cording to Hansen's researches, be prolonged for many years.

Preservation on Cotton Wool or Filter Paper (after Hansen). When saccharomycetes are to be preserved for a shorter time, cotton wool or filter paper can, according to Hansen, be used, especially when a pure culture has to be sent away. A small quantity of freshly cultivated

yeast sediment is placed on a little sterile hygroscopic cotton wool in a Freudenreich or Hansen flask. When filter paper is used, a small piece is folded once, then wrapped up in four or five thicknesses of filter paper, and the whole sterilised. After this a few drops of the thick yeast liquid are cautiously poured on the inner sides of the folded paper and one of the coverings wrapped round it; when the latter has absorbed the moisture it is removed and replaced by a second covering, and finally placed in the remaining coverings. This process must of course be performed carefully so as to prevent infection taking place. A pure culture may be transmitted in this way in an ordinary envelope; the duration of life is limited, however, to a few months. A pure culture may be preserved safely by the above-described preparation on cotton wool, and the cells also live longer than in the filter paper covers. Also most kinds of moulds may be kept alive for several years by these dry preservation methods.

Preservation of Bacteria. For preserving pure cultures of bacteria there is no method which corresponds to the Hansen saccharose method. In bacteriological laboratories pure cultures are preserved on or in the particular nutrient medium in such a way that they are always renewed after a certain time, a very troublesome process. Bacteria in the spore condition can, however, be preserved in the dry condition in many cases. According to Hansen's investigations acetic acid bacteria remain alive in beer for several years.

Preservation of Ordinary Brewery Yeast.-In connection with the preservation of pure cultures, a few words may also be said about the preservation of ordinary brewery yeast. Experiments were made and methods described more than 100 years ago concerning these more or less impure mixtures. Thus, the yeast was mixed with ashes and the moisture removed from the mixture by means of a cloth, or the yeast was mixed with sugar or with pulverised wood or animal charcoal and the mixture then dried. Beer in a cold cellar was also employed as a preserving medium.

O. Reinke described a method some years ago. The well-washed and quickly pressed yeast is very rapidly enclosed in two sheets of sterile filter paper. The yeast

is then pressed flat, rolled up again in a sheet of ordinary white blotting paper, sprinkled with traces of sterilised boric acid, and then pressed between sterilised asbestos plates to remove the water. The latter are subjected in a hermetically closing metal box to a strongly cooled air current, sterilised and dried by concentrated sulphuric acid. After thorough drying the packets are arranged in a metal receptacle in such a way that each is surrounded with a layer of cold sterilised burnt gypsum. Finally the metal boxes are soldered up.

Will has also made experiments of this kind. He washed and pressed the yeast and mixed it with one of the following substances: kieselguhr, asbestos, gypsum, scraps of filter paper, wood charcoal and wood shavings: the two latter gave the best result, especially the wood shavings. The drying was done as quickly as possible on an oven at a temperature between 25° and 48°, being begun at the lower temperature and continued at the higher. When the yeast was dry it was filled into tin boxes which were hermetically soldered and stored at a temperature of

2° to 7° C. Some of the specimens preserved in this way contained living cells after the course of eleven years, this happening both with culture yeasts and wild yeasts. Heron has also described a method quite recently.

In all these methods for preserving common brewery yeast the latter is washed and pressed. Different experiinenters perform the drying in somewhat different ways. Whichever it may be, the entrance of bacteria and other foreign organisms during these manipulations cannot be

FIG. 46.-Hansen's Flask for the transmission of pure yeast cultures.

avoided. Even when one begins with a pure yeast from the pure culture apparatus, a yeast is obtained in the end which is always more or less contaminated.

Transmission of Yeast.-In sending small samples of yeast the methods described on page 117 using cotton wool and filter paper are taken advantage of. If a larger quantity of pure yeast culture is to be sent, which will be used in the liquid condition in the pure culture apparatus, Hansen recommends the employment of a flask the appearance of

which may be seen from Fig. 46. The flask is made of strong thick glass and has a flat bottom. The yeast is passed into the flask through the side tube, after which the latter is closed. Fig. 47 gives a section of such a side tube when closed; c is the tube, the mouth of which is provided with a small collar, b a tightly closing rubber stopper, a a strong rubber cap tightly stretched over the stopper, being fastened at d by copper wire. The binding may be seen at a (Fig. 46). The rubber stopper must fit exactly and

FIG. 48.-Jörgensen's Metal Flask for the transmission of pure yeast cultures.

be easy to take out after the cap is removed. To add to the security the bent tube is divided into two parts, which are connected by a rubber tube; the latter is fastened to the glass tubes by wire; b is

a

pinchcock which can completely close up the tube, c is filled with cotton wool and used as an air filter when the yeast is poured off through the side tube. Both the rubber and glass tubing should be sterilised separately.

A glass flask naturally has as the contents are visible,

advantages over a metal flask which is of importance both in working with it and also in the transmission of yeast through the Customs, as is now so frequently done; but it is exposed to the risk of breakage during transmission if the packing is faulty. For this reason metal flasks have been frequently used of late as substitutes. Such for instance is the flask of Alfr. Jörgensen represented in Fig. 48. The principle is exactly the same as that of the Hansen flask.

The above-mentioned transmission flasks are constructed efly with regard to brewery requirements. Only small sses or bottles are necessary for wine fermentation, as it then generally a question of sending small quantities of

ast.

5.-Preparation of Spore Cultures.

Spore Cultures of Saccharomyces. Even at the present day the statement sometimes occurs that a spore formation will be produced merely by sowing a little yeast

a moist gypsum block, a potato, or slice of carrot. In so far as the conditions of spore formation are treated, quite incorrect statements are even now not infrequently made, e.g., that the yeast should be well washed beforehand, that the whole process depends upon a starving condition, etc. The old error of the formation of spores at a low temperature is now less frequently met with. Under the circumstances just mentioned it will, however, depend on chance, as regards most species of Saccharomyces, whether they form spores or not.

The essential part of the method does not consist in the use of any particular substratum, such as gypsum blocks, potato or carrot slices. The substratum on which the cultivation proceeds is in the main unimportant. A shallow layer of water in a culture flask, gelatine, etc., may be used with equal advantage, as will be shown later. The chief point lies simply in the use of a moist surface. But it is essential to the method that we should know the best conditions for favouring the function in question. These conditions were ascertained by Hansen and the essentials published in 1883, additions being made in later communications. (A more detailed explanation of the physiology of this function is given in the next section.) According to these investigations a copious formation of spores takes place, if (1) the