comparison). If the endpoint pH is 

 other than that of the uninoculated control, a titration is made of the 

 latter and its titration value is subtracted algebraically as a correction 

 or "blank", from that of the culture. The result is usually recorded 

 as ml. of 0.1 normal acid per 100 ml. of the culture fluid. If the 

 culture produces an alkaline reaction, the titration is performed with 

 0.1 A^ HCl, and recorded after correction, if any, in the same way but 

 as a minus quantity of titratable acid. Special precautions are 

 necessary if the titratable acidity is to include all of the volatile acids, 

 including COo and bicarbonate, that may be present in the culture 

 that is being titrated. 



It should be emphasized that, in most cases, the titratable acidity 

 is merely a measure of the buffering capacity (see below) of the 

 medium within the pH range observed. It does not permit further 

 interpretation without additional data on the components of the 

 culture. The titratable acidity is of some importance, along with 

 final pH, in the comparison of high acid producing organisms. For 

 such comparisons to be valid, it is necessary that the different organ- 

 isms be grown in the same medium. Different media which vary 

 in buffering capacity may yield misleading results. 



Buffer action. The titration curve of a weak acid has a sigmoid 

 shape, each end of the curve having a large (steep) slope, and the 

 main central portion having a small slope. This small slope ex- 

 presses the buffer action of the system, that is, the ability of the 

 system (comprising the weak acid and its salt) to resist large change 

 in pH on the addition of acid or alkali. The sigmoid shape of the 

 titration curve expresses, therefore, the fact that the buffer action of 

 such a system is maximal at the midpoint and decreases on either 

 side of this point, first gradually and then more extensively as either 

 end of the curve is approached. The limits of the pH zone of effec- 

 tive buffer action may be arbitrarily set at 1.5 pH units greater and 

 less than the pK' of the acid of the buffer system. It is obvious that 

 increasing the concentration of the buffer system will increase its 

 buffer action; therefore buffer action also depends upon the concen- 

 tration of the buffer system. 



The buffer action of a culture medium is dependent on its composi- 

 tion and may vary considerably in different regions of pH. Signifi- 



