2. Fix up the filtering apparatus as for the qualitative examination of water, and filter the soil water.

3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique similar to that described for the water sample, _vide_ pages 434-436).

Every cubic centimetre of suspension now contains the soil water from nearly 1 gramme of earth.

The methods up to this point are identical no matter which organism or group of organisms it is desired to isolate; but from this stage onward the process is varied slightly for each particular bacterium.

~I. The Coli-typhoid Group.~--

~II. Streptococci.~--

~III. Bacillus Anthracis.~--

~IV. Bacillus Tetani.~--

The methods adopted for the isolation of these organisms are identical with those already described under water (page 437 _et seq._).

~V. Bacillus Oedematis Maligni.~--Method precisely similar to that employed for the B. tetani.

~VI. The Nitrous Organisms.~--

1. Take ten tubes of Winogradsky's solution No I (_vide_ page 198) and number them consecutively from 1 to 10.

2. Inoculate each tube with varying quant.i.ties of the material as follows:

To tube No. 1 add 1.0 c.c. of the soil water.

To tube No. 2 add 0.1 c.c. of the soil water.

To tube No. 3 add 1.0 c.c. from Capsule I.

To tube No. 4 add 0.1 c.c. from Capsule I.

To tube No. 5 add 1.0 c.c. from Capsule II.

To tube No. 6 add 0.1 c.c. from Capsule II.

To tube No. 7 add 1.0 c.c. from Capsule III.

To tube No. 8 add 0.1 c.c. from Capsule III.

To tube No. 9 add 1.0 c.c. from Capsule IV.

To tube No. 10 add 0.1 c.c. from Capsule IV.

Label and incubate at 30 C.

~VII. The Nitric Organisms.~--

3. Take ten tubes of Winogradsky's solution No II, number them consecutively from 1 to 10 and inoculate with quant.i.ties of soil water similar to those enumerated in section VI step 2. Label and incubate at 30 C.

4. Examine after twenty-four and forty-eight hours' incubation. From those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at 30 C.

5. Make further subcultivations from such of those tubes as show growth, and again incubate.

6. If growth occurs in these subcultures, make surface smears on plates of Winogradsky's silicate jelly (_vide_ page 198).

7. Pick off such colonies as make their appearance and subcultivate in each of these two media.

TESTING FILTERS.

Porcelain filter candles are examined with reference to their power of holding back _all_ the micro-organisms suspended in the fluids which are filtered through them, and permitting only the pa.s.sage of germ-free filtrates. In order to determine the freedom of the filter from flaws and cracks which would permit the pa.s.sage of bacteria no matter how perfect the general structure of the candle might be, the candle must first be attached by means of a long piece of pressure tubing, to a powerful pump, such as a foot bicycle pump, fitted with a manometer. The candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump. Any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it.

The examination for permeability is conducted as follows:

_Apparatus Required_:

Filtering apparatus: The actual filter candle that is used must be the one it is intended to test and must be previously carefully sterilised; the arrangement of the apparatus will naturally vary with each different form of filter, one or other of those already described (_vide_ pages 42-48).

Plate-levelling stand.

Case of sterile plates.

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

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

Tubes of nutrient gelatine.

Flask containing sterile normal saline solution.

Sterile measuring flask, 1000 c.c. capacity.

METHOD.--

1. Prepare surface cultivations, on nutrient agar in a culture bottle, of the Bacillus mycoides, and incubate at 20 C., for forty-eight hours.

2. Pipette 5 c.c. sterile normal saline into the culture bottle and emulsify the entire surface growth in it.

3. Pipette the emulsion into the sterile measuring flask and dilute up to 1000 c.c. by the addition of sterile water.

4. Pour the emulsion into the filter reservoir and start the filtration.

5. When the filtration is completed, pour six agar plates each containing 1 c.c. of the filtrate.

6. Incubate at 37 C. until, if necessary, the completion of seven days.

7. If the filtrate is not sterile, subcultivate the organism pa.s.sed and determine its ident.i.ty with the test bacterium before rejecting the filter--since the filtrate may have been accidentally contaminated.

8. If the filtrate is sterile, resterilise the candle and repeat the test now subst.i.tuting a cultivation of B. prodigiosus--a bacillus of smaller size.

9. If the second test is satisfactory, test the candle against a cultivation of a very small coccus, e. g., Micrococcus melitensis, in a similar manner; in this instance continuing the incubation of cultivations from the filtrate for fourteen days.

TESTING OF DISINFECTANTS.