MICRO-ORGANISMS AND FERMENTATION. NET BOOK.— This book is supplied to the Trade on terms which will not allow of Discount to the Public. CHARLES GRIFFIN & CO., LTD. MICRO-ORGANISMS AND FERMENTATION. CHARLES GRIFFIN & CO.'S PUBLICATIONS. THIRD EDITION. In Handsome Cloth. Fully Illustrated. 21s. net. PRINCIPLES AND PRACTICE OF BREWING. FOR THE USE OF STUDENTS AND PRACTICAL MEN. BY WALTER J. SYKES. REVISED BY ARTHUR R. LING, F.I.C., F.C.S., Editor of the Journal of the Institute of Brewing. CONTENTS. —Physical Principles Involved.— The Chemistry of Brewing.— The Micro- scope.— Vegetable Biology. —Fermentation.— Water.— Barley and Halting.— Arrange- ment of Brewery Plant. — Quantities of Materials. — Fermentation. — Antiseptics. — Finings.— Characteristics of Beer. — Diseases of Beer. — INDEX. " A thorough and comprehensive text-book . . . up-to-date . . . a standard text-book." — Brewerg Journal. In Large 8vo. Complete in Two Volumes. Each Volume Complete in itself, and Sold Separately. TECHNICAL, MYCOLOGY: The Utilisation of Miero-Organisms in the Arts and Manufactures. BY DR. FRANZ LAFAR, Prof, of Fermentation-Physiology and Bacteriology in the Technical High School, Vienna. TRANSLATED BY CHARLES T. C. S ALTER. Vol. I.-SCHIZOMYCETIC FERMENTATION. Vol. II.-EUMYCETIC FERMENTATION. NOTE. — Part I. of Vol. II. was issued separately at 7s. 6d. Copies of Part II., Vol. II., have, therefore, been bound up to enable those possessing Part I. to complete their copies. The price of Vol. II., Part II., is 18s. net. " The first work of the kind which can lay claim to completeness in the treatment of a fascinating subject. The plan is admirable, the classification simple, the style is good, and the tendency of the whole volume is to convey sure information to the reader. " — La ncet. In Crown 8vo, Handsome Cloth. Price 7s. 6d. net. FERMENTS: AND THEIR ACTIONS. A Text-Book on the Chemistry and Physics of Fermentative Changes. BY CARL OPPENHEIMER, PH.D., M.D. TRANSLATED BY C. AINSWORTH MITCHELL, B.A., F.I.C., F.C.S. " Such a veritable multum inparvo has never yet appeared." — Brewers' Journal. SIXTH EDITION, Thoroughly Revised. Fully Illustrated. 21s. FOODS: THEIR COMPOSITION AND ANALYSIS. BY A. WYNTER BLYTH, M.R.C.S., F.I.C., F.C.S., Barrister-at-Law, Public Analyst for the County of Devon, and Medical Officer of Health for St. Marylebone. AND M WYNTER BLYTH, B.A., F.I.C., F.C.S. "A new edition of Mr. Wynter Blyth's standard work, ENRICHED WITH ALL THE RBCENT DISCOVERIES AND IMPROVEMENTS, will be accepted as a boon."— Chemical Sews. FOURTH EDITION. Thoroughly Revised. In Large 8vo, Cloth, with Tables and Illustrations. 21s. net. POISONS: THEIR EFFECTS AND DETECTION. BY A. WYNTER BLYTH, M.R.C.S., F.I.C., F.C.S., Barrister-at-Law, Public Analyst for the County of Devon, and Medical Officer of Health for St. Marylebone. "Undoubtedly THK MOST COMPLETE WORK on Toxicology in our language."— The Analyst. "As a PRACTICAL GUIDE, we know NO BETTER work."— The Lancet (on the Third Edition). LONDON: CHARLES GRIFFIN & CO., LIMITED, EXETER STREET, STRAND. MICRO-ORGANISMS AND FERMENTATION. BY ALFRED JORGENSEN, DIRECTOR OF THE LABORATORY FOR THE PHYSIOLOGY AND TECHNOLOGY OF FERMENTATION, COPENHAGEN. TRANSLATED BY S. H. DAVIES, M.Sc. I-'OURTH EDITION, COMPLETELY REVISED. LONDON: CHARLES GRIFFIN AND COMPANY, LIMITED; EXETER STREET, STRAND. 1911. [All Rights Reserved^ TRANSLATOR'S PREFACE. A TEXT-BOOK written by one of the foremost exponents of the honoured Danish School of Micro-Biological Research, and by a pioneer of world-wide reputation in the industrial applica- tion of selected types of yeast, is certain of a welcome from English readers. In comparison with the enormous output of works on the organisms of disease, little has yet been published in English on the technical applications of Micro-Biology. This book covers ground which is not fully surveyed in any existing treatise. The necessity of embodying the results of ten years' research has led to such a mass of additions and alterations in the last English edition that this must be regarded as a new work. It is based on the fifth German edition. S. H. DA VIES. YORK, December, 1910. PREFACE. THEjfirst edition of this work was issued in the form of a text-book in 1886. It was the first attempt to express the biological significance of the science of fermentation and of the fermentation industry, a field where the chemical point of view had hitherto prevailed. I was induced to give this form