a. _Adjustment of Reaction._
_aa._ _Phenol Red Method for adjustment to a hydrogen-ion concentration
of P_{H+} = 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.
The standard reaction for culture media for water analysis shall be +1.0
per cent., as determined by tests of the sterilized medium. As
ordinarily prepared, broth and agar will be found to have a reaction
between +0.5 and +1.0. For such media no adjustment shall be made. The
reaction of media containing sugar shall be neutral to phenolphthalein.
Whenever reactions other than the standard are used, it shall be so
stated.
b. _Sterilization._
All media and dilution water shall be sterilized in the autoclav at 15
lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. All air
must be forced out of the autoclav before the pressure is allowed to
rise. As soon as possible after sterilization the media shall be removed
from the autoclav and cooled rapidly. Rapid and immediate cooling of
gelatin is imperative.
Media shall be sterilized in small containers, and these must not be
closely packed together. No part of the medium shall be more than 2.5
cm. from the outside surface of the glass. All glassware shall be
sterilized in the dry oven at 170° C. for at least 1½ hours.
c. _Nutrient Broth. To Make One Liter._
1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of
distilled water.