1X43-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



in each of the solutions, and radiant power incident upon the systems 

 under comparison. Also, any inherent color in the unknown must 

 be compensated by an equivalent amount in the optical path through 

 the standard. These conditions are met by selecting clear, un- 

 scratched tubes of uniform bore, glass thickness, and color, by having 

 the same concentrations of indicator in the unknown and the stand- 

 ard, by dispersing the color uniformly in the solutions, and by employ- 

 ing 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 

 standards 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 be- 

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



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

 standards 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 stand- 

 ards are not available and there are no facilities for making standard 

 buffer solutions, color standards may be prepared by the drop-ratio 

 method as refined by Gillespie (1920). The method of preparing the 

 standards consists 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 4. 

 The preparation of the standards is explained in the next two para- 

 graphs and in Table 5. 



Although the alcoholic solutions of the indicator acids mentioned 

 in Table 2 may be used, Gillespie recommends for accurate work the 

 use of aqueous solutions of the indicator salts (the preparation of 

 which is specified in Table 2), except in the case of methyl red. 

 Table 5, lower half, gives specifications for the recommended con- 

 centrations of seven of the indicator stock solutions. The exact 

 concentration of the indicator solutions 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.). 



