METHODS FOR MICROSCOPIC STUDY OF BACTERIA 215 
Amount indicator, Amount 55 
gram dry stain. NaOH, cc. 
Brom phenol blue 0.1 3.0 
Methyl red 0,1 7.4 
Brom cresol purple 0.1 3.7 
Brom thymol blue 0.1 3.2 
Phenol red 0.1 5.7 
It has appeared better to dissolve the indicator in about 15 cc. of 
water containing the requisite amount of alkaU, and then to neutra- 
lize the alkali by the addition of the exact equivalence of -^ HCl. 
The volume is then made up to 25 cc. 
The Amnunt of Indicator to he Added to the Cultural Media.— It is 
absolutely essential that the same amounts of indicator be added 
to standard hydrogen-ion solutions and to the medium to be tested. 
P^or convenience, the volume of all solutions, both standard and 
unknown, should be exactly 10 cc. For this amount of fluid, the 
indicators, prepared as outlined, are added in the following con- 
centration.' Five drops are added to each tube of standard and 
unknown solution. The proper dilution, according to Clark, is as 
follows : 
Amount 
Amount Dilution, solution, 
indicator. per cent. Indicator. re. 
5 drops 0.04 Brom phenol blue 10 
5 " 0.02 Methyl red 10 
5 " 0.04 Brom cresol purple 10 
5 " 0.04 Brom thymol blue 10 
5 " . 02 Phenol red 10 
Standard Sohitions of Knoivn Hydrogen-i/m Concentration. — Prepa- 
ration of Standards. — \. Pure dihydrogen potassium phosphate, twice 
recrystallized from distilled water and dried to constant weight at 
100° C. 9.078 gm. are dissolved in redistilled water (conductivity 
water, pH about 6.75) and made up to a volume of 1000 cc. 
Preparations of Solutions of pn Values, 4.5 to 8.3. 
Cc. M Na!HP04. Cc. ^ KH2PO4. PH. 
0.0 "100.0 4.5(4.529) 
1.0 99 . 5.0 
2 5 97.5 5.3 
5 95.0 5.6 
10 90.0 5.9 
20.0 80.0 6.3 
.38 62.0 6.6 
43 5 56.5 6.7 
49 5 50.5 6.8 
55 5 44.5 6.9 
61 39.0 7.0 
66 5 33.5 7.1 
72 5 27.5 7.2 
76.5 23.5 7.3 
80.5 19.5 7.4 
84.0 16.0 7.5 
86 5 13.5 7.7 
90 3 9.7 7.8 
93.0 7.0 7.9 
94 . 5 5.5 8.0 
100.0 8.3(8.302) 
1 The concentration of the indicators is uniformly either 0.2 or 0.4 per cent when 
made up. 
