accelerate development. Instead of taking up the drop on filter paper, a little gelatine may be added; the whole is then taken up on a platinum wire or similar instrument and introduced into the wort. Lindner accordingly begins with a liquid, then uses gelatine, and only after a growth has formed on the gelatine are the mass cultures prepared in flasks containing nutrient liquid.

Schönfeld's Method. Of the dilution methods that of Schönfeld remains to be described. According to him a

dilution is prepared by means of liquefied culture gelatine, and small spots are placed on a cover glass from this gelatine mixture by means of a drawing pen. Each spot ought, as

When this method

far as possible, only to contain one cell. is further considered it will be seen that the spots ought only to be so large that the whole of each may be in the field of view when using a medium magnification, and it will be found necessary to add a little more gelatine so that the small gelatine spots may not dry up and in order that a growth may take place at all. The method is, as may be seen from the foregoing, a combination of those of Hansen and Lindner.

II. Physiological Methods.-It often happens that the organism which is to be cultivated in a pure state is present in comparatively small numbers, and the abovementioned dilution methods cannot then be applied. We must then resort to a physiological method. These methods, however, are by no means exact, and offer no certainty of obtaining a perfectly pure culture.

Fractionated Culture after Klebs and Others. A method in general use among the older physiologists and bacteriologists was a combination of an imperfect dilution method with a physiological method. Klebs's method, the so-called fractionated culture, is an example of this, as it consists in inoculating new sterile culture liquid with the

previous culture as soon as that has developed. In this way Klebs expected to obtain finally a pure culture of the species present in greatest numbers at the beginning. The preponderance of one species is therefore a condition for the use of the method; but that species which was in the majority to begin with may not survive best, so that it is questionable in the case where a pure culture is obtained whether it consists of the species looked for. The pure

culture obtained will, at any rate, belong to that species which increases most strongly under the prevailing conditions; but this will not in every case be the species of which it is desired to obtain a pure culture.

Pasteur's Method.-Pasteur (1876) also employs this physiological principle. He gives some indications as to the possibilities of obtaining a pure culture, by making use of the various physiological properties of micro-organisms, but chiefly of the greater or less capacity of increasing in different culture media, or, if the culture liquid is an unfavourable one, of the greater or less resisting power. A struggle between the species is thus brought about, in which possibly the weaker is killed, but this is not certain; those species, too, which are equally strong will be able to live peaceably together, and on this account also it is uncertain whether a pure culture is obtained. This holds in general for the addition of chemical substances to the culture medium, in order to favour the development of one organism at the expense of the other. Pasteur's use of tartaric acid in the preparation of pure yeast may be instanced; it affords a distinct proof of the uncertainty to which such a method may lead. Since the properties of that species. of which it is desired to make a pure culture are in most cases unknown, the method is also less attractive for the reason that it simply assumes that the properties of the species are known. The above holds also for the employment

of certain temperatures for the same purpose. The physiological method is only important in so far that it is a preparatory one; single cell culture must be employed for the preparation of a genuine pure culture.

Pure Cultures of Bacteria. In the foregoing we have had chiefly in view the pure culture of yeast cells; with regard to bacteria, the physiological method may be employed in general for a preparatory cultivation, otherwise Koch's plate culture is the most suitable means, since bacteria are too small to be recognised with certainty in an isolated state in gelatine or liquids. The plate culture is repeated several times, the starting point each time being from a colony in the previous plate culture. The probability of obtaining a pure culture increases with the number of plate cultures. Sometimes the appearance of a colony affords a means of determining whether a pure culture has been reached, as this is often characteristic of the single species; microscopical examination of the cells also assists, of course, in the elucidation.

Pure Cultures of Mould Fungi.-In order to prepare a pure culture of mould fungi, a single sporangium (e.g., of Mucor) or the conidia of a single conidiophore (e.g., of Penicillium) is touched with a sterile needle. The adhering sporangium spores or conidia are then inoculated into a nutrient medium such as wort or wort gelatine, or an additional step may be taken, the spores being distributed in water and a plate culture in wort gelatine prepared. In the foregoing, reference has been made to Brefeld's investigations on the development of mould fungi. In his treatises are to be found very valuable directions on this point.

4. Methods of Preservation.

Hansen's Saccharose Method for the Preservation of Yeasts and Moulds.-It is of great importance in physiological fermentation laboratories to be able to keep pure cultures of the various micro-organisms in such a way that it is not necessary to make frequent additions of new culture medium in order to keep them living. Hansen has elaborated a method of preserving yeast fungi and many mould fungi, viz., by storing in a 10 per cent. cane sugar solution. As regards yeasts the process is as follows: A strong young growth of the species of yeast to be preserved is cultivated for twenty-four hours in wort at 25° C. The top liquid is all poured off from the settled yeast and a small quantity of the latter placed in a 10 per cent. aqueous solution of saccharose, which is set away in a flask at not too high a temperature. Ordinary room temperature is the highest working temperature, and if the flask used is a Freudenreich or a Hansen flask, it must be kept in a dry place so that moulds may not grow through the tube of the cap. The evaporation of liquid in a Pasteur flask is quite insignificant; some cultures in saccharose have been kept in Pasteur flasks in the Carlsberg laboratory for more than twenty years, as previously mentioned, without showing any noteworthy evaporation of liquid. The evaporation from the other flasks is also tolerably small, if the cotton wool in the cap tube is not kept too loose, the tube being of the proper length and the cap fitting well. Flasks of the latter kind can also be kept for several years without a renewal of liquid being necessary, if they are well filled at the beginning. (Cf. pp. 61, 62.)

So far as the saccharomycetes are concerned the vitality appears to be almost unlimited if the preserving takes place in the above manner. Numerous cultures of these have been

kept alive for more than twenty years. Death has seldom taken place, and then only in a few species. This method has thus given excellent results. It has been recommended that the pure cultures should be allowed to stand in the fermented wort, that is in beer, and preserved in this way. But it has been shown that there is then no certainty that the growths will be kept alive, the duration of life when such a method of preservation is used being subject to great variations. The same species-this holds for all saccharomycetes-lives in some cases in beer only for a few months, in other cases for several years. Beer is therefore of little utility as a preserving medium, since the cultures must be renewed every two or three months; it is, of course, almost impossible to do this in an ordinary laboratory.

It has been advanced against the saccharose preservation method that the saccharomycetes increase in it and form yeast rings and films, of which the cells vary in morphological and physiological characteristics from the original seeding, and give a progeny which inherit these new properties. On investigating the numerous old saccharose cultures of the Carlsberg laboratory, the author arrived at the result that yeast ring and film formation only take place in saccharose solutions when the seeding, and therefore also the increase, has been too great. According to Hansen's researches the increase from a scanty seeding in a saccharose solution is limited, but if a larger amount of yeast is seeded, a vigorous increase, as mentioned, takes place, accompanied by the formation of films and yeast rings; even when the yeast is washed beforehand this can occur, the stronger cells living at the expense of the weaker. Under these circumstances it is possible that numerous generations are cultivated under abnormal conditions and that thereby a variation may ensue. The author obtained proof that the