to my work by the fact that in 1881 I had established an institute in which my first aim was to treat the problems of the fermentation industries from a micro-biological point of view. This necessitated a short course for technologists and chemists who wished to study the science of fermentation on new lines, and as both older and younger students were attracted to my laboratory, the subject-matter had to be arranged so that the book could serve as a guide, even to those who had no special preliminary knowledge. During my co-operation with E. C. Hansen in that early period, the principles of the practical application of pure cultures were broadly outlined, and accordingly an explanation of the principles underlying the resulting technical reforms formed the essence of the treatise. The new editions which appeared in rapid succession, showed that the attempt had been successful. At the same time, I had the good fortune to introduce into breweries in many countries properly selected pure cultures of bottom yeasts, following the precedent of E. C. Hansen, who shortly before had intro- duced similar yeasts into the Carlsberg Brewery in Copen- hagen. At this early stage, I successively described pure cultures of yeast types which are used in other branches of the fermentation industry, and I introduced them into top- fermentation breweries, as well as into distilleries, yeast fac- tories, fruit-wine factories, etc. Thus a beginning was made in the application of this new principle to new fields of industry. Consequently, to keep pace with these developments, together with the results of research in the science of fermentation, my book had to be so largely modified that each new edition became to some extent a new book, whilst the growing mass of material considerably increased the size of the book. Having to keep within certain limits, I was reluctantly compelled to VI PREFACE. leave various problems undiscussed. On the other hand, repetition could not always be avoided for the sake of young students, when similar subjects were discussed in different sections. Among the many new questions demanding fuller treatment in this, the fourth English edition, I may specially mention the fermentation of milk and other dairy products ; the enzymes, and the conditions of yeast nutriment. As regards the technical part of the work, a new section deals with methods of cleansing and disinfection in the fermentation industry, and the section dealing with the application of pure cultures to the various branches of the fermentation industry has been considerably enlarged. In its present form the book will, I hope, serve as a manual, not only to zymo-technologists, but also to analysts and physiologists.* For convenience, the old names of yeast species have been retained along with the new, in the systematic treatment given in the fifth chapter. As the work is concerned with the micro-organisms of the fermentation industry, due regard has been paid to the practical application of research work, and the description of both useful and injurious species has received special attention. The classification of the yeasts has been partially based on the same requirements. The description of each species em- phasises those characteristics that are of special importance to the industry. I am glad to take the opportunity of expressing my thanks to the head of the students' department in my laboratory, Herr J. C. Holm ; the systematic part of Chap, v., together with the very full bibliography, are essentially his work. Finally, I wish to express my warm thanks to many authors who have kindly sent me reprints of their published works. I regret that the limited extent of my book has in many cases prevented me from making use of valuable publications. COPENHAGEN, January, 1909. ALFRED JORGENSEN. * A short description of ths most important practical conditions found in the fermentation industry and in the laboratory is to be found in my short work, Practical Management of Pure Yeast. London, 1903. CONTENTS. CHAPTER I. Microscopical and Physiological Examination. PAGES 1. Microscopical Preparations ; Staining, and Microscopical Examination, 1-10 2. Biological Research ; the Microscope ; Moist Chambers, . . . 10-13 3. Sterilisation 13-31 (a) Sterilisation of Glass and Metal Articles, .... 14-15 (b) Sterilisation of Liquids and Solid Nutritive Substrata, . . 15-31 4. Disinfection in Practice, 31-36 5. Flasks 36-40 6. Nutritive Substrata, . 