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:[[212]]
| 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: