former. Cells with an ordinary thin wall may also keep alive a very long time and thus appear as resting cells.

Colonies formed from saccharomycetes on solid nutrient substrata also present some marks in their appearance which serve to distinguish between species, but by no means in every case, and here also we have to reckon on variations. Hansen in his first communication on the six species of

FIG. 79.-Saccharomycetes which form ascospores.

1. Sacch. cerevisia I. 2. Sacch. Pastorianus I. 3. Sacch. Pastorianus II. 4. Sacch. Pastorianus III. 5. Succh, ellipsoideus I. 6. Sacch. ellipsoideus II. a, cells with septa; b, cells with more than normal number of spores; c, cells with distinct indications of spore formation. About 9. (After Hansen.)

Saccharomyces already mentioned, drew attention to the fact that differences exist among them in this respect which may be brought about by the culture medium and by temperature. He found, for instance, that Sacch. ellipsoideus I., cultivated on wort gelatine at 25° C., is very different from the other five species, the surface of the colonies assuming a net-like structure; further, that Sacch. Pas

torianus II. in streak cultures in yeast water gelatine at 15° C. develops, in sixteen days, colonies with smooth edges, whereas the latter are hairy in Sacch. Pastorianus III., under the same conditions of cultivation. Aderhold, Lindner and others have made subsequent communications on differences among the species in the above respect. For this purpose Lindner sows drops of yeast on nutrient gelatine and thus obtains the so-called giant colonies.

Spore Formation.-Besides vegetative increase by budding, saccharomycetes, like all other ascomycetes, form endospores, the cell being transformed into an ascus (Fig. 79). Schwann, in 1839, first observed spores in yeast. They are not mentioned again until 1868, by de Seynes, and were described for several species by Reess in 1870. The most contradictory views prevailed with regard to the conditions of their formation until Hansen published his researches in 1883. For example, the belief up till then had been that only enfeebled cells washed in water were capable of forming spores, and that the most favourable temperature lay near the freezing point, whereas Hansen showed that this view was entirely erroneous. He found, on the contrary, that spore formation does not, as a rule, take place under these conditions, and that young, well-nourished cells must be sown if a strong spore formation is to be obtained. According to him, further essential conditions. are: abundant moisture, plenty of fresh air and a comparatively high temperature (for most species yet investigated, 25° C. is a good temperature). He carried out thorough and comprehensive experiments, particularly on temperature conditions; these will be described later on. His methods of spore culture are described in Section II., p. 121.

There are species of which the young cells form spores under almost all conditions of cultivation, even under such

conditions as are very unfavourable for the majority of them. A growth consisting of old cells gives a less vigorous spore formation; if the growth is very old it does not sporulate at all.

Quite recently Hansen made additional researches on this point, and has, among other things, defined the difference between spore formation and budding. The result of these investigations is comprised in the following: If young, vigorous cells are brought into a thin layer of water to which air has free access, colonies are formed (even if all the culture liquid has been removed from the cells) at first by budding; hereafter spore formation takes place, begin

f

FIG. 80.-Saccharomyces cerevisia I., Hausen.

Spores at commencement of germination. Formation of a septum may be seen at a, d, e and g. In e, ƒ and g the walls of the mother cells have burst; g shows a septum formed by the coalescing of three spores into a three-winged spore body; the enclosing wall of the latter is burst in three places. 1999. (After Hansen.)

ning first in the mother cell and extending from this to the younger members of the colony. After several days spores are generally also found in the youngest cells, i.e., such cells as have not put forth buds. It may be seen from this that the yeast cell can produce spores directly without previously forming buds. This also happened when cultivating a wine yeast in a solution of calcium sulphate. But the most remarkable fact brought out by Hansen in this connection is that the spore itself can occur as a spore mother cell. This happens when the spore, after being a short time in a culture liquid containing sugar, has swelled up and is then

transferred to an aqueous solution of calcium sulphate. No buds are then produced, and instead spores are formed in the interior of the swollen spore.

Spores, like vegetative cells, consist of a membrane which encloses protoplasm and a cell nucleus. They have the same soft consistency as the latter, but possess greater power to resist drying, heating, etc. Their shape is varied; they are most frequently spherical (e.g., Sacch. cerevisia I., Figs. 79 (1), 80, 81, 82 and 89), with greater or less tendency to an ellipsoidal shape; in particular species they are kidney-shaped (e.g., Sacch. Marxianus), in others "hatshaped" (e.g., Sacch. anomalus, Figs. 83 and 102), i.e., shaped

FIG. 81.-Saccharomyces cerevisia I., Hansen. Germination of old spores. 1929. (After Hansen.)

like the segment of a sphere with a projecting rim round the edge. In some a highly refractive body is found in the middle of the spore (e.g., Sacch. hyalosporus). The number of spores in a cell varies from one to eleven (Fig. 79).

Hansen found that there was a difference between culture yeast and wild yeast in the structure of the spore plasma. The spores of culture yeast appear to be empty, while, on the other hand, the spores of wild yeast are strongly refractive. This difference is of importance in the analysis of brewery yeast, although the details have not yet been specified.

An appearance often observed in the spore-bearing cell

is the growing together of the spore walls, a septum being thus formed (Fig. 80, g). The cell can thus be changed into a many-winged spore body, its walls forming one unit. Sometimes also pseudo-septa are formed between the spores by the latter compressing the plasma which lies between them (Figs. 79, a, and 80, a, d, e).

FIG. 82.--Saccharomyces cerevisiae I., Hansen. Germinating spores. The series e-e"""" was cultivated on wort gelatine, the others in wort. Temperature about 20° C.; a and b dried some time beforehand. Time data reckoned from beginning of experient. a, Three spores connected, no mother-cell wall; a' after 19 hrs., a" after 22, a"" after 30. b, A cell with four spores; b' after 18 hrs. c, A cell with four spores; c' after 9 hrs., c" after 10. d, A cell with three spores; d' after 10 hrs., d" after 13, d" after 17, d""" after 21, d'"""" after 25. e, A cell with two spores; e'-e'"""" after 71, 81, 11, 20 and 50 hrs. respectively. ƒ and g, Two cells with spores; f, g' after 22 hrs., f", g" after 25. h, A cell with two spores; h' after 9 hrs., h" after 13; in h" the wall between the two spores has disappeared, and both have grown into one. 1990. (After Hansen.)

Spores free themselves from the mother-cell by swelling up and causing the wall of the mother-cell to burst.

Hansen found the following two types of germination: