First type germination takes place like ordinary budding, and can occur at any point of the surface of the spore (Figs. 80, 81, 82 and 83).

Sometimes it begins while the spore still lies in the mother-cell. Septum formation usually takes place. Sometimes, when the spores grow together, one takes nourishment from the others, and accordingly acts as a parasite. Examples: Sacch. cerevisiæ I. (Figs. 80, 81 and 82), Sacch. Pastorianus I., II. and III., Sacch. ellipsoideus I. and II., Sacch. anomalus (Fig. 83).

FIG. 83.—Saccharomyces anomalus, Hansen. Spores germinating from an old, partially dried gypsum block culture. Cultivation took place in dilute wort: a at 28° C., b and c at 23° C. a'-a""" after 7, 12, 15 and 20 hrs. respectively; b'-b''''' after 10, 21, 24, 25 and 27 hrs. respectively; c' after 8, c" after 10, and c"" after 21 hrs. 1909. (After Hansen.)

Second type (Figs. 84 and 85): Two or more spores generally grow together in the very first stages of germination, yet old spores are able to germinate individually without growing together. Germination begins with a wart-like or sausage-shaped lengthening which grows on and often occurs as germ threads or bunches. Only from this promycelium is the development of the yeast cells effected later on, a partition wall being first formed between the promycelium and the young yeast cell; the latter is detached by fission of the wall. Example: Sacch. Ludwigii (Figs. 84 and 85).

FIG. 84.-Saccharomyces Ludwigii, Hansen. Germination of spores from a gypsum block culture: a, b and c were 12 days old at 25° C., d, e, ƒ and g were 1 months old at the room temperature. The cultivation was made in wort, a at 25 C., the others at 18° to 20° C. a, A cell with four spores; a' after 8 hrs., a" after 25, a"" after 26. b, A cell with four spores in two groups ; b' after 91 hrs., b′′ after 12. c, A cell with four spores in two groups; c'-c'""' after 12, 15. 20, 24 and 27 hrs. respectively. d, Two free spores; d'-d'"""""""' after 18, 20, 26, 28, 29, 304 and 33 hrs. respectively. f, Four free spores; after 19 hrs. gh, A group of three spores, of which the two lowermost, h, were connected, but are separated from one another by fission: the uppermost spore, g, has separated itself in the same way from a fourth spore; g'h' after 17 hrs., g'h" after 21, g""h"" after 23, g'""'h""" after 261⁄2, g''''' after 28. The lowermost spore in this group did not develop. 1909. (After Hansen.)

Just as Hansen's investigations on film formation have been incorporated in the system of yeast analysis, so also have his above experiments on spore formation been fruitful

as regards important characteristics for distinguishing groups and single species; he based on this his method of analysing brewery yeast described in Section II., p. 134. It was found, first, that in different species at the same temperature spores begin to form after different time intervals, and, secondly, that the temperature limits of spore formation are different for different species. Hansen thus determined the spore curves for six species by observing for a series of different temperatures the times at which the formation of spores first began. The cardinal points, viz., the maximum, optimum and minimum temperatures, have special significance, and of these

a

E

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FIG. 85.-Saccharomyces Ludwigii, Hansen.

Germination of spores from an old

gypsum block culture. The germination took place in dilute wort. a and b, Groups of spores in which each spore has developed its own germ thread. a represents the first stages of germination, b a further development; in the group e various forms of coalescence may be seen. 90 to $90. (After Hansen.) particularly the first and last. The two following examples may serve as illustrations of such spore curves :—

Sacch. cerevisiæ I.

At 37° C. no spores develop.

36-37° C. first indications appear after 29 hours.

10 days.

C. no spores develop.

14

Other investigators have published similar curves for other species. Those given by Will for four species of brewery bottom yeasts are especially noteworthy and will be considered more fully in the systematic description.

FIG. 86.—Schizosaccharomyces octosporus, Beijerinck. Formation of the ascus. 1,

a round cell shortly before the formation of the septum.

II., III, IV., V.

and VI., after 1, 3, 6, 10 and 17 hrs. respectively. The times are reckoned from the commencement of the observation. 1999. (After Schiönning.!

It may be seen from the above two series of numbers that spore formation proceeds very slowly at low temperatures, but more quickly as the temperature rises until certain point, namely the optimum, is reached, after which spore formation proceeds the more slowly the nearer the maximum temperature is approached.

him to state the general law that the maximum temperature Hansen's experiments on the effect of temperature led for the formation of spores in Saccharomycetes always lies several degrees lower than that for bud formation and the minimum temperature a few degrees higher. He determined the temperature limits for budding and spore formation in

eleven species. In these species the maximum temperatures for budding lie between 47° and 34° C., the minimum temperatures between 3° and 1° C., the maximum temperatures for spore formation between 37° and 28° C., and the minimum temperatures between 11° and 3° C. Those species which have the highest temperature maxima for budding and spore formation have the same also for film formation.

Schiönning has observed a peculiar mode of ascus formation, in a single species belonging to the genus Schizosaccharomyces, viz., Schizosacch. octosporus.

This takes place in the following manner: The cell I. (Fig. 86) enlarges in one direction and forms a septum (II.). After a certain time fission takes place. The two new cells now lie either touching one another or connected together at one point (III.). In a further stage both cells gradually coalesce so as to form a cell shaped like an hour glass (IV.). The cell grows and the hour glass shape disappears (V.); finally it assumes an ellipsoidal shape and the ascus is formed (VI.). The spores are then formed in the latter.

4.-The Chemical Constituents of the Cell.

The cell membrane consists, according to Casagrandi, probably of pectose or perhaps of an analogous pectin substance, and the chief constituents of the protoplasm consist of albuminoids. Glycogen and fat are frequently present in large quantity.

Errera (1885) first showed that yeast cells contain glycogen. Kayser and Boullanger found that, with a plentiful air supply, there is always less glycogen formed than with a small air supply. The greater the amount of sugar present or the weaker the acidity of the substratum the more glycogen is formed. Large doses of tartaric acid are said to be very effective in preventing formation of glycogen. Otherwise the constituents of yeast cells are substantially