The method which has been adopted, therefore, is to employ either a secondary phosphate saturated with carbon dioxide at the temperature of the experiment, or a mixture of five molecular proportions of the secondary phosphate with one molecular proportion of a primary phosphate, in which the amount of bicarbonate formed is negligible. In the former case it is necessary to ascertain whether any of the carbon dioxide evolved is derived from the bicarbonate by the action of acid originally present or produced in the yeast-juice or by a disturbance of the original equilibrium owing to the chemical change which occurs. This is done by acidifying duplicate samples with hydrochloric acid before and after the fermentation and measuring the gas evolved in each case. Any necessary correction can then be made. The calculation of the extra amount of carbon dioxide evolved from yeast-juice containing sugar when a phosphate is added involves an estimation of the amount which would have been evolved in the absence of added phosphate, and this is a matter of some difficulty. Since the final steady rate of fermentation attained is often slightly different from the initial rate, the practice has been adopted of ascertaining this final rate and then calculating the total evolution corresponding to it for the whole period from the time of the addition of the phosphate to the end of the observations. This amount deducted from the observed total leaves the extra amount of carbon dioxide formed, and it is this quantity which is equivalent to the phosphate added. Alcohol is simultaneously produced in the normal ratio. The justification for this method of calculation will be found later (p. 54). The following table, containing the results of experiments with [Pg 044] glucose, fructose, and mannose, indicates very clearly the nature of the method of calculation and also of the agreement between observation and theory. Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing 1 gram of the sugar to be examined (a large excess) were incubated with toluene at 25° for one hour, in order to remove all free phosphate, and to each were then added 5 c.c. of a solution of sodium phosphate corresponding to 0·1632 gram of Mg{2}P{2}O{7} and equivalent to 32·6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were then observed until they had passed through the period of acceleration and had fallen and attained a steady value, the gases being measured moist at 19·3° and 760·15 mm.