40-43 7. Preparation of Pure Culture, 43-55 (a) Physiological Methods, 45-47 (b) Dilution Methods, 47-55 8. Counting the Yeast Cells, 55-58 CHAPTER II. Biological Examination of Air and Water. Introduction, ............ 59-60 Air Analyses — Miquel's Methods and Results, 60-62 Methods of Hesse, Hueppe, von Schlen, Frankland, Miquel, Petri, andFicker, 62-63 Sampling Air, ........... 64 Miquel's Criticism of the Gelatine Process, ..... 64 E. C. Hanson's Researches on Air, . . . . . . 65-69 Saito's Researches, 69 Water Analyses — Holm's and Jorgensen's Examination of Water, .... 69-71 Examination of Ice, 71 Methods of Hansen, Wichmann, Lindner, and Jorgensen, . . 71-75 viii CONTENTS. CHAPTER III. Bacteria. PAGES Forms, 76-78 Anatomical Structure, Colouring Matters, Phosphorescent Bacteria, . 78-80 Chemical Composition, 81 Nutrition, 81 Anaerobiosis, 82 Locomotion, 83 Propagation, Spore- Formation, . . 83-85 Influence of Temperature, Light, Pressure, Antiseptics, and Mechanical Vibration, 85-87 Classification, 88-92 1. Acetic Acid Bacteria, 92-105 2. Lactic Acid Bacteria, 106-125 3. Butyric Acid Bacteria, 125-132 4. Bacteria Fermenting Cellulose, 132-133 5. Alcohol-forming Bacteria, . . . . . ' . . 133-135 6. Kephir, Koumiss, Mazun, Leben, Yoghourt, Ginger-Beer, . . 135-141 7. Slime-forming Bacteria, . 142-151 8. Bacteria with Inverting, Diastatic, and Proteolytic Enzymes, . 151-153 9. Sarcina, • . . 153-160 10. The Fermentation of Tobacco, 160-161 11. Iron and Sulphur Bacteria. Nitrifying Bacteria, .... 162-164 CHAPTER IV. Moulds. Introduction, . 165-166 Anatomical Structure, .......... 167-171 Classification of the Fungi, ......... 171 Nutritive Physiology, 17-1-175 Action of External Influences, 175-178 Chemical Constituents ; Enzymes, ........ 178-183 Botrytis cinerea (Sclerotinia Fuckeliana), 183-187 Penicillium glaucum, ...'...... 187-191 Aspergillus, 191-197 Mucor, 197-210 Monilia, 210-214 Oidium lactis, 214-216 Fusarium, ... ........ 217 Chalara 217-218 Dematium, 218-220 Cladosporium, 221-222 Oidium (Erysiphe) Tuckeri, 222-223 Peronospora • . . . . 223-224 CONTENTS. IX CHAPTER V. Yeasts. PAGES Introduction, 225 Nutrition of Yeasts, , 225-229 Theories of Fermentation, 229-255 The Enzymes of Yeast, 255-256 The Action of the Saccharomycetes and similar Fungi on Carbo- hydrates and other Constituents of Nutritive Liquids — Diseases in Beer, 256-266 The Products of Alcoholic Fermentation, . . . . . . 267-269 Auto-Fermentation, 269-270 Fermenting Power, Fermentative Energy, Raising Power, . . . 270 The Biological Relationship of Yeast, . 270-280 Variations in the Saccharomycetes, ....... 280-289 M orphology and Anatomy of Yeast Cells, 290-313 Yeast Deposits, . -290-292 Film Formation . . . . 292-297 Cultures on Solid Substrata, 297-298 Structure and Character of Yeast Cells, ...... 29S-3< 2 Ascospore Formation, . . . . . . . ... 302-310 Analysis of Yeast, 310-313 I. Saccharomyces, ........... 314-385 Classification of the Saccharomycetes, ...... 315-317 1. Species used Industrially (Culture Yeasts), . .... 317-344 (a) Brewery Yeasts, ......... 317-341 Classification, 318-319 The use of Pure Bottom Yeasts in Practice, . . . 319-320 Carlsberg Bottom Yeasts 1 and 2, ..... 320-323 Low-Fermentation Yeasts described by Will, . . . 324 " Saaz" and "Frohberg" (P. Lindner), .... 325 Low-Fermentation Yeasts described by Schonfeld and Rommel, 325 Saccharomyces cerevisias or Sacch. cer. I. (Hansen), . . 326-328 Top-Fermentation Yeasts described by JOrgensen and Holm, 329-339 Top-Fermentation Yeasts described by Regensburger, . 339-341 The use of Pure Top- Fermentation Yeasts in Practice. . 341 (b) Distillery and Press Yeasts, 342-343 (c) Wine Yeasts .... 343-344 2. Yeasts not yet applied Industrially, ...... 345-385 Saccharomyces Pastorianus or S. Past. I.. . . . . 345-347 S. intermedius or S. Pastorianus II. , . . . . . 347-349 S. validus or S. Pastorianus III. , 349-351 S. ellipsoideus or S. ellips. I., 351-353 S. turbidans or S. ellips. II 354-355 S. Willianus, 355 S. Bayanus, 356 X CONTENTS. PAGES S. Logos, . 356-357 S. thermantitonum, ......... 357 S. Ilicis 358 S. Aquifolii, . . 358 S. Vordermanni, 358 S. Sake, 359 S. Batatse, 359 S. cartilaginosus 359-360 S. multisporus, 360 S. mali Risler Kayser, ........ 360 S. Marxianus, . 360-361 S. exiguus, 361-362 S. Jorgensenii, .......... 362 S. Zopfii, . 362-363 S. Bailii, 363 S. hyalosporus, 363 S. Rouxi, 364 S. Soya 364 S. mali, Duclaux Kayser, 365 S. unisporus, 365 S. flava lactis, 365 " Levure de sel " a, 365-366 S. Hansenii, 366 S. minor, 366 Pichia membransefaciens or S. membr., ..... 