ix«-U MANUAL OF METHODS FOR PURE CULTURE STUDY 



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 place 8 drops, and so on to the last tube 

 which should contain 1 drop. In the back row of tubes place 1 drop 

 in the left hand tube, 2 in the next, etc., up to 9 in the last. Make 

 up approximately N/20 stock solutions of NaOH and HCl (i.e., 0.2% 

 NaOH; and 1 ml. concentrated HCl (sp. gr. 1.19) diluted to 240 ml.). 

 Then, except in the case of those indicators for which different direc- 

 tions are given in Table 5, add one drop of the stock acid solution 

 to each tube in the front row and 1 drop of the stock alkali solution 

 to each tube in the back row; add enough distilled water to each tube 

 to bring its total contents to 5 ml., thoroughly mix the contents of 

 each tube and return to its place in the rack. It will be seen from 

 Table 5 that two of the indicators, namely thymol blue and brom- 

 phenol blue, require more of the alkali or the acid, respectively, than 

 the other standards in order to insure the appearance of full alkaline 

 or acid color. In the case of thymol blue (alkaline range) and cresol 

 red, the production of the required acid color (yellow) requires not a 

 strong acid but a weaker one such as mono-potassium phosphate or, 

 in the case of thymol blue, distilled water alone. 



The arrangement of tube-pairs indicated in Table 4 produces pro- 

 gressively different colors corresponding to steps of 10% in the 

 transformation of the indicator from its acid to its alkaline color. 

 That is, each pair of tubes, when aligned between the eye and a 

 source of white light, will show a color mixture corresponding to a 

 definite pH. This pH can be computed by means of equation 5 

 which can be rewritten as 



drops of alkalinized indicator 



pH = pK'+log (5a) 



drops of acidified indicator 



The fraction on the right side of the above equation is called the 

 drop-ratio. The values of the standards for seven of the indicators 

 are given in Table 5. They may be computed for the other indica- 

 tors by using the above equation and the pK' values in Table 2. 



For approximate work it is often possible to compare the Gillespie 

 standards with the unknown by merely holding the two tubes of the 

 standard in the hand between the eye and a source of light. For 

 accurate work, however, a comparator block must be used, but one 

 with six holes instead of four, so that a tube of the unknown solution 

 (without indicator) can stand behind the pair of tubes of the standard. 

 The tube of the unknown for comparison with the standard should 

 contain the same amount of indicator as the sum of those in the two 

 standard tubes, i.e., ten drops per 5 ml.; and, of course, this tube 

 must be backed by two tubes of water to equalize the optical path 

 through the standard pair. 



Indicator Papers. Passing mention may be made of these laboratory aids for the 

 approximate measurement of pH. Red and blue litmus papers for the detection of 

 alkalinity and acidity are well known. Papers impregnated with other indicators, 

 singly or in various combinations, can be made or obtained on the market. Those 

 with a single indicator may be of use to detect roughly, (about ±0.3 to 0.4 pH), values 

 within a relatively narrow zone of pH; those with indicator combinations enable one to 



