Part 41

In reporting a single test, it is preferable merely to record results as observed, indicating the amounts tested and the result in each, rather than to attempt expression of the result in numbers of B. coli per cc. In summarizing the results of a series of tests, however, it is desirable, for the sake of simplicity, to express the results in terms of the numbers of B. coli per cc., or per 100 cc. To convert results of fermentation tests to this form, the result of each test is recorded as indicating a number of B. coli per cc. equal to the reciprocal of the smallest decimal or multiple fraction of a cubic centimeter giving a positive result. For example, the result: 10 cc. +; 1 cc. +; 0.1 cc. -; would be recorded as indicating one B. coli per cc. An exception should be made in the case where a negative result is obtained in an amount larger than the smallest portion giving a positive result; for example, in a result such as: 10 cc. +; 1 cc. -; 0.1 cc. +. In such case, the result should be recorded as indicating a number of B. coli per cc. equal to the reciprocal of the dilution next larger than the smallest one giving a positive test, this being a more probable result.

Where tests are made in amounts larger than 1 cc., giving average results less than one B. coli per cc., it is more convenient to express results in terms of the numbers of B. coli per 100 cc.

The following table illustrates the method of recording and averaging results of B. coli tests:

Result of Tests in Amounts Designated. Indicated Number of B. coli. 10 cc. 1 cc. 0.1 cc. .01 cc. per cc. per 100 cc. + − − − 0.1 10. + + − − 1.0 100. + + + − 10.0 1,000. + + + + 100.0 10,000. + + − + 10.0 1,000. ————— ————— Totals (for estimating averages) 121.1 12,110. Average of 5 tests 24.0 2,422.

The above method of expressing results is not mathematically altogether correct. The average number of B. coli per cc., as thus estimated, is not precisely the most probable number calculated by application of the theory of probability. To apply this theory to a correct mathematical solution of any considerable series of results involves, however, mathematical calculations so complex as to be impracticable of application in general practice. The simpler method given is therefore considered preferable, since it is easily applied and the results so expressed are readily comprehensible.

In order that results as reported may be checked and carefully valuated, it is necessary that the report should show not only the average number of B. coli per cc., but also the number of samples examined; and, for each dilution, the total number of tests made, and the number (or per cent.) positive.

10. INTERPRETATION OF RESULTS.

While it is not within the province of this report to suggest the proper interpretation of results obtained by the use of the methods herein specified as standard, the committee feels that a word of caution should be given regarding the significance of the presence in a water of members of the B. coli group as defined in this report. Recent work seems to indicate that the B. coli group as herein defined consists of organisms of both fecal and non-fecal origin. Therefore care must be exercised in judging the sanitary quality of a water solely from the determination of the presence of members of the group.

11. DIFFERENTIATION OF FECAL FROM NON-FECAL MEMBERS OF THE B. COLI GROUP.

(1) At least 10 cultures should be used. If possible these should be subcultured from plates made direct from the water since all of the cultures obtained by plating from fermentation tubes may be descendants of a single cell in the water. If cultures from water plates are not available those obtained from plates made as prescribed under B (p. 101) may be used.

(2) Inoculate each culture into dextrose potassium phosphate broth, adonite broth, and gelatin. For additional confirmatory evidence inoculation may be made into tryptophane broth, and saccharose broth. The dextrose broth must be incubated at 30°. Other sugar broths may be incubated at 30° or 37° as convenient. Gelatin should be incubated at 20°.

Footnote H:

(a) Peptone Medium for the Methyl Red Test. To Make One Liter.

1. To 800 cc. of distilled water add 5 grams of Proteose-Peptone, Difco., or Witte’s Peptone (other peptones should not be substituted), 5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate (K{2}HPO{4}). A dilute solution of the K{2}HPO{4} should give a distinct pink with phenolphthalein.

2. Heat with occasional stirring over steam for twenty minutes.

3. Filter through folded filter paper, cool to 20° C. and dilute to 1,000 cc. with distilled water.

4. Distribute 10 cc. portions in sterilized test-tubes.

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.

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.