Part 39

6. INCUBATION.

All gelatin plates shall be incubated for 48 hours at 20 C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture.

All agar plates shall be incubated for 24 hours at 37° C. in a dark, well-ventilated incubator in an atmosphere practically saturated with moisture. Glass covered plates shall be inverted in the incubator. Any deviation from the above described method shall be stated in making reports.

7. COUNTING.

In preparing plates, such amounts of the water under examination shall be planted as will give from 25 to 250 colonies on a plate; and the aim should be always to have at least two plates giving colonies between these limits. Where it is possible to obtain plates showing colonies within these limits, only such plates should be considered in recording results, except where the same amount of water has been planted in two or more plates, of which one gives colonies within these limits, while the others give less than 25 or more than 250. In such case, the result recorded should be the average of all the plates planted with this amount of water. Ordinarily it is not desirable to plant more than 1 cc. of water in a plate; therefore, when the total number of colonies developing from 1 cc. is less than 25, it is obviously necessary to record the results as observed, disregarding the general rule given above.

Counting shall in all cases be done with a lens of 2½ diameter’s magnification, 3½X. The Engraver’s Lens No. 146 made by the Bausch & Lomb Optical Company fills the requirements, and is a convenient lens for the purpose.

8. THE TEST FOR THE PRESENCE OF MEMBERS OF THE B. COLI GROUP.

It is recommended that the B. coli group be considered as including all non-spore-forming bacilli which ferment lactose with gas formation and grow aërobically on standard solid media.

The formation of 10 per cent. or more of gas in a standard lactose broth fermentation tube within 24 hours at 37° C. is presumptive evidence of the presence of members of the B. coli group, since the majority of the bacteria which give such a reaction belong to this group.

The appearance of aërobic lactose-splitting colonies on lactose-litmus-agar or Endo’s medium plates made from a lactose broth fermentation tube in which gas has formed confirms to a considerable extent the presumption that gas-formation in the fermentation tube was due to the presence of members of the B. coli group.

To complete the demonstration of the presence of B. coli as above defined, it is necessary to show that one or more of these aërobic plate colonies consists of non-spore-forming bacilli which, when inoculated into a lactose broth fermentation tube, form gas.

It is recommended that the standard tests for the B. coli group be either (A) the Presumptive, (B) the Partially Confirmed, or (C) the Completed test as hereafter defined, each test being applicable under the circumstances specified.

A. PRESUMPTIVE TEST.

1. Inoculate a series of fermentation tubes with appropriate graduated quantities of the water to be tested. In every fermentation tube there must always be at least three times as much medium as the amount of water to be tested. When necessary to examine larger amounts than 10 cc. as many tubes as necessary shall be inoculated with 10 cc. each.

2. Incubate these tubes at 37° C. for 48 hours. Examine each tube at 24 and 48 hours, and record gas-formation. The records should be such as to distinguish between:

(a) Absence of gas-formation.

(b) Formation of gas occupying less than ten per cent. (10%) of the closed arm.

(c) Formation of gas occupying more than ten per cent. (10%) of the closed arm.

More detailed records of the amount of gas formed, though desirable for purposes of study, are not necessary for carrying out the standard tests prescribed.

3. The formation within 24 hours of gas occupying more than ten per cent. (10%) of the closed arm of fermentation tube constitutes a positive presumptive test.

4. If no gas is formed in 24 hours, or if the gas formed is less than ten per cent. (10%), the incubation shall be continued to 48 hours. The presence of gas in any amount in such a tube at 48 hours constitutes a doubtful test, which in all cases requires confirmation.

5. The absence of gas formation after 48 hours’ incubation constitutes a negative test. (An arbitrary limit of 48 hours’ observation doubtless excludes from consideration occasional members of the B. coli group which form gas very slowly, but for the purposes of a standard test the exclusion of these occasional slow gas-forming organisms is considered immaterial.)

B. PARTIALLY CONFIRMED TEST.

1. Make one or more Endo’s medium or lactose-litmus-agar plates from the tube which, after 48 hours’ incubation, shows gas formation from the smallest amount of water tested. (For example, if the water has been tested in amounts of 10 cc., 1 cc., and 0.1 cc., and gas is formed in 10 cc., and 1 cc., not in 0.1 cc., the test need be confirmed only in the 1 cc. amount.)

2. Incubate the plates at 37° C., 18 to 24 hours.

3. If typical colon-like red colonies have developed upon the plate within this period, the confirmed test may be considered positive.

4. If, however, no typical colonies have developed within 24 hours, the test cannot yet be considered definitely negative, since it not infrequently happens that members of the B. coli group fail to form typical colonies on Endo’s medium or lactose-litmus-agar plates, or that the colonies develop slowly. In such case, it is always necessary to complete the test as directed under “C” 2 and 3.

C. COMPLETED TEST.