Close the mouth of the bulb with a rubber stopper.

Alternately invert and revert the tube six or eight times, to bring the soda solution into intimate contact with the gas.

Return the residual gas to the end of the closed branch, and measure.

The loss in volume of gas = carbon dioxide.

The residual gas = hydrogen.

Transfer gas to the bulb of the tube, and explode it by applying a lighted taper.

(B) _Sulphuretted Hydrogen._--

_Media Required_:

Iron peptone solution (_vide_ page 185).

Lead peptone solution.

1. Inoculate tubes of media, and incubate together with control tubes.

2. Examine from day to day, at intervals of twenty-four hours.

The liberation of the H_{2}S will cause the yellowish-white precipitate to darken to a brownish-black, or jet black, the depth of the colour being proportionate to the amount of sulphuretted hydrogen present.

Quant.i.tative: For exact quant.i.tative a.n.a.lyses of the gases produced by bacteria from certain media of definite composition, the methods devised by Pakes must be employed, as follows:

[Ill.u.s.tration: FIG. 153.--Gas-collecting apparatus.]

_Apparatus Required_:

Bohemian flask (300 to 1500 c.c. capacity) containing from 100 to 400 c.c. of the medium. The mouth of the flask is fitted with a perforated rubber stopper, carrying an L-shaped piece of gla.s.s tubing (the short arm pa.s.sing just through the stopper). To the long arm of the tube is attached a piece of pressure tubing some 8 cm. in length, plugged at its free end with a piece of cotton-wool. Measure accurately the total capacity of the flask and exit tube, also the amount of medium contained. Note the difference.

Gas receiver. This is a bell jar of stout gla.s.s, 14 cm. high and 9 cm. in diameter. At its apex a gla.s.s tube is fused in.

This rises vertically 5 cm., and is then bent at right angles, the horizontal arm being 10 cm. in length. A three-way tap is let horizontally into the vertical tube just above its junction with the bell jar.

An iron cylinder just large enough to contain the bell jar.

About 15 kilos of metallic mercury.

Melted paraffin.

An Orsat-Lunge working with mercury instead of water, provided with two gas tubes of extra length (capacity 120 and 60 c.c. respectively and graduated throughout, both being water-jacketed) or other gas a.n.a.lysis apparatus, capable of dealing with CO_{2}, O_{2}, H_{2}, and N_{2}.

METHOD.--

1. Inoculate the medium in the flask in the usual manner, by means of a platinum needle, taking care that the neck of the flask and the rubber stopper are thoroughly flamed before and after the operation.

[Ill.u.s.tration: FIG. 154.--Orsat-Lunge gas a.n.a.lysis apparatus.]

2. Fill the iron cylinder with mercury.

3. Place the bell jar mouth downward in the mercury--first seeing that there is free communication between the interior of the jar and the external air--and suck up the mercury into the tap; then shut off the tap.

4. Plug the open end of the three-way tap with melted wax.

5. Connect up the horizontal arm of the culture flask with that of the gas receiver by means of the pressure tubing (after removing the cotton-wool plug from the rubber tube), as shown in Fig. 153.

6. Give the three-way tap half turn to open communication between flask and receiver, and seal _all_ joints by coating with a film of melted wax. When the tap is turned, the mercury in the receiver will naturally fall.

7. Place the entire apparatus in the incubator. (Two hours later, by which time the temperature of the apparatus is that of the incubator, mark the height of the mercury on the receiver.)

8. Examine the apparatus from day to day and mark the level of the mercury in the receiver at intervals of twenty-four hours.

9. When the evolution of gas has ceased, remove the apparatus from the incubator; clear out the wax from the nozzle of the three-way tap (first adjusting the tap so that no escape of gas shall take place) and connect it with the Orsat.

10. Remove, say, 100 c.c. of gas from the receiver, reverse the tap and force it into the culture flask. Remove 100 c.c. of mixed gases from the culture flask and replace in the receiver.

Repeat these processes three or four times to ensure thorough admixture of the contents of flask and receiver.

11. Now withdraw a sample of the mixed gases into the Orsat and a.n.a.lyse.

In calculating the results be careful to allow for the volume of air contained in the flask at the commencement of the experiment.

For the collection of gases formed under anaerobic conditions a slightly different procedure is adopted:

1. Fix a culture flask (500 c.c. capacity) with a perforated rubber stopper carrying an ~L~-shaped piece of manometer tubing, each arm 5 cm.

in length.

2. Prepare a second ~L~-shaped piece of tubing, the short arm 5 cm. and the long arm 20 cm., and connect its short arm to the horizontal arm of the tube in the culture flask by means of a length of pressure tubing, provided with a screw clamp.

3. Fill the culture flask completely with boiling medium and pa.s.s the long piece of tubing through the plug of an Erlenmeyer flask (150 c.c.

capacity) which contains 100 c.c. of the same medium.

4. Sterilise these coupled flasks by the discontinuous method, in the usual manner.

Immediately the last sterilisation is completed, screw up the clamp on the pressure tubing which connects them, and allow them to cool.

As the fluid cools and contracts it leaves a vacuum in the neck of the flask below the rubber stopper.

5. To inoculate the culture flask, withdraw the long arm of the bent tube from the Erlenmeyer flask and pa.s.s it to the bottom of a test-tube containing a young cultivation (in a fluid medium similar to that contained in the culture flask) of the organism it is desired to investigate.

6. Slightly release the clamp on the pressure tubing to allow 4 or 5 c.c. of the culture to enter the flask.

7. Clamp the rubber tube tightly; remove the bent gla.s.s tube from the culture tube and plunge it into a flask containing recently boiled and quickly cooled distilled water.

8. Release the clamp again and wash in the remains of the cultivation until the culture flask and tubing are completely filled with water.

9. Clamp the rubber tubing tightly and take away the long-armed gla.s.s tubing.