ends, and in some species slightly constricted in one or two places. Cells of this kind were noticed by PASTEUR (XII.) during his researches on wine. They were also found by REESS (I.) in the secondary fermentation of certain wines examined by him; and this worker named them Sacch. Pastorianus in honour of the French scientist. Cells of this type have been more frequently observed by subsequent investigators, and this specific name has been gradually modified into a morphological term. When it is said of a yeast that it exhibits pastorianus forms, or is of the Pastorianus type, the term merely implies that under normal conditions the sedimental yeast of the species in question chiefly forms cells that are sausage-shaped, and not globular, oval, or elliptical. An example of this kind is afforded by Sacch. Pastorianus I., illustrated in Fig. 129. This species was discovered by HANSEN (II. and XII.), in 1880 and 1881, in the air at the Alt-Carlsberg brewery, Copenhagen, and was introduced into the literature, under the above name, by him in 1882, after he had succeeded in proving that it had also crept into the stock yeast of this brewery, imparting to the resulting beer an obnoxious bitter by-flavour and a smoky smell. It is therefore a virulent pathogenic yeast (in the technical sense).

It would be a great mistake on the part of the reader to assume, from the foregoing sketch of the three main typical forms of yeast cells, that each species of yeast invariably assumes the same form. This is not the case, a powerful influence on the form being exerted by the conditions of cultivation. This last fact was unknown, and indeed undiscoverable by the methods in use, at the time Reess set up his specific classification based solely on the form of the cells. Thus, up to the year 1882, it was thought that the bottom-fermentation yeast used in brewing always consisted of the one single species Sacch. cerevisiae; and it was not until 1883 that E. CHR. HANSEN (XII.), showed that we have to reckon with a large number of species, and that consequently the names Sacch. cerevisia, Sacch. Pastorianus, Sacch. ellipsoideus, &c., could henceforth be merely used as group names. Since that time, no small portion of this worker's investigations has been devoted to the question of the dependence of cell form on the conditions of cultivation, and to the elucidation of the fact that, morphologically, the character of a species of yeast does not reside in the form of the cell alone, but also in the manner of its dependence on the external conditions of which it is the result. If these conditions be known to a certain extent, then the form of the cells constitutes a very valuable and fairly reliable indication. Since, like other manifestations of vitality, the form of the cells is a resultant of two components, namely, inherited properties and the sum of all the external influences, it is evident that, even if the uniformity of the latter conditions could be made absolutely certain, the

former reason alone would preclude the expectancy of perfect regularity in the cells of a culture. Moreover, owing to the imperfect state of our knowledge, both in chemistry and physics, the production of absolutely identical conditions of existence in two cultures started at different times, is unattainable. Again, even when working with a single cell, it will be found that the daughter-cells, grown in one and the same nutrient medium, differ among themselves; in one of them one of the inherited properties latent in the mother-cell makes its appearance, whilst in another of the cells other inherited properties preponderate.

Cells from the sedimental yeast of a young culture in beer wort. Magn. 1000. (After Hansen.)

It is necessary to lay emphasis on this point, because it only too often happens that a beginner in the cultivation of yeast feels compelled to lose all faith in his knowledge when he observes that a pure culture, prepared from a single cell strictly according to all the rules of the method, consists of cells all more or less different in size and form. Now there is nothing special about such variability, which is exhibited by bacteria and all other living organisms, and that too in no less a degree than with the yeasts. Nevertheless, within the limits afforded by the aforesaid difficulty of control, one and the same species grown under the same conditions will give cells of approximately uniform shape, say, for instance, that of the sedimental yeast of the Cerevisiæ type.

That under these conditions, it is actually possible to trace differential characteristics, may be explained by the aid of the two drawings shown below, both of them representing samples from the sedimental yeast of a culture in beer wort, at the close of the primary fermentation. Fig. 130 is the so-called Carls

berg Bottom-Yeast No. 1, the first yeast prepared by the pure-
culture method, and by means of which E. Chr. Hansen
introduced his pure-culture method into practice, at the Alt-
Carlsberg brewery, in 1883. It was isolated as the principal
constituent of the same stock yeast, which was found to be in-
fected with the aforesaid Sacch. Pastorianus I. A characteristic
feature of this yeast is the preponderance of pointed oval cells,
those of purely globular form being very much in the minority.
Elongated cells also are very rare. On the other hand, the
Carlsberg Bottom-Yeast No. 2, shown in Fig. 131, is character-
ised by the more rounded appearance of its cells, and by the
occurrence of unusually large, or giant, cells, one of which can
be seen on the left of the illustration. The appearance of these

FIG. 130.-Carlsberg Bottom-Yeast No. 1.

Cells from the sedimental yeast at the close of primary fermentation. Magn. 1000. (After Hansen.)

FIG. 131.-Carlsberg Bottom-Yeast No. 2.

Cells from the sedimental yeast at the close of primary fermentation. Magn. 1000. (After Hansen.)

giant cells is specially remarkable in some species, and then
forms a good indication. Thus, BEYERINCK (XX.) found large
cells attaining as much as 20 μ in diameter, in old agar-agar
cultures of a budding fungus known as Sacch. Kefyr, which he
had isolated from the Kephir to be described in our final
chapter, whereas the other cells measured only 5 to 6 μ.