366-368 Willia anomala or S. anomalus, . 368-370 Willia Saturnus or S. Sat., ....... 370 S. acidi lactici, 370-371 S. fragilis, 371-372 Zygosaccharomyces Barkeri, 372-374 Saccharomycodes Ludwigii or Sacch. L., . . . . . 374-377 Schizosacch. or Sacch. comesii, 377 Schizosacch. octosporus, 377-379 Schizosacch. Pombe, 380-381 Schizosacch. mellacei, . 381-383 Saccharomycopsis or Saccharomyces guttulatus, . . . 383-384 Saccharomycopsis capsularis, . 384-385 JI. Budding Fungi without Spore-Formation, 385 Torula 385 Hansen's species 385-388 Torula Novae Carlsbergia, 388 Torula a = T. Holmii 388 Torula A and B, Schionning 389-391 Torulas described by Will, 391-392 Torulas described by van Hest, 392-393 Torulas described by Meissner, 393 Torula colliculosa, . 393 CONTENTS. xi PAGES Torulas described by Adametz, ....... 393-394 "Salt Yeast," 394 Torula epizoa, ........... 394 " Levure de sel" /3 and y, 394-395 Sacch. brassicae, .......... 395 Torula (Awamori), ....... ... 395 Torula b = mucilaginosa, 395-396 Torula c = cinnabarina, 396-397 Torula in Beer or Green Malt, ..... ... 397-398 Blastoderma salmonicolor, ........ 398 Mycoderma humuli and rubrum, ....... 398 Torula in Milk and Must, 398 Black Yeast, 398 Torula Yeasts fermenting Lactose, ....... 399 Sacch. lactis 399-400 Sacch. Kephyr and Tyrocola, ....... 400 Lactomyces inflans caseigrana, ....... 400 Torula in Butter, Cheese and Milk, 400-401 Torula amara . 401 Saccharorayces apiculatus 402-407 Mycoderma cerevisiae and vini, . 407-413 CHAPTER VI. The Pure Culture of Yeast on a Large Scale. Industrial Application, 414 Propagating Apparatus, .......... 415-418 Despatch of Pure Cultures, 419 Preservation of Pure Cultures, ........ 420 BIBLIOGRAPHY, 421 INDEX, 481 MICRO-ORGANISMS AND FERMENTATION. CHAPTER I. MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 1. Microscopical Preparations, Staining, and Microchemical Examination. THE Microscope will always be the chief means for investigating micro-organisms, for these are, as individuals, almost always invisible to the naked eye. The microscope is made up of a mechanical and an optical part. The mechanical part, or stand, consists of the foot, the Stage, the tube carry- ing the lenses, and the adjustment for regulating the distance between the lens and the object lying on the stage. The adjustment is partly " coarse " (a screw engaging in a toothed gear attached to the tube) whereby the tube can be rapidly raised or lowered, partly " fine " (a finely cut screw) by means of which the tube can be gradually raised or lowered, after finding the object with the coarse adjustment. The tube commonly consists of two telescoping parts. A table, which usually accom- panies the instrument and gives the scale of magnification, records the cor- responding length Of tube either in Fig. 1. millimetres (usually 180 mm.) or in inches (usually 10"). The optical part consists of the lenses and illuminating apparatus (a mirror and Abbe condenser). 1 MICRO-ORGANISMS AND FERMENTATION. The lenses form the most important part of the microscope ; the system turned towards the eye is called the eyepiece, and that turned towards the object is called the objective. When a bundle of parallel rays of light strikes a convex lens, the rays are refracted and collect at a point on the other side of the lens, called the f OCUS. The distance between this point and the lens is called the focal distance. If a small object is placed on the stage at a slight distance beyond the focal length of the lens and illuminated by the mirror, the rays passing through the lenses of the objective will not be parallel, but will diverge, and so form a magnified image of the object. The smaller the focal length, the greater the magnification. This real inverted and magnified image formed by the objective, which must fall exactly in the focus of the eyepiece, is seen through the latter as an imaginary, magnified image of the picture formed by the objective. The whole magnification secured by the microscope is, therefore, a product of the magnification due to the objective and that due to the eyepiece. The magnification is always expressed as a linear and not as a quadratic dimension — i.e., the relationship between the length of a line as seen through the microscope and its length as seen by the naked eye. From any given point of the object on the stage of the microscope