In such experiments we encounter another difficulty. If the yeast sown in the non-aerated fermentable liquid is in the least degree impure, especially if we use sweetened yeast-water, we may be sure that alcoholic fermentation will soon cease, if, indeed, it ever commences, and that accessory fermentations will go on. The vibrios of butyric fermentation, for instance, will propagate with remarkable facility under these circumstances. Clearly then, the purity of the yeast at the moment of impregnation, and the purity of the liquid in the funnel, are conditions indispensable to success. To secure the latter of these conditions, we close the funnel, as shown in Fig. 60, by means of a cork pierced with two holes, through one of which a short tube passes, to which a short length of india-rubber tubing provided with a glass stopper is attached; through the other hole a thin curved tube is passed. Thus fitted, the funnel can answer the same purposes as our double-necked flasks. A few cubic centimetres of sweetened yeast-water are then put in it and boiled, so that the steam may destroy any germs adhering to the sides. When cold the liquid is impregnated by means of a trace of pure yeast, introduced through the glass-stoppered tube. If these precautions are neglected it is scarcely possible to secure a successful fermentation in our flasks, because the yeast sown is immediately held in check by a development of anaërobian vibrios. For greater security, we may add to the fermentable liquid, at the moment when it is prepared, a very small quantity of tartaric acid, which will prevent the development of butyric vibrios. [Illustration: Fig. 62.] The variation of the ratio between the weight of the yeast and that of the sugar decomposed by it now claims special attention. Side by side with the experiments which we have just described, we conducted a third lot by means of the flask C (Fig. 62), holding 4·7 litres (8-1/4 pints), and fitted up like the usual two-necked flasks, with the object of freeing the fermentable liquid from foreign germs, by boiling it to begin with, so that we might carry on our work under conditions of purity. The volume of yeast-water (containing 5 per cent. of sugar) was only 200 c.c. (7 fl. oz.), and consequently, taking into account the capacity of the flask, it formed but a very thin layer at the bottom. On the day after impregnation the deposit of yeast was already considerable, and forty-eight hours afterwards the fermentation was completed. On the third day we collected the yeast, after having analyzed the gas contained in the flask. This analysis was easily accomplished by placing the flask in a hot-water bath, whilst the end of the curved tube was plunged under a cylinder of mercury. The gas contained 41·4 per cent. of carbonic acid, and, after the absorption, the remaining air contained—