(D) + = indol production. = slight indol production. - = no indol formed.

(E) + = acid production. - = alkali production. O = no change in reaction. C = clot.

21. Determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated.

On the facing insert page is reproduced a blank from the author's Laboratory Water a.n.a.lysis Book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined.

B. ~Concentration Method.~

The remaining organisms referred to on page 426 are more conveniently sought for by the concentration method.

_Collection of the Sample._--The quant.i.ty of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue gla.s.s Winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. The bottle may be packed in a wooden box or in an ordinary wicker case. The method of collecting the sample is identical with that described under the heading of Quant.i.tative Examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory.

_Apparatus required_:

Sterile Chamberland or Doulton "white" porcelain open mouth filter candle, fitted with rubber washer.

Rubber cork to fit mouth of the filter candle, perforated with one hole.

Kitasato serum flask, 2500 c.c. capacity.

Geryk air pump or water force pump.

Wulff's bottle, fitted as wash-bottle, and containing sulphuric acid (to act as a safety valve between filter and pump).

Pressure tubing, clamps, pinch-c.o.c.k.

Retort stand, with ring and clamp.

Rubber cork for the neck of Winchester quart, perforated with two holes and fitted with one 6 cm. length of straight gla.s.s tubing, and one V-shaped piece of gla.s.s tubing, one arm 32 cm. in length, the other 52 cm., the shorter arm being plugged with cotton-wool. The rubber stopper must be sterilised by boiling and the gla.s.s tubing by hot air, before use.

Flask containing 250 c.c. sterile broth.

Test-tube brush to fit the lumen of the candle, enclosed in a sterile test-tube (and previously sterilised by dry heat or by boiling).

Case of sterile pipettes, 10 c.c. in tenths.

Case of sterile pipettes, 1 c.c. in tenths.

Case of sterile pipettes, 1 c.c. in hundredths.

Tubes of various nutrient media (according to requirements).

Twelve Buchner's tubes with rubber stoppers.

Pyrogallic acid tablets.

Caustic soda tablets.

[Ill.u.s.tration: Sample form]]

[Ill.u.s.tration: FIG. 209.--Water filtering apparatus. That portion of the figure to the left of the vertical line is drawn to a larger scale than that on the right, in order to show details of Sprengel's pump.]

METHOD.--

1. Fit up the filtering apparatus as in the accompanying diagram (Fig.

209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle.

[Ill.u.s.tration: FIG. 210. Sterile test-tube brush.]

2. Filter the entire 2000 c.c. of water through the filter candle.

3. When the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing.

4. Reverse the position of the gla.s.s tubes in the Wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid.

5. Slowly open the metal clamps and allow air to gradually pa.s.s through the acid, and enter filter flask, and so restore the pressure.

6. Unship the apparatus, remove the cork from the mouth of the candle.

7. Pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (Fig. 210) emulsify the slimy residue which lines the candle, with the broth.

Practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (Some bacteria will of course be left behind on the walls of the filter and in its pores.)

Up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria.

The Coli-Typhoid Group.--

1. Number nine tubes of bile salt broth (_vide_ page 180), consecutively from 1 to 9.

2. To No 1 add 1 c.c. } of the original water sample 2 add 2 c.c. } before the nitration is commenced.

3 add 5 c.c. }

3. To the remaining tubes of bile salt broth add varying quant.i.ties of the suspension by means of suitably graduated sterile pipettes, as follows:

No. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample).

No. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample).

No. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample).

No. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample).

No. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample).

No. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample).

4. Put up each tube anaerobically in a Buchner's tube and incubate at 42 C.

5. The subsequent steps are identical with those described under the Enrichment method (see page 428 to 431; Steps 8 to 18).

_Alternative Methods._--