7. Litmus.—Reagent litmus of highest purity (not litmus cubes) or azolitmin (Kahlbaum) shall be used for all media requiring a litmus indicator.
8. General Chemicals.—Special effort shall be made to have all the other ingredients used for culture media chemically pure.
III. METHODS.
1. PREPARATION OF CULTURE MEDIA.
a. Adjustment of Reaction.
aa. Phenol Red Method for adjustment to a hydrogen-ion concentration of PH+ = 6.8–8.4. Withdraw 5 cc. of the medium, dilute with 5 cc. of distilled water, and add 5 drops of a solution of phenol red (phenol sulphone phthalein). This solution is made by dissolving 0.04 grams of phenol red in 30 cc. of alcohol and diluting to 100 cc. with distilled water.
Titrate with a 1:10 dilution of a standard solution of NaOH (which need not be of known normality) until the phenol red shows a slight but distinct pink color. Calculate the amount of the standard NaOH solution which must be added to the medium to reach this reaction. After the addition check the reaction by adding 5 drops of phenol red to 5 cc. of the medium and 5 cc. of water.
bb. Titration with phenolphthalein. (For the convenience of those who wish to retain the use of this method for the present it is given here, but it is recommended that as soon as possible the more accurate method of determining the hydrogen-ion concentration be substituted.)
In a white porcelain dish put 5 cc. of the medium to be tested, add 45 cc. of distilled water. Boil briskly for one minute. Add 1 cc. of phenolphthalein solution (5 grams of commercial salt to one liter of 50 per cent. alcohol). Titrate immediately with a n/20 solution of sodium hydrate. A faint but distinct pink color marks the true end-point. This color may be precisely described as a combination of 25 per cent. of red (wave length approximately 658) with 75 per cent. of white as shown by the disks of the standard color top made by the Milton Bradley Educational Co., Springfield, Mass.
All reactions shall be expressed with reference to the phenolphthalein neutral point and shall be stated in percentages of normal acid or alkali solutions required to neutralize them. Alkaline media shall be recorded with a minus (-) sign before the percentage of normal acid needed for their neutralization and acid media with a plus (+) sign before the percentage of normal alkali solution needed for their neutralization.