more uniformly distributed owing to the use of a liquid nutrient medium. In Koch's plate culture a colony very frequently showed itself to have been derived from more than one cell; the possibility consequently arises that several species may be mixed together in one colony.

Shortly after Koch had communicated his plate culture method, Hansen published his second pure culture method (XIX. 4, 5). The dilution, in this case, is carried out in nutrient gelatine, but the starting point is from the single cell which is controlled under the microscope. With respect to their accuracy, Hansen's two methods are equally good, the substitution of gelatine being made as it appreciably lightens the work connected with the preparation of the pure culture.

From the above it is plain that Koch's method does not conform with the requirements strictly necessary for a starting point, as does that of Hansen, since the latter starts from a single cell. With bacteria, however, this cannot be entirely carried out. Koch's method is much more suitable for separating the various elements of mixed cultures, so that one does not isolate only that kind which occurs most frequently, but also most of the others. His method also acquired the distinction of bringing nutrient gelatine into general use as a culture medium; from the appearance which the growths have on this medium, the characters of the species can to some extent be made out.

In course of time the number of species-belonging to various divisions of the fungus system-which were treated in Hansen's investigations on the organisms occurring in beer and beer worts became very large. His first treatise (XIX. 1) on fermentation organisms was published in 1879. Among bacteria, he made a special study of those of vinegar. He explains hitherto unknown differences in form, and shows how to recognise the conditions causing these variations.

What we know of the morphology of these species is due chiefly to him (XIX. 9). He further studied mould fungi and the fungi of alcoholic fermentation generally, but more especially the Mucors and the Saccharomycetes. His experimental researches on this subject are contributions to the general biology of the whole of the fungi (XIX. 8). As an example we might cite his researches on the circulation in nature, on the life cycle and on the conditions for spcre and film formation. He gives definite methods for bringing into play the last-named functions, so that what was formerly entirely beyond control can now be brought about with certainty. There might be named, in addition, his researches on the germination of spores, on the relation between the form of the cell and the conditions of culture, on the limits of life of cells, using different methods of preservation, on the behaviour of species towards the sugars, etc. These investigations became likewise of immediate importance for the recognition of species; new points of view were here brought to light, and this question was sifted to a depth not hitherto attained. The above-mentioned group of investigations showed that the Saccharomycetes, under certain methods of treatment, appear with constant characteristics, and that we can here, as with other fungi, make a separation into species, a point which was doubted by several investigators at the time when Hansen began his work. Another part of Hansen's work treats of variation (XIX. 8). He shows how, under certain conditions of culture, the characteristics can vary, that these variations are either temporary (variation of the cell form, variation of alcohol production) or permanent, and how the latter retain their characteristics through endless generations and under all methods of treatment (sporeless and filmless varieties). Theoretically these investigations are of special interest as showing that, in the apparent irregularity of the variations, conformability to

law prevails. For the fermentation industries their importance lies in the fact that they show how new and permanent races can be prepared with certainty.

Having mentioned, in the foregoing, chiefly the theoretical works of Hansen, we will now give a review of his practical investigations. But, in fact, we can draw no sharp line, since here theory and practice constantly go hand in hand. Practical difficulties, with which the two Carlsberg breweries and also the Tuborg brewery in Copenhagen had to contend, induced Hansen, confident of success, to strive with all his energy to effect a fundamental reform. The practical consequence of his theoretical investigations was, on the one hand, the elimination of the disease yeasts; on the other, the separation of the culture yeast (the Saccharomyces cerevisiae of former investigators) into several species and races, and, finally, systematic choice from the latter. This choice forms the most substantial part of Hansen's pure culture system, and was based on the study of these species from new points of view. Finally, he worked out a new method for the analysis of brewery yeast (XIX. 10). It was then made clear how very different these species and races are, and how each gives a special character to the liquids fermented by it. The great differences of the yeast showed themselves in a surprising manner, especially when the so-called wine yeast, Saccharomyces ellipsoideus, was separated into its systematic units.

Hansen published, in 1883, not only theoretical considerations, but, at the same time, the results of experiments which he had carried out in the old and new Carlsberg breweries in Copenhagen. His new system was worked out to the smallest details, and had also been tested in practice, so that it could be applied at once without any preliminary experimenting. As one may gather from the above, Hansen himself introduced his system for the bot

tom fermentation breweries. This system spread quickly into different countries and found an entrance not only into the breweries, but gradually also into the spirit and pressed yeast industries, and the manufacture of wine.

The relation between Pasteur's and Hansen's work in this respect was clearly and forcibly expressed by Delbrück in a lecture (XXIV.) delivered in Berlin in 1895: "Looking back on the last twenty-five years, there are two greatepochs marking the scientific development of brewing; Pasteur's work, which was done after 1870, and which is adopted in principle when we nowadays strive, by the setting up of cooling vessels, to ward off external infection, forms one epoch; Hansen's, the other. But Pasteur's attempts could not lead to a fruitful issue, because one link was missing which was furnished by Hansen in his systematic choice of pure yeast. These two men and their discoveries have been the moving forces of the last decade, and have brought brewing to what it is to-day."

By Hansen's discoveries, the subject of micro-biology and fermentation technique here treated was given new life, and an impulse was imparted to the formation of a rich literature. From that time onwards, the interest of technical fermentation laboratories is claimed especially by the Saccharomycetes; and now new laboratories for promoting the industry are being erected in which biologists work side by side with chemists, where formerly the latter monopolised the whole field of work. An ever increasing body of distinguished investigators has taken up the subject, and their names and work will be given in the following sections, where the subject, as reviewed in this introduction, will now be examined more closely and treated in fuller detail.

SECTION II.

THE LABORATORY.1

MICRO-BIOLOGY has, during its work in the service of the alcoholic fermentation industries, developed a special technique and elaborated special methods; its research has assumed a character of its own, as may indeed be seen in the fitting up and in the apparatus of the laboratories which are now to be found in many places, sometimes as private laboratories, sometimes as state institutions. Some are

purely for research, as, for example, that at Carlsberg, and their work is to promote the science of fermentation organisms by scientific investigation in a theoretical and practical direction; others, and the most belong to this class, are designed to serve practical men by furnishing them with analyses, and providing them with pure cultivations of selected species and varieties of yeast. Laboratories for the study of fermentation organisms are now also to be found attached to a number of the technical colleges (Hochschulen). These were set up for educational purposes after it had been recognised what an important influence the study has on the scientific instruction and on the industrial activity of the manufacturer.

As the number of laboratories increased, and according as they were constructed for the study of special branches of the fermentation industry, the outfit which had been

The bibliography will be found at the end of the book.