a bundle of rays may pass through the lens. The angle which the outermost rays of the bundle form is called the angular aperture. It is not customary to allow all the rays of the angiilar aperture to pass through the system of lenses which make up the objective. A larger or smaller number of the peripheral rays are excluded by means of diaphragms consisting of metal discs with round apertures, which are of various sizes, and fit into the opening on the stage. In this way the Fi£- 2- actual angular aperture of the objective is reduced. The peripheral rays would give an indistinct picture, but the picture would, on the other hand, lose in clearness if too many of the outer rays were excluded. For this reason the objective is so constructed that the aperture shall be as great as possible. An expression for the value of the latter is given by the numerical aperture (the sine of half the angular aperture multiplied by the index of refraction of the medium in front of the lens — ah*, water, oil, etc.). When the rays of light are refracted in the objective some of them are immedi- ately split up into the component coloured rays, and the image shows coloured edges (chromatic aberration). To avoid this the objective is made up of several different lenses prepared from various kinds of glass (crown glass, flint glass) which possess different refractive powers. It is thus possible to prevent any indistinctness of outline. MICROSCOPICAL PREPARATIONS. 3 To secure greater magnification a system of immersion is used, the space between the front lens of the objective, which is made of crown glass, and the cover glass being filled with a strongly refractive medium, water or oil. The immersion is homogeneous if, as is often the case, the oil has the same index of refraction as the cover glass and the front lens. In this way an increase of the numerical aperture is secured, and, therefore, a greater resolving power — the limit of definition for the smallest perceptible details. The eyepiece consists of two lenses, the upper, which comes into direct contact with the eye, and the lower, called the collective lens, which collects the rays of light so that the field Of vision is reduced, and is, therefore, more easily surveyed. Between the two lenses a diaphragm is inserted in order to further reduce the field. The collective lens is also of importance in securing, along with the upper lens, the exclusion of the coloured edges of the microscopical picture. The greater the curvature of the lenses in the eyepiece, the more do they magnify the image projected by the objective, which at the same time becomes darker and less distinct. To obtain a well-lighted field when working with high magnifications, it is necessary to use strong objectives and weak eyepieces. It is well known that the lenses of the human eye alter their shape according to the distance of the objects that are under observation. They can accommodate themselves, and, by projecting, shorten the focal length, and thereby produce a sharp image on the retina. On the other hand, by a reduction of the convexity the focal length is increased, and thus a clear image of distant objects may be thrown on to the retina. If the eye is short-sighted or long-sighted, the tube of the microscope must be set to suit the focal length of the eye, and the size of the image will differ for abnormal eyes. The normal focal distance is considered to be 250 mm., and tables Of magnification are based on this. The actual magnification for any individual eye must be established by a special calculation with the help of a glass micrometer. The illumination of the object is secured by a mirror placed below the stage which is capable of movement in all directions, so that the object can receive direct or oblique illumination, and so that the mirror can be fixed at different distances from the object. For low powers the plane side of the mirror is used, for high powers the concave side. With ordinary magnifications it is of import- ance to secure suitable illumination, as the eye soon tires if the light is too strong. Instead of the usual diaphragm in the stage, an iris diaphragm may be used, enabling the aperture to be reduced or enlarged by means of a number of sickle- shaped leaves sliding over each other. To give illumination over a large surface (e.g., in the examination of coloured substances), a combination of lenses known as a condenser is introduced between the mirror and the preparation. When