colonies or islets of yeast which have originated on the surface of the liquid itself. Other conditions being equal, the dimensions attained by the film vary according to the species of yeast.

The form of the cells from which the film is constructed differs in general from that of the sedimental cells, by attaining greater longitudinal development (up to 150 μ and over), whilst the transverse measurement is often less than in the cells of sedimental yeast. The second characteristic is a more or less abundant branching. An example is shown in Fig. 132.

The time required for the development of the film to become manifest differs,

other conditions being equal, with the species of the yeast, and is longer the lower the temperature of the culture. According to a series of determinations made on species of Saccharomyces by HANSEN (XVI.), the time required for Saccharomyces cerevisia I. at 33° to 34° C. is about 9 to 18 days; at 20 to 28° C., about 7 to II days; at 13° to 15° C.,

FIG. 132.-Saccharomyces ellipsoideus I. Hansen.
Cells and chains of cells from the film of an old culture

about 15 to 30 days; in beer wort. Magn. 1000. (After Hansen.)

at 6° to 7° C., about 2 to 3 months. occurred above 34° C. or below 5° C.

No formation of film

These limits of temperature, which also apply to all the other species examined, are therefore more restricted than those wherein the yeast is able to bud and incite fermentation. Film formation is therefore precluded in the fermenting cellars of bottom-fermentation breweries, the temperature here being, wherever possible, maintained between o° and 2° C. It has already been shown by Hansen, and confirmed by H. WILL (VIII.), R. ADERHOLD (I.), and other workers, that the time elapsing before the film makes its appearance, and the dimen

Cells of film grown on beer wort at 20° to 28° C. Magn. 1000. (After Hansen.)

sions attained by the film, are very greatly dependent on the conditions of cultivation (composition of the nutrient solution, and also in a high degree on the method of sterilisation, supply of air, &c.).

In some cases morphological peculiarities in the film cells afford a means for differentiating the species. With Sacch. Past. II. and Sacch. Past. III. this can be recognised in a beautiful manner, and at the same time a fresh instance is afforded of the dependence of cell form on temperature. The first-named, weak, harmless, top-fermentation species was isolated by Hansen from the air of the Carlsberg brewery. The other, which is of decidedly top-fermentation character, was obtained as a pure culture from a Copenhagen lager-beer suffering from haze, and was recognised as the cause of the malady. It is difficult to distinguish between them by the form of the cells present in the sedimental yeast, both being very similar to Sacch. Past. I. (see Fig. 129). The same also applies to the component members of their films grown at 20° to 28° C. and illustrated in Figs. 133 and 134. The case is, however,

different-as HANSEN (XVI.) has shown-when the two have been allowed to form films at 13° to 15° C., since under these con

Cells in film grown on beer wort at 18° to 15° C. Magn. 1000. (After Hansen.)

ditions one of them, Sacch. Past. III., exhibits a number of highly elongated cells (Fig. 135) with lateral buds, whilst those of Sacch. Past. II.,

on the other hand, retain very much the same form as when grown at 20° to 28° C., i.e. only slightly elongated, and even globular. An essential precaution for this comparison is the use of young films, since when the films are very old the No. II. species exhibits elongated cells just like those of No. III. Similar conditions in this respect obtain between Sacch. ellipsoideus I. and Sacch. ellipsoideus II. Hansen.

FIG. 136.-Saccharomyces Past. II. Hansen.

Cells in film grown on beer wort at 13° to 15° C. Magn. 1000. (After Hansen.)

The faculty of developing a film on the surface of suitable nutrient solutions is shared by nearly all the budding fungi,

both Saccharomycetes and non-Saccharomycetes. Of these latter we shall deal (in the last section), under the generic name Mycoderma, with a special group, the species of which are widely distributed and grow spontaneously on the surface of wine or beer when the latter are exposed to the air, a quick-growing, wrinkled skin (mould film) being formed. Air being highly essential to Mycoderma, these organisms normally grow exclusively in the form of a superficial film on the nutrient solution. Owing to this peculiarity they are the cause of disturbance in researches into film-formation in the true yeasts, when the latter are not grown as pure cultures but contaminated with the very abundant Mycoderma. On this account objection may be raised against the reports of Reess (the first to observe the production of films by true Saccharomycetes), and also those of PASTEUR (III.). In 1876 the latter characterised as ærobic or mould yeasts the film developing on the surface of fully fermented wort. He halted between two opinions with respect to this phenomenon, one being that it was a special (i.e. ærobic) condition of development of the beer yeast residing at the bottom of the fermented liquid; whilst the other looked on the film as composed of extraneous cells undesirably present with the sowing. It was not until Hansen applied pure cultures to these investigations that a decision could be formed on this point.

The question of the convertibility of bottom-fermentation yeast into top-fermentation yeast, and vice versa, is also touched by the foregoing explanations. Pasteur was of opinion that the "robic" cells constituting the film that had developed it at the close of primary fermentation in his cultures inoculated with (impure) bottom-fermentation yeast, were capable of exciting top-fermentation when transferred to a fresh. nutrient solution. He even gave a recipe by means of which the brewer could prevent any such undesirable conversion of the stock yeast. This theoretically and practically important question was afterwards taken up by Hansen, who found that the descendants of the films of all the species of bottom yeasts examined by him in this connection, invariably produced nothing but bottom-yeast cells when inoculated into fresh nutrient solutions, even when exposed to a temperature (26° C.) very favourable to the progress of top-fermentation.

The true Saccharomycetes can be separated-though not very sharply-into two groups: one of which does not form films until the primary fermentation is terminated and the sedimental yeast has all come down; whilst in the other group growth proceeds on the surface from the commencement, and indeed in many cases exclusively so. An instance of this latter kind is afforded by the Saccharomyces membranæfaciens, first discovered by E. CHR. HANSEN (VIII.) in the mucinous

discharge (248) from elm, and later in well-water by J. KOEHLER (I.).

The separate stages of the development of film were closely investigated in four species of bottom-fermentation yeast by H. WILL (VIII.). At the outset no difference can be detected between the cells of the sedimental yeast and those retained floating on the surface by flakes of albumin and residual fragments of the "head," and from which the development of the

FIG. 137.-Cells of sedimental FIG. 138.-Film cells of the first yeast from a wort culture generation, and permanent cells offrom the film of a wort culture ofBottom-fermentation beer yeast No. 93 of the Munich Brewing Station. Magn. 750. (After Will.)

yeast islets originates. Later on, however, it is observed that these floating cells produce daughter-cells, the chief feature of which as may be seen from a comparison of Figs. 137 and 138-is that, instead of appearing singly or in pairs on the mother-cell, as they do in the sedimental yeast, a number are formed simultaneously thereon. They are also much smaller (e.g. only 7 μ as compared with 10 μ) than those in the sedimental yeast, are oval or sausage-shaped, and in turn produce similar daughter - cells, the whole remaining connected