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 was by no means the case, for it is more difficult to separate the cells from each other in the gelatin than in the liquid. To obtain an absolutely pure culture with certainty it is necessary, even when the gelatin method is employed, to start from a single cell. To effect this some of the nutrient gelatin containing yeast cells is placed on the under-surface of the cover-glass of the moist chamber. Those cells are accurately marked, the position of which is such that the colonies, to which they give rise, can grow to their full size without coming into contact with other colonies. The growth of the marked cells is kept under observation for three or four days, by which time the colonies will be large enough to be taken out of the chamber and placed in flasks. The contents of the flasks can then be introduced into larger flasks, and finally into an apparatus suitable for making enough yeast for technical purposes. Such, in brief, are the methods devised by that brilliant investigator Hansen; and these methods have not only been the basis on which our modern knowledge of the Saccharomycetes is founded, but are the only means of attack which the present-day observer has at his disposal.

From the foregoing it will be seen that the term fermentation has now a much wider significance than when it was applied to such changes as the decomposition of must or wort with the production of carbon dioxide and alcohol. Fermentation now includes all changes in organic compounds brought about by ferments elaborated in the living animal or vegetable cell. There are two distinct types of fermentation: (1) those brought about by living organisms (organized ferments), and (2) those brought about by non-living or unorganized ferments (enzymes). The first class include such changes as the alcoholic fermentation of sugar solutions, the acetic acid fermentation of alcohol, the lactic acid fermentation of milk sugar, and the putrefaction of animal and vegetable nitrogenous matter. The second class include all changes brought about by the agency of enzymes, such as the action of diastase on starch, invertase on cane sugar, glucase on maltose, &c. The actions are essentially hydrolytic.

Biological Aspect of Yeast.—The Saccharomycetes belong to that division of the Thallophyta called the Hyphomycetes or (q.v.). Two great divisions are recognized in the Fungi: (i.) the Phycomycetes or Algal Fungi, which retain a definitely sexual method of reproduction as well as asexual (vegetative) methods, and (ii.) the Mycomycetes, characterized by extremely reduced or very doubtful sexual reproduction. The Mycomycetes may be divided as follows: (A) forms bearing both sporangia and conidia (see ), (B) forms bearing conidia only, e.g. the common mushroom. Division A comprises (a) the true Ascomycetes, of which the moulds Eurotium and Penicillium are examples, and (b) the Hemiasci, which includes the yeasts. The gradual disappearance of the sexual method of reproduction, as we pass upwards in the fungi from the points of their departure from the Algae, is an important fact, the last traces of sexuality apparently disappearing in the ascomycetes.

With certain rare exceptions the Saccharomycetes have three methods of asexual reproduction:—

1. The most common.—The formation of buds which separate to form new cells. A portion of the nucleus of the parent cell makes its way through the extremely narrow neck into the daughter cell. This method obtains when yeast is vigorously fermenting a saccharine solution.

2. A division by fission followed by Endogenous spore formation, characteristic of the Schizosaccharomycetes. Some species show fermentative power.

3. Endospore formation, the conditions for which are as follows: (1) suitable temperature, (2) presence of air, (3) presence of moisture, (4) young and vigorous cells, (5) a food supply in the case of one species at least is necessary, and is in no case prejudicial. In some cases a sexual act would appear to precede spore formation. In most cases four spores are formed within the cell by free formation. These may readily be seen after appropriate staining.

In some of the true Ascomycetes, such as Penicillium glaucum, the conidia if grown in saccharine solutions, which they have the power of fermenting, develop single cell yeast-like forms, and do not—at any rate for a time—produce again the characteristic branching mycelium. This is known as the Torula condition. It is supposed by some that Saccharomyces is a very degraded Ascomycete, in which the Torula condition has become fixed.

The yeast plant and its allies are saprophytes and form no chlorophyll. Their extreme reduction in form and loss of sexuality may be correlated with the saprophytic habit, the proteids and other organic material required for the growth and reproduction being appropriated ready synthesized, the plant having entirely lost the power of forming them for itself, as evidenced by the absence of chlorophyll. The beer yeast S. cerevisiae, is never found wild, but the wine yeasts occur abundantly in the soil of vineyards, and so are always present on the fruit, ready to ferment the expressed juice.

Chemical Aspect of Alcoholic Fermentation.—Lavoisier was the first investigator to study fermentation from a quantitative standpoint. He determined the percentages of carbon, hydrogen and oxygen in the sugar and in the products of fermentation, and concluded that sugar in fermenting breaks up into alcohol, carbonic acid and acetic acid. The elementary composition of sugar and alcohol was fixed in 1815 by analyses made by Gay-Lussac, Thénard and de Saussure. The first-mentioned chemist proposed the following formula to represent the change which takes place when sugar is fermented:— This formula substantially holds good to the present day, although a number of definite bodies other than carbon dioxide and alcohol occur in small and varying quantities, according to the conditions of the fermentation and the medium fermented. Prominent among these are glycerin and succinic acid. In this connexion Pasteur showed that 100 parts of cane sugar on inversion gave 105.4 parts of invert sugar, which, when fermented, yielded 51.1 parts alcohol, 49.4 carbonic acid, 0.7 succinic acid, 3.2 glycerin and 1.0 unestimated. A. Béchamp and E. Duclaux found that acetic acid is formed in small quantities during fermentation; aldehyde has also been detected. The higher alcohols such as propyl, isobutyl, amyl, capryl, oenanthyl and caproyl, have been identified; and the amount of these vary according to the different conditions of the fermentation. A number of esters are also produced. The characteristic flavour and odour of wines and spirits is dependent on the proportion of higher alcohols, aldehydes and esters which may be produced.

Certain yeasts exercise a reducing action, forming sulphuretted hydrogen, when sulphur is present. The “stinking fermentations” occasionally experienced in breweries probably arise from this, the free sulphur being derived from the hops. Other yeasts are stated to form sulphurous acid in must and wort. Another fact of considerable technical importance is, that the various races of yeast show considerable differences in the amount and proportion of fermentation products other than ethyl alcohol and carbonic acid which they produce. From these remarks it will be clear that to employ the most suitable kind of yeast for a given alcoholic fermentation is of fundamental importance in certain industries. It is beyond the scope of the present article to attempt to describe the different forms of budding fungi (Saccharomyces), mould fungi and bacteria which are capable of fermenting sugar solutions. Thus, six species isolated by Hansen, Saccharomyces cerevisiae, S. Pasteurianus I., II., III., and S. ellipsoideus, contained invertase and maltase, and can invert and subsequently ferment cane sugar and maltose. S. exiguus and S. Ludwigii contain only invertase and not maltase, and therefore ferment cane sugar but not maltose. S. apiculatus (a common wine yeast) contains neither of these enzymes, and only ferments solutions of glucose or laevulose.

Previously to Hansen’s work the only way of differentiating