studying an object, the separate parts of which can only be distinguished by differences in their refractivity, a narrow bundle of rays must be used, and this is secured by placing a diaphragm with a smaller opening in the aperture of the stage. The microscopical examination of the organisms of fer- mentation throws light upon their size, form, colour, the 4 MICRO-ORGANISMS AND FERMENTATION. refractive indices of different parts of the cell, and, generally, of all those conditions which are the object of morphological research. As we are dealing with living forms, we can only arrive at a real knowledge of them by studying their life conditions, through biological and physiological research. Biological research is concerned with the investigation of life phenomena under the conditions existing in nature ; thus, such conditions as the distribution of single species ; their occurrence ; the numbers present in different localities at different seasons ; their sensitiveness to Jight, to heat, to the moisture of the atmosphere, etc. Physiological research has for its object the study of the life history of the organism, the conditions of nourishment and propagation. It is also concerned with the different kinds of fermentative activity, so far as these can be estab- lished by studying the influence of organisms on the liquids in which they are growing, and with the nature of the sub- stances or forces causing fermentation (enzymes). Specially constructed apparatus is available for such investigations, and many of these lines of research are closely allied with chemical studies. One essential condition of any exact investigation into the life history of micro-organisms must be secured — the cer- tainty that we are working with a single cell or with one vegetation, consisting of a single species, and, therefore, derived from one cell. We shall see in the following pages how the technique has been slowly developed, and how this goal has been reached, as the result of many scientific and technical attempts to prepare absolutely pure cultures. For the ordinary examination of yeasts and moulds, a clear magnification of 600 suffices. For the examination of the fine details of these organisms and of bacteria, higher powers are required. Although an immersion lens is of great service, it is not essential for ordinary technical work. It is of real importance that the organisms of fermentation should be examined, as much as possible, in a living state, and either in a drop of water or of a liquid in which they have been growing. The drop is placed on an object glass. MICROSCOPICAL PREPARATIONS. 5 and spread out to form a thin layer, by placing a cover glass on top of it, or else the drop may be placed inside a moist chamber (described later) in which the growth and propaga- tion of the cells can be followed. For instance, by such observations of the living cell, the development of spores in the yeast cell may be observed, and the difference in shape of the ripe spore in individual species, the thickness of the wall of the mother-cell, etc., may be noted. Certain characters, however, of the detailed construction of these organisms can only be detected by the use of special drying and staining methods. To do this, the cells are sub- jected to a thorough treatment with concentrated dyes, some of a poisonous character. They are killed, and the possibility exists that the characters brought out by staining may differ somewhat from those of the living cell. Drying may also modify the length and breadth of the bacteria. On the other hand, the staining process has explained many phenomena which were not apparent by observation of the living cell. Dilute dyes (e.g., eosin, methylene blue) are used in the technical examination of yeasts to obtain an idea of the proportion between dead and living cells in a vegetation, since the dead cells alone absorb the dye. As an example of a method of staining, we may instance the treatment to which yeast cells are subjected in order to observe the cell-nucleus and its subdivision. Hoffmeister proceeds as follows : — The young, vigorous yeast growth is washed several times with distilled water, and then before the actual staining it is subjected to the process of fixing, according to one of the recognised methods. For instance, the yeast is stirred up with Rath's solution (consisting of a litre of a concentrated, aqueous solution of picric acid, together with 4 c.c. of glacial acetic acid and 1 gramme of