_Reagents Required_: Sulphuric acid (25 per cent.). Metaphenylene diamine, 5 per cent. aqueous solution. METHOD.-- 1. Prepare tube cultivations and incubate together with control tubes (i. e., uninoculated tubes of the same medium, placed under identical conditions as to environment). This precaution is necessary as the medium is liable to take up nitrites from the atmosphere, and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube. Test both the culture tube and the control tube for the presence of nitrites. 2. Add a few drops of sulphuric acid to the medium in each of the tubes. 3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube. Brownish-red colour = nitrites. The depth of colour is proportionate to the amount present. ~8. Gas Production.~-- (A) _Carbon Dioxide and Hydrogen._-- _Apparatus Required_: Fermentation tubes (_vide_ page 161) containing sugar bouillon (glucose, lactose, etc.). The medium should be prepared from inosite-free bouillon (_vide_ page 183). _Reagent Required_: n/2 caustic soda. METHOD.-- 1. Inoculate the surface of the medium in the bulb of a fermentation tube and incubate. 2. Mark the level of the fluid in the closed branch of the fermentation tube, at intervals of twenty-four hours, and when the evolution of gas has ceased, measure the length of the column of gas with the millimetre scale. Express this column of gas as a percentage of the entire length of the closed branch. 3. To analyse the gas and to determine roughly the relative proportions of CO_{2} and H_{2}, proceed as follows: Fill the bulb of the fermentation tube with caustic soda solution. 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. Quantitative: For exact quantitative analyses of the gases produced by bacteria from certain media of definite composition, the methods devised by Pakes must be employed, as follows: [Illustration: FIG. 153.--Gas-collecting apparatus.] _Apparatus Required_: