together. These cells form a new and special generation, which Will terms the first generation of true film cells, since with their appearance the formation of the film begins. In addition to these there quickly form other cells, which are characterised by considerably thicker membrane and an abundant content of glycogen and fat. These prove to be true permanent cells, both on account of their anatomical structure and their physiological behaviour, since they alone retain vitality and are capable of development when the cultures are old and all the other cells and the film have perished. The yeast ring, which grows about the same time, is specially rich in these permanent cells. They soon produce highly elongated, sausageshaped or tubular cells, which in turn behave in a similar manner and also produce analogous lateral daughter-cells, the result being the formation of many-membered chains of the kind shown in Fig. 132. Will terms the members of these bands, film cells of the second generation. The older the film the more luxuriant do they grow, and the farther do the film cells of the first generation retire to the background. At a later period, partition walls are formed-more or less abundantly in the different species-in these elongated cells; and similar septa are also found in the chains of buds resulting from the germination of the permanent cells in wort. A view of these is given in Fig. 139.

These chains of elongated cells partake of the character of an articulated mycelium. The capacity for producing such was first positively demonstrated by Hansen, and proves that the Saccharomycetes belong to the Mycomycetes, or Eumycetes with septate mycelia. Their position within this sub-kingdom was then, as already stated, indicated by their capacity for producing ascospores, which will be discussed in the next paragraph. In coloured nutrient media, such as beer wort and wine, the progress of film development is accompanied by a bleaching action, i.e. the disappearance of colouring matters. In this manner the colour of a wort can be slowly changed from dark brown to straw yellow.

The film cells also differ strongly in their chemico-physiological behaviour from those of the sedimental yeast. The latter still develop in presence of an extremely low oxygen tension, and devote their chief energy to the decomposition of sugars. On the other hand, the metabolism of the film cells is indissolubly connected with the presence of a copious supply of oxygen. According to the results of investigations conducted on this point by B. RAYMANN and K. KRUIS (I.), they oxidise, to carbon dioxide and water, the alcohol continuously formed in the fermenting underlying solution, and degrade the albuminoids therein to amides and ammoniun salts of organic acids. Formic acid and valerianic acid are also formed. Hence

in this case the fermentative action of the sedimental yeast is replaced by respirative activity.

When submerged in fresh nutrient solution, these film cells produce vegetations, which finally behave just like normal sedimental yeast. The rate of morphological change and adaptation of physiological character differ with the species of yeast. In some, as was ascertained by

H. WILL (VIII.), the characteristics appertaining to the film cells remain unimpaired in

the first new generations, and in specially conspicuous cases several recultivations (repeated tranferences of the crop to fresh nutrient solution) are required in order to produce a sedimental yeast equal in all respects to the original ancestors of the film cells used. Notice should also be taken, e.g. of an observation on this point by ED. KAYSER (VI.). Further consideration will be given in subsequent paragraphs to this behaviour, from the standpoint of the theory of variation.

To

FIG. 139.-Pair of Permanent Cells from the yeast ring of a six-months-old wort culture of Munich bottom-yeast No. 2, and germinated to a well-developed chain of buds in a drop of wort on a microscope slide in sixty-four hours at 10° C. A septum has formed inside three of the members of the chain. Nearly all the cells exhibit one or two vacuoles, and the two permanent cells (D) show an even larger number. Magn. 750. (After Will.)

At present we have to deal with the consequences connected with the practical cultivation of yeast, namely the restriction of film vegetation and the exclusion of cells derived therefrom. effect this object it is necessary to keep the stock yeast in the laboratory under such conditions as are unfavourable to the development of film, without being at the same time inimical to the sedimental yeast. The appearance of the former may be counteracted by frequent transferences of the cultures to fresh nutrient solutions, and by keeping the culture at low temperatures. According to HANSEN, the best storage medium for prolonged use is a 10 per cent. solution of saccharose. In this event, however, the sowing should not amount to more than a trace. When, from any cause, the only yeast culture available for fulfilling an order is one that is already covered with a film, the same is suitable for direct transference to the large repro

duction vessel, but must be first freshened up by preparing a re-inoculation, which in turn is used to inoculate a fresh nutrient solution as soon as development is in full swing. The operation is several times repeated, according to circumstances, until one is able to assume that the film cells present in the first inoculation of sedimental yeast have been entirely suppressed. The beginner cannot be too, strongly advised not to regard the task of yeast cultivation as completed by the preparation of the pure cultures, but rather to keep the latter under constant supervision, examination and care. Neglect of these precautions, and, in the case under consideration, the use of sedimental yeast containing film cells, may, under certain circumstances, lead to irregularities in the progress of fermentation on the large scale, diminution in the quality (flavour, &c.) of the product, and hence to unpleasant consequences for the yeast cultivator. Cases in point have been recorded by A. JOERGENSEN (VII.).

This, however, must not be held to imply that the film cells are the cause of all the unwelcome alterations that may appear in beer yeasts. On the contrary, other forces are here in operation; and from this side also, as already mentioned, we arrive at the wide field of variation in the yeast cell, a domain in which, as will be shown in a later chapter, Hansen, by his extensive experimental researches, has been our pioneer. Moreover, it should be recalled that RAYMANN and KRUIS (I.) were able, by means of yeast derived from old film cells, to produce good beer that could not be distinguished from that obtained by the aid of normal yeast. This harmonises with the results obtained by Alb. Klocker (privately communicated to the author) with Carlsberg bottom-yeast No. I. and No. II., Sacch. cerevisiæ I. Hansen, Marienthal yeast, and Will's No. 2 stock yeast.

