80 MANUAL OF MICROBIOLOGICAL METHODS 



ranges. The last column and footnote h of the table give specifications 

 for the preparation of stock solutions of the monosodium salt of each oi 

 the sulfonphthaleins. 



It will be noted from footnote a that ethanolic solutions are ordinarily 

 satisfactory. For precise work, however, aqueous solutions of the indi- 

 cator salts are preferable to the alcoholic solutions of the free acids. To 

 obviate the labor of preparing the neutralized solutions, some makers 

 now offer the soluble salts of the sulfonphthaleins. They are ammonium, 

 sodium, or possibly other salts of these compounds. In ordinary use, the 

 indicator salts contribute negligibly to the total ions present in a test 

 solution, and the nature of the cation may be of no consequence. How- 

 ever, in some studies of bacterial nutrition, the kind of cation and even 

 the small amounts thus added may be of significance. In such cases, it 

 is advisable to learn from the maker what cations (Na, NH4, etc.) are 

 present in the indicator salt in order to make due allowance for their 

 possible effects. 



The colorimetric method of pH determination depends on matching 

 the color of a suitable indicator in the unknown solution with that of the 

 same indicator in a standard. The standards can be set up in two differ- 

 ent ways: by means of buffer standards or by means of ''drop ratios." 

 These will be considered in detail presently. In brief outline, the 

 colorimetric method includes these major steps: 



1. Selection of the appropriate indicator 



2. Preparation of color standards 



3. Color comparison for pH determination 



Later paragraphs will outline essential specifications that must be 

 observed in each of these steps in order to assure reliable results. 



Selection of the appropriate indicator. Test successive small portions 

 (1 ml) of the unknown with a drop of bromthymol blue (BTB). If the 

 color produced is orange or red, then the unknown is probably in the 

 range of pH covered by thymol blue (acid range). If the BTB color is 

 yellow, repeat the test with the indicators of successively lower pK^ (see 

 Table 6) until that indicator is found which gives a color within its sensi- 

 tive or useful range. If the BTB color is blue, proceed in like manner 

 with indicators of higher pK' until the appropriate indicator is found. 

 Of course, if the unknown is more acid than pH 1 or more alkaline than 

 pH 10, none of the indicators listed in Table 6 will serve. 



If the unknown solution is unbuffered (e.g., water or saline) or very 

 weakly buffered, the buffering effect of the added indicator may prevail 

 and significantly change the pH of the unknown. In such cases, special 

 methods are required (see Clark, 1928). 



It is plain that a rough idea can be obtained as to the pH value of a 

 sufficiently buffered solution by simply finding which indicators give their 



