Part 40

1. From the Endo’s medium or lactose-litmus-agar plate made as prescribed under “B,” fish at least two typical colonies, transferring each to an agar slant and a lactose broth fermentation tube.

2. If no typical colonies appear upon the plate within 24 hours, the plate should be reincubated another 24 hours, after which at least two of the colonies considered to be most likely B. coli, whether typical or not, shall be transferred to agar slants and lactose broth fermentation tubes.

3. The lactose broth fermentation tubes thus inoculated shall be incubated until gas formation is noted; the incubation not to exceed 48 hours. The agar slants shall be incubated at 37° C. for 48 hours, when a microscopic examination shall be made of at least one culture, selecting one which corresponds to one of the lactose broth fermentation tubes which has shown gas-formation.

The formation of gas in lactose broth and the demonstration of non-spore-forming bacilli in the agar culture shall be considered a satisfactory completed test, demonstrating the presence of a member of the B. coli group.

The absence of gas-formation in lactose broth or failure to demonstrate non-spore-forming bacilli in a gas-forming culture constitutes a negative test.

APPLICATION OF PRESUMPTIVE, PARTIALLY CONFIRMED, AND COMPLETED TESTS.

A. The Presumptive Test.

1. When definitely positive, that is showing more than 10 per cent. (10%) of gas in 24 hours, is sufficient:

(a) As applied to all except the smallest gas-forming portion of each sample in all examinations.

(b) As applied to the smallest gas-forming portion in the examination of sewage or of water showing relatively high pollution, such that its fitness for use as drinking water does not come into consideration. This applies to the routine examinations of raw water in connection with control of the operation of purification plants.

2. When definitely negative, that is showing no gas in 48 hours, is final and therefore sufficient in all cases.

3. When doubtful, that is showing gas less than 10 per cent. (10%) (or none) in 24 hours, with gas either more or less than 10 per cent. in 48 hours, must always be confirmed.

B. The Partially Confirmed Test.

1. When definitely positive, that is, showing typical plate colonies within 24 hours, is sufficient:

(a) When applied to confirm a doubtful presumptive test in cases where the latter, if definitely positive, would have been sufficient.

(b) In the routine examination of water supplies where a sufficient number of prior examinations have established a satisfactory index of the accuracy and significance of this test in terms of the completed test.

2. When doubtful, that is, showing colonies of doubtful or negative appearance in 24 hours, must always be completed.

C. The Completed Test.

The completed test is required as applied to the smallest gas-forming portion of each sample in all cases other than those noted as exceptions under the “presumptive” and the “partially confirmed” tests.

The completed test is required in all cases where the result of the confirmed test has been doubtful.

9. EXPRESSION OF RESULTS.

In order to avoid fictitious accuracy and yet to express the numerical results by a method consistent with the precision of the work, the numbers of colonies of bacteria per cubic centimeter shall be recorded as follows:

Number of bacteria per cc. From 1 to 50 shall be recorded as found " 51 " 100 " " " to the nearest 5 " 101 " 250 " " " " " " 10 " 251 " 500 " " " " " " 25 " 501 " 1,000 " " " " " " 50 " 1,001 " 10,000 " " " " " " 100 " 10,001 " 50,000 " " " " " " 500 " 50,001 " 100,000 " " " " " " 1,000 " 100,001 " 500,000 " " " " " " 10,000 " 500,001 " 1,000,000 " " " " " " 50,000 " 1,000,001 " 10,000,000 " " " " " " 100,000

This applies to the gelatin count at 20° C. and to the agar count at 37° C.

SUMMARY OF STEPS INVOLVED IN MAKING PRESUMPTIVE, PARTIALLY CONFIRMED AND COMPLETED TESTS FOR B. COLI.

────────────────────────────────────────────────────────────┬───────── Steps in procedure. │ Further │procedure │required. ────────────────────────────────────────────────────────────┼───────── I. Inoculate lactose broth fermentation tubes; incubate 24 │ hours at 37° C.; observe gas-formation in each tube. │ 1. Gas-formation, 10 per cent. or more; constitutes │ positive presumptive test. │ (a) For other than smallest portion of any sample │ showing gas at this time, and for all portions, │ including smallest, of sewage and raw water this│ test is sufficient. │None (b) For smallest gas-forming portion, except in │ examinations of sewage and raw water. │III 2. Gas-formation less than 10 per cent. in 24 hours; │ inconclusive. │II II. Incubate an additional 24 hours, making a total of 48 │ hours’ incubation; observe gas-formation. │ 1. Gas-formation, any amount; constitutes doubtful │ test, which must always be carried further. │III 2. No gas-formation in 48 hours; constitutes final │ negative test. │None III. Make plate from smallest gas-forming portion of sample │ under examination; incubate 18 to 24 hours; observe │ colonies. │ 1. One or more colonies typical in appearance. │ (a) If only “partially confirmed” test is required│None (b) If completed test is required, select two │ typical colonies for identification. │V 2. No typical colonies. │IV IV. Replace plate in incubator for an additional 18 to 24 │ hours; then, whether colonies appear typical or not, │ select at least two of those which most nearly resemble B.│ coli. │V V. Transfer each colony fished to: │ 1. Lactose broth fermentation tube; incubate not more │ than 48 hours at 37° C. Observe gas-formation. │None 2. Agar slant; incubate 48 hours at 37° C. │ (a) If gas formed in lactose broth tube inoculated│ with corresponding culture │VI (b) If no gas formed in corresponding lactose │ broth tube, test is completed and negative. │None VI. Make stained cover-slip or slide preparation, and │ examine microscopically. │ 1. If preparation shows non-spore-forming bacilli in │ apparently pure culture, demonstration of B. coli is │ completed. │None 2. If preparation fails to show non-spore-forming │ bacilli or shows them mixed with spore-bearing forms │ or bacteria of other morphology. │VII VII. Replate, to obtain assuredly pure culture, select │ several colonies of bacilli and repeat steps V and VI. │ ────────────────────────────────────────────────────────────┴─────────

In order that tests for B. coli may have quantitative significance, the following general principles and rules should be observed:

Ordinarily not less than three portions of each sample should be tested, the portions being even decimal multiples or fractions of a cubic centimeter; for example, 10 cc., 1 cc., 0.1 cc., .01 cc., etc. It is essential that the dilutions should be such that the largest amount gives a positive test (unless the water is such as to give negative tests in 10 cc.), and the smallest dilution, a negative result. To insure this result, it is often necessary to plant four or five dilutions, especially in the examination of a sample of entirely unknown quality. The quantitative value of a series of tests is lost, unless all or at least a large proportion of the smallest dilutions tested have given negative results.