15. Label tubes of bile salt broth and inoculate with the following amounts of diluted oysters:

No. 6 with 10 c.c. cylinder fluid = 0.1 oyster.

No. 5 with 1 c.c. cylinder fluid = 0.01 oyster.

No. 4 with 1 c.c. capsule I fluid = 0.001 oyster.

No. 3 with 1 c.c. capsule II fluid = 0.0001 oyster.

No. 2 with 1 c.c. capsule III fluid = 0.00001 oyster.

No. 1 with 1 c.c. capsule IV fluid = 0.000001 oyster.

16. Transfer 100 c.c. cylinder fluid (= 1 oyster) to an Erlenmeyer flask and add 50 c.c. double strength bile salt broth, and label 7.

17. Duplicate all the above indicated cultures.

18. Put up the tube cultures in Buchner's tubes and incubate anaerobically at 42 C.

If growth occurs in tube 1 the organism finally isolated, e. g., B.

coli, must have been present to the extent of one million per oyster.

19. Complete the examination for members of the Coli-typhoid group and sewage streptococci, as directed under Water Examination, page 429 (steps 11-21).

20. Inoculate a series of 6 tubes of litmus milk with quant.i.ties of the material similar to those indicated in step 15; heat to 80 C. for ten minutes, and incubate under anaerobic conditions at 37 C. Examine for the presence of B. enteritidis sporogenes as directed under Water Examination, page 438 (steps 7-10).

EXAMINATION OF SEWAGE AND SEWAGE EFFLUENTS.

Quant.i.tative.--

_Collection of the Sample._--As only small quant.i.ties of material are needed, the samples should be collected in a manner similar to that described under water for quant.i.tative examination and transmitted in the ice apparatus used in packing those samples.

_Apparatus Required._--As for water (_vide_ page 420).

METHOD.--

1. Arrange four sterile capsules in a row and number them I, II, III, IV.

2. Pipette 9 c.c. sterile bouillon into capsule No. I.

3. Pipette 9.9 c.c. sterile bouillon into capsules II, III, and IV.

4. Add 1 c.c. of the sewage to capsule No. I by means of a sterile pipette, and mix thoroughly.

5. Take a fresh sterile pipette and transfer 0.1 c.c. of the mixture from No. I to No. II and mix thoroughly.

6. In like manner transfer 0.1 c.c. from No. II to No. III, and then 0.1 c.c. from No. III to No. IV.

Now 1 c.c. of dilution No. I contains 0.1 c.c. of the original sewage.

1 c.c. of dilution No. II contains 0.001 c.c. of the original sewage.

1 c.c. of dilution No. III contains 0.00001 c.c. of the original sewage.

1 c.c. of dilution No. IV contains 0.0000001 c.c. of the original sewage.

7. Pour a set of gelatine plates from the contents of each capsule, three plates in a set, and containing respectively 0.2, 0.3, and 0.5 c.c. of the dilution. Label carefully; incubate at 20 C. for three, four, or five days.

8. Enumerate the organisms present in those sets of plates which have not liquefied, probably those from dilution III or IV, and calculate therefrom the number present per cubic centimetre of the original sample of sewage.

Qualitative.--The qualitative examination of sewage is concerned with the identification and enumeration of the same bacteria dealt with under the corresponding section of water examination; it is consequently conducted on precisely similar lines to those already indicated (_vide_ pages 426 to 441).

EXAMINATION OF AIR.

Quant.i.tative.--

_Apparatus Required_:

Aspirator bottle, 10 litres capacity, fitted with a delivery tube, and having its mouth closed by a perforated rubber stopper, through which pa.s.ses a short length of gla.s.s tubing.

Erlenmeyer flask, 250 c.c. capacity (having a wide mouth properly plugged with wool), containing 50 c.c. sterile water.

Rubber stopper to fit the mouth of the flask, perforated with two holes, and fitted as follows:

Take a 9 cm. length of gla.s.s tubing and bend up 3 cm. at one end at right angles to the main length of tubing. Pa.s.s the long arm of the angle through one of the perforations in the stopper; plug the open end of the short arm with cotton-wool.

Take a gla.s.s funnel 5 or 6 cm. in diameter with a stem 12 cm. in length and bend the stem close up to the apex of the funnel, in a gentle curve through a quarter of a circle; pa.s.s the long stem through the other perforation in the rubber stopper.

A battery jar or a small water-bath to hold the Erlenmeyer flask when packed round with ice.

Supply of broken ice.

Rubber tubing.

Screw clamps and spring clips, for tubing.

Water steriliser.

Retort stand and clamps.

Apparatus for plating (as for enumeration of water organisms, _vide_ page 420).

METHOD.--

1. Fill 10 litres of water into the aspirating bottle and attach a piece of rubber tubing with a screw clamp to the delivery tube. Open the taps fully and regulate the screw clamp, by actual experiment, so that the tube delivers 1 c.c. of water every second. The screw clamp is not touched again during the experiment.

At this rate the aspirator bottle will empty itself in just under three hours. Shut off the tap and make up the contents of the aspirator bottle to 10 litres again.

2. Sterilise the fitted rubber cork, with its funnel and tubing, by boiling in the water steriliser for ten minutes.

3. Remove the cotton-wool plug from the flask, and replace it by the rubber stopper with its fittings. Make sure that the end of the stem of the funnel is immersed in the bouillon.

4. Place the flask in a gla.s.s or metal vessel and pack it round with pounded ice. Arrange the flask with its ice casing just above the neck of the aspirator bottle.

[Ill.u.s.tration: FIG. 216.--Arrangement of apparatus for air a.n.a.lysis.]