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though variable, yet, as estimated by weight of product formed, is out of all proportion to the weight of their own substance. These are facts of so great importance, and so intimately connected with the theory of fermentation, that it is indispensable to endeavour to establish them experimentally, with all the exactness of which they will admit.

The question before us is whether yeast is in reality an anaërobian plant, and what quantities of sugar it may cause to ferment, under the various conditions under which we cause it to act.

The following experiments were undertaken to solve this double problem:-We took a double-necked flask, of three litres (five pints) capacity, one of the tubes being curved and forming an escape for the gas; the other one, on the right hand side (FIG. 1), being furnished with a glass tap. We filled this flask with pure yeast water, sweetened with 5 per cent. of sugar candy, the flask being so full that there was not the least trace of air remaining above the tap or in the escape tube; this artificial wort had, however, been itself aerated. The curved tube was plunged in a porcelain vessel full of mercury, resting on a firm support. In the small cylindrical funnel above the tap, the capacity of which was from 10 cc. to 15 cc. (about half a fluid ounce) we caused to ferment, at a temperature of 20° or 25° C. (about 75° F.), five or six cubic centimetres of the saccharine liquid, by means of a trace of yeast, which multiplied rapidly, causing fermentation, and forming a slight deposit of yeast at the bottom of the funnel above the tap. We then opened the tap, and some of the liquid in the funnel entered the flask, carrying with it the small deposit of yeast, which was sufficient to impregnate the saccharine liquid contained in the flask. In this manner it is possible to introduce as small a quantity of yeast as we wish, a quantity the weight of which, we may say, is hardly appreciable. The yeast sown multiplies rapidly and produces fermentation, the carbonic gas from which is expelled into the mercury. In less than twelve days all the sugar had disappeared, and the fermentation had finished. There was a sensible deposit of yeast adhering to the sides of the flask; collected and dried

2 Capable of living without free oxygen-a term invented by Pasteur.-ED.

it weighed 2.25 grammes (34 grains). It is evident that in this experiment the total amount of yeast formed, if it required oxygen to enable it to live, could not have absorbed, at most, more than the volume which was originally held

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FIG. I

in solution in the saccharine liquid, when that was exposed to the air before being introduced into the flask.

Some exact experiments conducted by M. Raulin in our laboratory have established the fact that saccharine worts, like water, soon become saturated when shaken briskly with an excess of air, and also that they always take into solution a little less air than saturated pure water contains under the same conditions of temperature and pressure. At a temperature of 25° C. (77° F.), therefore, if we adopt the coefficient of the solubility of oxygen in water given in Bunsen's tables, we find that I litre (134 pints) of water saturated with air contains 5.5 cc. (0.3 cubic inch) of oxygen. The three litres of yeast-water in the flask, supposing it to have been saturated, contains less than 16.5 cc. (I cubic inch) of oxygen, or, in weight, less than 23 milligrammes (0.35 grains). This

was the maximum amount of oxygen, supposing the greatest possible quantity to have been absorbed, that was required by the yeast formed in the fermentation of 150 grammes (4.8 Troy ounces) of sugar. We shall better understand the significance of this result later on. Let us repeat the fore

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going experiment, but under altered conditions. Let us fill, as before, our flask with sweetened yeast-water, but let this first be boiled, so as to expel all the air it contains. To affect this we arrange our apparatus as represented in the accompanying sketch. (FIG. 2.) We place our flask, A, on a tripod above a gas flame, and in place of the vessel of mercury substitute a porcelain dish, under which we can put a gas flame, and which contains some fermentable, saccharine liquid, similar to that with which the flask is filled. We boil the liquid in the flask and that in the basin simultaneously, and then let them cool down together, so that as the liquid in the flask cools some of the liquid is sucked from the basin into the flask. From a trial experiment which we

conducted, determining the quantity of oxygen that remained in solution in the liquid after cooling, according to M. Schützenberger's valuable method, by means of hydrosulphite of soda, we found that the three litres in the flask, treated as we have described, contained less than one milligramme (0.015 grain) of oxygen. At the same time we conducted another experiment, by way of comparison (FIG. 3). We took a flask, B, of larger capacity than the former one, which we filled about half with the same volume as before of a saccharine liquid of identically the same composition. This liquid had been previously freed from alterative germs by boiling. In the funnel surmounting A, we put a few cubic centimetres of saccharine liquid in a state of fermentation, and when this small quantity of liquid was in full fermentation, and the yeast in it was young and vigorous, we opened the tap, closing it again immediately, so that a little of the liquid and yeast still remained in the funnel. By this means we caused the liquid in A to ferment. We also impregnated the liquid in B with some yeast taken from the funnel of A. We then replaced the porcelain dish in which the curved escape tube of A had been plunged, by a vessel filled with mercury. The following is a description of two of these comparative fermentations and the results they gave. The fermentable liquid was composed of yeast-water sweetened with 5 per cent. of sugar-candy; the ferment employed was sacchormyces pastorianus.

FIG. 3

B

The impregnation took place on January 20th. The flasks were placed in an oven at 25° (77° F.).

Flask A, without air.

January 21st-Fermentation commenced; a little frothy liquid issued from the escape tube and covered the mercury.

NaHSO,, now called Sodium hyposulphite.-D. C. R.

The following days, fermentation was active.

Examining the yeast mixed with the froth that was expelled into the mercury by the evolution of carbonic acid gas, we find that it was very fine, young, and actively budding.

February 3rd.-Fermentation still continued, showing itself by a number of little bubbles rising from the bottom of the liquid, which had settled bright. The yeast was at the bottom in the form of

a deposit.

February 7th.-Fermentation still continued, but very languidly. February 9th.-A very languid fermentation still went on, discernible in little bubbles rising from the bottom of the flask.

Flask B, with air.

January 21st.-A sensible development of yeast.

The following days, fermentation was active, and there was an abundant froth on the surface of the liquid.

February 1st.-All symptoms of fermentation had ceased.

As the fermentation in A would have continued a long time, being so very languid, and as that in B had been finished for several days, we brought to a close our two experiments on February 9th. To do this we poured off the liquids in A and B, collecting the yeasts on tared filters. Filtration was an easy matter, more especially in the case of A. Examining the yeasts under the microscope, immediately after decantation, we found that both of them remained very pure. The yeast in A was in little clusters, the globules of which were collected together, and appeared by their well-defined borders to be ready for an easy revival in contact with air.

As might have been expected, the liquid in flask B did not contain the least trace of sugar; that in the flask A still contained some, as was evident from the non-completion of fermentation, but not more than 4.6 grammes (71 grains). Now, as each flask originally contained three litres of liquid holding in solution 5 per cent. of sugar, it follows that 150 grammes (2,310 grains) of sugar had fermented in the flask B, and 145.4 grammes (2,239.2 grains) in the flask A. The weights of yeast after drying at 100° C. (212° F.) were—

For the flask B, with air....1,970 grammes (30.4 grains).
For the flask A, without air..1,368 grammes".

This appears to be a misprint for 1.638 grammes=25.3 grains.—D. C. R

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