Will's observations bring to mind the flying yeast (Flughefe) so dreaded by the brewer, i.e. yeast cells which are of smaller size than those of the sedimental yeast, and, instead of settling, continue to swim in the beer, and thus retard clarification. This presumptive relation has not yet been more closely investigated, but the researches of Hansen and others have placed beyond doubt that this phenomenon is in many cases attributable to the presence of wild yeasts. Another point that requires closer examination is the part played, in the maturing of beer, by the film cells produced within the liquid. Finally, investigation from this point of view is also desirable on the problem of the cause of flocculence in the process of making pressed yeast by the new, so-called aeration or wort process, which differs chiefly from the Viennese method (§ 255) in the thick mash being replaced by a clear mash as nutrient medium; this, after pitching, being well roused by aeration, whereby the reproduc

tion of the cells is strongly stimulated, accelerated, and increased. When fermentation is at an end the contents of the fermenting vessel are cooled and drawn off into large, flat clarifying pans, where the yeast crop settles down, and, after the removal of the supernatant liquid for distillation, is washed with water and finally forced into filter presses, where it is brought into saleable condition. Occasionally the deposition of the yeast crop in the clarifying pans, and therefore its separation from the liquid, is obstructed by a so-called flocculence, which is characterised by the continued re-ascension of flocculent aggregations of cells from the deposit. The phenomenon has been described by STENGLEIN and JOERRES in "Alcohol (1892, p. 218), and also by O. DURST (I.).

§ 247.-The Ascospores.

The first observation of the ascospores in yeast cells was made by TH. SCHWANN (II.). He pointed out that these fungi reproduce in two ways: one being by the process known as budding, and the other by the formation, within the parent-cell, of daughter-cells, which are liberated when the membrane of the parent-cell opens. After this phenomenon had been described more closely by J. DE SEYNES (I.) in 1868, it was also observed a year later by M. REESS (III.) in cultures of beer yeast on boiled sections of carrots, &c. He found the process of development coincide with that of certain low Ascomycetes, and therefore classed these forms as ascospores, calling the mother-cells asci, and for this reason relegating the Saccharomycetes (in 1870) to the position of the lowest family among the Ascomycetes.

The earliest accurate investigations into the conditions under which this sporulation occurs were carried on by E. CHR. HANSEN (XII.), and, apart from the general biological results, led to the important fact that we have here a reliable means, hitherto lacking, of separating the genus Saccharomyces into its species.

The conditions influencing the production of ascospores in the Saccharomycetes are given below: (1) To obtain energetic sporulation, it is necessary that the sample should consist of young and well-nourished cells. (2) The supply of air must be abundant. (3) The medium must be moist. (4) The temperature of the environment must be maintained within certain limits. (5) Within these limits the time required for the occurrence of sporulation is a function of the temperature. (6) Between the two extreme limits at which sporulation is still possible is an optimum temperature corresponding to a time minimum. The maximum temperature for sporulation is some

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what lower, and the minimum temperature rather higher, than for the phenomenon of budding.

To examine the individual conditions more closely. That the time within which sporulation occurs should be a function of the temperature, requires no further analysis; but careful attention should be bestowed on the point (in 1) as to the condition of the cells, this being the prime factor determining the time limit. The time required for ascospores to be developed by any given species of Saccharomyces, kept at any given temperature, differs according to the physiological condition of the cells themselves. Hence, if it be desired to produce sporulation (unconditionally) in any given species, all that is necessary is to take cells that are in vigorous condition—a state attainable by repeated preliminary transferences into fresh nutrient solution. The case is, however, different when it is a question of determining the time required for sporulation to make its appearance at one or another temperature. In such event, it must be borne in mind that this time limit is a function, not merely of the temperature, but also of the physiological condition of the species under examination; consequently this latter factor must be eliminated in order to enable the influence of the former to be determined. Experience has shown that sporulation occurs earliest and most certainly when the cells have reached the culminating point of their reproductive (budding) and fermentative activity; and it is therefore in this condition that they should be employed for the experiment in question. On this account the cells to be examined for the time limit of sporulation should be subjected to the following preliminary treatment: the sample is sowed in sterilised beer wort and left to stand for several days at room temperature, Pasteur flasks being the best vessels for the purpose. A portion of the resulting sedimental yeast is transferred to fresh sterile beer wort and kept therein for twenty-four hours at 25° C., the fresh deposit being afterwards freed as carefully as possible from the supernatant liquid, and employed for starting the spore cultures.

One example will suffice to show the necessity of taking the condition of the cells into consideration. It is afforded by Hansen's experience, and relates to Sacch. Pastorianus I. The culture was first conducted for a few days at room temperature, after which the sedimental yeast was retransferred, in the above manner, to two flasks, one of which was kept for twenty-four hours, the other for forty-eight hours, at 26° to 27° C. before starting the spore cultures. The following figures show the time required for the commencement of sporulation in the two

cases: