94 MANUAL OF MICROBIOLOGICAL METHODS 



limitation that definite and significant potentials are possible only in the 

 presence of finite ratios of oxidant to reductant. In addition, the total 

 concentration of the reversible system may be decreased to and beyond a 

 level at which traces of electromotively active contaminants attain 

 dominance and an observed potential becomes unstable and difficult to 

 interpret. 



In contrast to the above-mentioned reversible processes which are 

 readily amenable to Eh measurement, there are a great many oxidation 

 reductions that proceed by a variety of mechanisms that do not permit 

 formulation and precise measurement in terms of equilibrium states. 

 Electrode potentials in such cases are difficult to interpret and of uncer- 

 tain significance. 



In cell suspensions and bacterial cultures, especially when deprived of 

 free access to oxygen, there develops with time a progressively more nega- 

 tive potential which traverses the zones characteristic of reversible 

 oxidation-reduction indicators (see next section). Polarization of the 

 electrode or a small dose of an oxidant may reverse the trend of reduction 

 potential temporarily, but the trend is resumed after a while to levels of 

 potential that may sometimes be associated with the type of cell and the 

 various metabolites in the suspension or culture. Duplicate electrodes in 

 such systems may not be in good agreement at the start, but they will 

 reach about the same limiting value in time. For examples, see Clark, 

 Cohen, et al. (1928), Allyn and Baldwin (1932), and Hewitt (1936). 



The Colorimetric Method 



Introduction. The empirical use of substances such as litmus or 

 methylene blue as indicators of reduction in bacterial cultures is well 

 known. For the determination of various degrees of reduction intensity 

 an appropriate series of indicators is necessary. Among those available 

 are reversible oxidation-reduction systems, the oxidants of which are usu- 

 ally colored and the reductants practically colorless. A number of such 

 indicator systems have been characterized and may be employed, with 

 due precautions, in determining an oxidation-reduction potential 

 colorimetrically. 



A selection of such indicators^ is listed in Tables 10 and 11. Similar 



^ A special comment is necessary in regard to neutral red (compound t in Tables 10 

 and 11). It undergoes reversible reduction in the usual manner, and the colorless 

 solution of reductant formed upon rapid reduction reoxidizes very rapidly when 

 exposed to air. However, the reductant on standing in solution at pH 4-6 for a little 

 time undergoes transformation to a fluorescent substance which is stable for days in the 

 presence of air but reoxidizes rapidly upon acidification. As an oxidation-reduction 

 indicator, therefore, neutral red must be employed with due caution and can be used 

 only for rough comparisons. 