The sedimental yeast found deposited at the bottom of the
fermenting vessels at the close of primary fermentation, in
bottom-fermentation breweries, is drawn off—after the removal
of the young beer-through an orifice in the bottom of the tun,
and is collected in a vessel wherein it is washed with water, to
be afterwards stored under ice water until required for pitching
a subsequent brew. This yeast, it may be stated, is a highly
diversified mixture. Apart from the possible presence of
several species of yeast and bacteria (sarcina, &c.), it also
contains sundry other ingredients, the most important of
which are: First, salts of lime, chiefly the oxalate, immediately
recognisable under the microscope by its octahedral, rhombo-
hedral or flat tabular crystals. These have been precipitated
during fermentation. Their origin is only to a small extent

attributable to the metabolism of the yeast; they also occur in unfermented wort, and in wort-gelatin, and, it may be remarked in passing, disturb the beginner when he examines such a gelatin plate under a low power (30 to 100) before inoculating with micro-organisms, or employing it for starting a single-cell culture. Secondly, the yeast mixture contains precipitated hop resins, in the shape of very small globules, sometimes united as aggregations; they give the resin reactions, and therefore assume a handsome red coloration in presence of alcoholic tincture of alkanna root. Thirdly, there are the so-called glutin bodies, which are fine globules of an albuminous nature, originating in the malt and precipitated from the wort at the low temperature of the fermenting cellar. They have formed the subject of some deep researches by H. WILL (III.). Fourthly, certain dark brown fragments, which are mostly looked on as hop resins by practical brewers, but in reality are said by H. WILL (IV.) to give all the reactions for albumin. When present in large quantity they form a source of trouble in brewing, by enveloping the yeast cells and rendering these latter inoperative. The upper layer of the sedimental yeast in the vat containing the beer in condition for racking, is specially rich in such extraneous admixtures, and is consequently rather dark in colour. This portion is generally removed in advance and thrown away, before the underlying "white" yeast is drawn off. Fifthly come mucilaginous matters of different kinds (§§ 254 and 255), which have been excreted by or extracted from the yeast cells. Sixthly are residual fragments of the mashed materials, hops, lupulin granules, and not infrequently aphides, and the like.

It has already been stated that all the samples examined were taken from fresh cultures, i.e. cultures in which the primary fermentation was just terminated and the deposited yeast crop was of recent date. On the other hand a different appearance is presented by the cells of a sediment that has lain for some time under the fermented liquid-that is to say, in old laboratory cultures, or the sludge found at the bottom of lagerbeer storage vats, and therefore consisting of cells that have been exposed to the influence of the supernatant beer for some considerable time (often several months). Under these circumstances a large number of elongated cells of the Pastorianus type are produced, even in yeasts that are ordinarily of decidedly cerevisiæ type. This is a very useful thing for the beginner to know, in order that he may not be disheartened on finding a number of cells of the suspicious Pastorianus type among the sludge of a vat, the beer in which was fermented with a pure culture yeast. Provided the brewing operations have been conducted in a cleanly manner, the process of spore analysis, described in § 247, will quickly reassure him of the absence of infection.

Up to the present the composition of the deposit in wine and the "wine yeast," in the sense used by tartaric acid manufacturers, has not been so thoroughly examined as is the case with the sedimental yeast of breweries. Next to yeast, the chief constituent is cream of tartar, which is gradually precipitated in the crystalline form by the increasing amount of alcohol in the fermenting wine.

When exposed to very unfavourable conditions, the yeast cell assumes a true involution form. Cells of this kind, resembling amœbæ in contour, are found, for example, in very old gypsum-plate cultures, or similar cultures on wort gelatin. They have been drawn by P. LINDNER (VI. and X.). Many cells that have become exposed to unfavourable conditions in sedimental yeast are able to withstand the same and survive uninjured, because they have previously laid up a considerable store of material and greatly strengthened their membrane. Under these circumstances they become permanent cells, which may be regarded as gemmæ (§ 223). These forms will be further discussed in SS 246, 249, and 251.

§ 246.-Film Formation.

Yeast cultures, started in the manner foreshadowed at the commencement of the preceding paragraph, are able to exhibit another form of development. If the samples have been taken with every care, all infection being prevented, and the vessels again closed with their germ-proof plugs of cotton-wool—which, however, do not exclude air-all that is then necessary is to store them in a quiet corner of the laboratory for some time (several weeks or even months), to find that film has developed on the surface of the liquid. The structure and physiological behaviour of this film will now be described.

This form of development originates in cells, which, thanks either to the presence of some fatty excretion adhering to the outside of the membrane, or to some albuminous or resinous deposit acquired from the nutrient medium, are able to float on the surface of the liquid when the latter becomes quiescent after fermentation has terminated. A good opportunity for these cells to remain afloat is afforded at the places where the surface of the liquid is in contact with the walls of the (round) culture vessel. Hence, it is in such positions that the development of the film will be earliest observed, in the form of a whitish ring. In many instances this ring is by no means complete at first, but appears in the form of a series of patches, i.e. colonies of cells, which gradually increase and then meet, coalescing to a closed ring. In other cases the annular formation is less fully developed, and the growth of the film proceeds from small