5. Sterilize by the intermittent method for 20 minutes on three
successive days.
Footnote I:
_Tryptophane Broth for Indol Test._
To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams
dipotassium hydrogen phosphate (K_{2}HPO_{4}), and 1 gram peptone.
Heat until ingredients are thoroughly dissolved, tube (6 to 8 cc.),
and sterilize in autoclave for 15 minutes after the pressure reaches
15 pounds. Some American peptones are standardized to contain a
uniform amount of tryptophane. If such peptone is used the tryptophane
in the above formula may be omitted and the peptone increased to 5
grams.
(3) After 48 hours record gas formation in adonite and saccharose
broths. Determine indol formation in tryptophane broth by adding drop by
drop, to avoid mixing with the medium, about 1 cc. of a 2 per cent.
alcoholic solution of p-dimethyl amido-benzaldehyd, then a few drops of
concentrated hydrochloric acid. The presence of indol is indicated by a
red color which is soluble in chloroform. There may be some unconverted
tryptophane still present which will give a distinctly blue color which
is insoluble in chloroform. A mixture of the two will be either blue or
violet. If from such a mixture of colors the red of indol be extracted
with chloroform proof of the presence of indol will be complete.
(4) After 5 days apply methyl red test and Voges-Proskauer test to
dextrose broth.
_Methyl Red Test._[J]
Indicator solution.—Dissolve 0.1 gram methyl red in 300 cc. alcohol and
dilute to 500 cc. with distilled water.
Footnote J:
(b) _Synthetic Medium for the Methyl Red Test._ To Make One Liter.
Dissolve 7 grams Na_{2}HPO_{4} (anhydrous) or 8.8 grams
Na_{2}HPO_{4}.2H_{2}O, 2 grams KHphthalate, 1 gram aspartic acid, and
4 grams dextrose in about 800 cc. of warm distilled water. When
solution is complete, cool and make up to 1 liter at room temperature.
Heat in an autoclave for 15 minutes after the pressure has reached 15
pounds, provided the total time of exposure to heat is not more than
one-half hour. The hydrogen-ion concentration of the medium is fixed
by the composition. It should be very close to P_{H} 7.0, slightly red
with phenol red. All materials should be recrystallized or if used
from stock furnished by manufacturers, should be carefully examined.
The di-sodium hydrogen phosphate may be used either as the anhydrous
salt obtained by dessication in vacuo at 100° C. or else as the salt
containing two molecules of water of crystallization. This is obtained
by exposing the recrystallized Na_{2}HPO_{4}.12H_{2}O for two weeks.
Use 0.88 per cent. of Na_{2}HPO_{4}.2H_{2}O.
Procedure in test.—1. To 5 cc. of each culture add 5 drops of methyl red
solution.
2. Record distinct red color as methyl red +, distinct yellow color as
methyl red -, and intermediate colors as ?.
_Voges-Proskauer Test._[216]
To the remaining 5 cc. of medium add 5 cc. of a 10 per cent. solution of
potassium hydroxide. Allow to stand over night. A positive test is
indicated by an eosin pink color.
(5) Gelatin tubes should not be pronounced negative until they have been
incubated at least 15 days.
The following group reactions indicate the source of the culture with a
high degree of probability:
Methyl red + │B. coli of fecal origin.
Voges-Proskauer − │
Gelatin − │
Adonite − │
Indol, usually + │
Saccharose, usually −│
Methyl red − │B. aërogenes of fecal origin.
Voges-Proskauer + │
Gelatin − │
Adonite + │
Indol, usually − │
Saccharose + │
Methyl red − │B. aërogenes, probably not of fecal
│ origin.
Voges-Proskauer + │
Gelatin − │
Adonite − │
Indol, usually − │
Saccharose + │
Methyl red − │B. cloacae, may or may not be of fecal
│ origin.
Voges-Proskauer + │
Gelatin + │
Adonite + │
Indol, usually − │
Saccharose + │