84 MANUAL OF MICROBIOLOGICAL METHODS 



the standard, by dispersing the color uniformly in the solutions, and by 

 employing proper illumination. 



The color comparison is conveniently made in a comparator block of the type 

 described by Clark (1928, 1948). Various forms of this are obtainable from supply 

 houses. Two pairs of tubes are arranged in the comparator as follows: (1) a tube 

 containing buffer standard plus indicator behind which is placed a tube containing 

 the unknown solution to compensate for inherent color and (2) a tube containing the 

 unknown solution plus indicator backed by a tube containing distilled water. The 

 two pairs of tubes are viewed against a uniform source of white light so placed that the 

 beams incident upon the two systems are of the same radiant power. The color stand- 

 ards are successively compared with the unknown until a match is obtained, thereby 

 establishing the pH of the unknown. If the color of the unknown falls between those 

 of two adjacent standards, an interpolated pH number may be estimated. 



Systems of fixed or "permanent" color standards are also available. These stand- 

 ards consist of colored glasses or other transparent material. Since the spectral 

 absorptions of such standards would hardly be expected to be exactly the same as 

 those of the indicators that they are supposed to match, the applicability and accuracy 

 of these fixed standards must be determined in each case before they are placed in 

 service. Acceptable sets of such standards can be of great convenience in the bac- 

 teriological laboratory, especially for approximate determinations. 



The drop-ratio standards of Gillespie. If commercial color standards are 

 not available and there are no facilities for making standard buffer solu- 

 tions, color standards may be prepared by the drop-ratio method as 

 refined by Gillespie (1920). The method of preparing the standards con- 

 sists in setting up pairs of tubes, containing stepwise proportions, of the 

 full alkaline color and the full acid color of an indicator in such a manner 

 that the resulting color of each pair, when properly viewed, represents a 

 definite pH within the sensitive range of that indicator. 



A general notion of the arrangement and composition of the drop-ratio 

 color standards may be obtained from inspection of Table 8. The 

 preparation of the standards is explained in the next two paragraphs and 

 in Table 9. 



Although the alcoholic solutions of the indicator acids mentioned in 

 Table 6 may be used, Gillespie recommends for accurate work the use of 

 aqueous solutions of the indicator salts (the preparation of which is speci- 

 fied in Table 6), except in the case of methyl red. Table 9, lower half, 

 gives specifications for the recommended concentrations of seven of the 

 indicator stock solutions. The exact concentration of the indicator solu- 

 tions is not very significant in much bacteriological work. 



Select 18 test tubes of approximately the same bore (between 12 and 

 15 mm). They can be selected by adding 10.0 ml of water to a large 

 number of test tubes and choosing a lot in which the columns of water 

 come to approximately the same height (i.e., ±1.5 mm). Place these 

 18 tubes in two rows in a rack, 9 tubes in each row. To the left-hand tube 

 in the front row add 9 drops of the indicator solution, in the second tube 



