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carefully cooled worts was not pure in the sense in which we, in the light of Hansen's' work, now use this expression. The replacement of the old cooling vessel by the new apparatus could only be of real use when a yeast was obtained which could be depended on.
Pasteur had (XI. 2, 3) from the beginning of his career combated the mechanical decomposition theory of Liebig. His investigations had led him, like Schwann, to the result that alcoholic fermentation only begins when yeast cells are present and that it is not possible to bring about fermentation by the mere use of a constituent of the yeast or even by means of chemical agents. “Fermentation,” said Pasteur,“ is life without air,” and he believed that it is the want of oxygen that makes the yeast cells exciters of fermentation, these then taking oxygen from the sugar and thereby producing the peculiar decomposition. Pasteur's theory has not, as we shall see later, stood the test of time.
Nägeli (1879) in the main supports Liebig (XVIII.). He expresses his molecular-physical theory in the following words : “Fermentation is the transference of the conditions of motion of the molecules, atomic groups and atoms of the various compounds constituting the living plasma, to the fermenting material, in consequence of which, equilibrium in the molecules of the latter is destroyed, the result being their disintegration”.
Traube's enzyme theory (1858) may be referred to here (XII.). According to this theory fermentation is explained as an effect due to the various enzymes contained in yeast and not to the yeast cell itself. This theory has lately been confirmed by the discoveries made by Emil Fischer and Ed. Buchner in the chemistry of fermentation.
E. Fischer's investigations (XXIII. 1, 2) on enzymes have not only brought to light new and important facts, but have also pointed to quite new views as to the nature of the processes concerned, and Ed. Buchner (XXV.) by submitting yeast cells to high pressure, succeeded in obtaining an extract capable of producing fermentation in solutions containing sugar. Thus the actual processes of fermentation are now, like enzyme action, included in the domain of organic chemistry.
Pasteur closed his studies in fermentation with his book, Études sur la Bière (1876), and proceeded to other fields of investigation where, as is well known, he gained still greater renown.
A few years previously (1870) the descriptive botanist and microscopist, Max Reess, had carried out a research (XIV.), which, considering the then state of the science, must be regarded as of importance. The spore formation discovered (VI. 2) by Schwann (1839), and observed later (1868) by Jules de Seynes (XIII.) in some of the fungi of alcoholic fermentation, was found by him to occur in many different species. He regarded this as the most important distinguishing characteristic of the genus Saccharomyces. Later investigations have confirmed the correctness of this view. On the other hand his statements of the conditions of this spore formation must be regarded as erroneous. He distinguished the species according to the appearance of the cells. He did not recognise the pure culture and could not therefore deal experimentally with the question of species. He used the form of the cell as the distinguishing character of the species, calling the ellipsoidal cells “Sacch. ellipsoideus,” the sausage-shaped, “Sacch. Pastorianus,” etc. It was proved later by Hansen that one and the same species of yeast can occur in all these different forms, and that, consequently, the shape of the cell cannot be applied in this way. The Reess species have therefore not found acceptance in modern experimental science.
We have now reached that point where interest was. lost in the theoretical as well as in the practical side of the question. The technologists felt themselves deceived by the expectations aroused by the above researches on yeast. The facts taught them by science did not hold good in practice, and often, indeed, their position became precarious. Large sums of money were lost in the breweries on account of accidents during fermentation, accidents the causes of which could not be explained, and against which precautions could not be taken. The yeast was spoken of as something mystical. The view of the science at that time (1884) was depicted in the following expression of Thausing's (XXII.): “Science has given us fine researches on fermentation organisms and on the nature of fermentation, but it has yielded almost nothing of direct value to the brewery; now, as before, the process of fermentation, so far as practical application is concerned, is veiled by a mystic darkness. The investigations of Hansen on the culture of pure yeast entitle us to great hopes; if they do not lie we are near the attainment of an end the importance of which cannot be sufficiently valued. In the first place, however, we have to reckon with the state of affairs as they stand at present.” Similar pronouncements had already been made by Holzner (XVII.) and Lintner (XX.).
Some years before this Hansen had published some of his investigations; but only now was attention directed to him. As botanist Hansen began and completed the reform which inaugurated the new era in the biology of the fungi of alcoholic fermentation, and also in fermentation technique as a consequence of the practical results achieved.
In 1880 and 1881, he conducted experiments on the micro-organisms occurring in air at various times of the year. During these researches he observed a characteristic which enabled him to decide whether a flask contains a pure culture of a yeast fungus or not, and on this he founded his first pure culture method. At the same time he began his experiments on the diseases produced in beer by yeast fungi, and expressed the belief that the wild yeast forms sometimes produce as great disturbances in fermentation industries as bacteria do. A little later he arrived at new points of view for the investigation of species, and indicated the outlines of spore analysis. The results of these pioneer investigations are to be found as short notes interspersed throughout his second treatise on the micro-organisms of the air (XIX. 2) which appeared at the beginning of 1882, but remained unnoticed at the time. His treatises published in 1883 formed, however, the real turning point (XIX. 3). At this time he had probed these questions to such a depth, that he was able to inaugurate a reform in theoretical as well as in practical relations.
In connection with pure culture methods, we have mentioned in the foregoing that Mitscherlich (IX. 2) had, in 1843, observed the budding of single yeast cells under the microscope. Of his successors Brefeld (1874) deserves particular mention as the one who brought to a high degree of perfection this method for studying the morphology and life history of different fungi (XV. 1, 2). But the procedure followed by these investigators did not suffice when absolutely pure cultures of micro-organisms were required in large quantities such as are necessary for physiological experiments ; the requirements are then quite different, and accordingly the efforts of the subsequent investigators were specially aimed at working out a process to suit this case.
Here we must place in the front rank Lister (1878), who sought to prepare pure cultures of lactic acid bacteria by distributing them in the culture liquid (XVI.). He diluted down until only some of the culture flasks contained a
growth, and from this he then infers that the flasks which show development each contain a pure culture.
But this method affords no security, and Hansen therefore, in 1880-81, worked out his first method, which has been referred to above. He made the important observation that the yeast cells, after they have been well shaken up in the flask containing nutrient liquid, sink to the bottom, and form there distinct and well-separated spots of yeast. Examination showed, as was to be expected, that those flasks, in which only a single yeast spot had developed, contained a pure culture. This observation was a considerable step forward. With this method Hansen combined cell-counting by means of a cover glass divided into squares. This rendered it possible to sow a single cell in each flask, and an exact method of preparing pure cultures in large quantities was thus obtained.
At the same time Robert Koch published his investigations on pathogenic bacteria, and, like Hansen, he felt the need of a satisfactory pure culture method for the preparation of mass cultures. Nutrient gelatine was brought by him into extensive use in bacteriology (XXI. 1). His first method (1881) for pure culture consisted in dilution in nutrient gelatine. Before the viscous gelatine had completely set, it was stroked with the point of an inoculation needle which had previously been in contact with the growth from which the required pure culture was to be prepared. The last streak made in this way may contain isolated colonies. The method was, as one can see, a very imperfect one, and Koch soon introduced another, viz., that of plate cultures (1883) (XXI. 2). In this method the germs are distributed in liquefied gelatine, and are, by this means, more thoroughly dispersed. Like Hansen, Koch also observed the single spot; but the last-named method is not so sure as that of Hansen, in which the cells can be