osmic acid) ; the cells are thus coloured yellow. After allowing the mordant to react for 24 hours, the cells are again washed with water, spread out in a thin layer on a cover-glass, and allowed to dry. The preparation is then treated according to Heidenhain's method. The cover-glass preparation is allowed to float in a Petri dish, on the surface of a solution containing 2-5 per cent, of iron alum ; after 6 to 24 hours the cover-glass is washed once with C MICRO-ORGANISMS AND FERMENTATION. water, and then placed in an aqueous haematoxylin solution. After a further 24 hours the cells, as seen under the micro- scope, are stained deep black. They are then treated for a few minutes with a 0-25 per cent, solution of iron alum, after which the yeast cells appear colourless with violet or greyish- black cell nuclei. The preparation is mounted in undiluted glycerine. One of the more advanced of recent workers in this field, A. Guilliermond, makes use of the method of staining just described to prove the presence of the cell- nucleus in the yeast cells, but for fixing he prefers picroformol (Bouiri). To prove the presence of the fine hairs, which serve the bacteria as organs of movement, the flagella or cilia, which can seldom be detected by direct microscopical examination of the living bacteria, the following method (Ldffler) is adopted : — A small quantity of a very young growth of bacteria (developed for five to eight hours in an incubator) is placed in a drop of water — the ordinary supply is preferable to distilled water — and the contents of this drop are divided amongst a number of drops of water, placed on a series of carefully cleaned cover-glasses. They are air-dried, and are then passed through a flame in order to fix the bacteria. Care must be taken that the preparation is not heated too strongly. The simplest means of avoiding this is to hold the cover-glass between the fingers, and not to heat it more strongly than they can bear. A large drop of a mordant is now spread over the heated cover-glass. The mordant, which is applied to render the bacteria absorbent to the actual stain, consists of 10 c.c. of tannic acid solution (20 per cent.) mixed with 5 c.c. of a cold saturated ferrous sulphate solution and 1 c.c. of a saturated aqueous or alcoholic fuchsine solution. The cover-glass is warmed for about half a minute until steam is given off, but violent boiling must be avoided. The prepara- tion is washed with a powerful stream of distilled water, and afterwards with absolute alcohol until the cover-glass is clear, and only the spot on which the water drop has been evaporated appears cloudy. The staining fluid is now poured over the surface of the cover-glass. It consists of a neutral saturated fuchsine solution in aniline. The preparation is warmed again for a minute until steam rises, washed with a stream of water, MICROSCOPICAL PREPARATIONS. 7 and is then ready for examination. It should be noted that all motile bacteria do not show their flagella when they are treated with a mordant of the above composition. One must proceed experimentally, for some bacteria require a mordant to which a few drops of a 10 per cent, soda solution have been added, whilst others require an addition of sulphuric or acetic acid in place of soda. Loffler found that several of the acid-forming bacteria require an alkaline mordant, whereas a number of alkali-forming organisms require an acid mordant. By such careful means beautiful pictures have been obtained, which show that bacteria are supplied with these organs of movement arranged in various ways ; they often cover the whole surface of the cell. A similar treatment brings out clearly the coating of slime which, for instance, surrounds the cells of acetic acid bacteria, but is invisible in an ordinary microscopical examination. In a few cases staining has proved of value in determining the species ; this is the case with the acetic acid bacteria investigated by E. C. Hansen, Bacterium aceli, B. Pasteurianum, and B. Kiitzingianum. Staining is most readily done by treating a vigorous growth with an aqueous solution of iodine in potass, iodide, or an alcoholic solution of iodine. The slime surrounding the cells of B. aceti is coloured yellow, whereas that of the other two species is coloured blue. The latter reaction is brought out more clearly when the slime is forced out to the sides by pressure on the cover-glass. Blue coloured flecks are thus formed, while the cells themselves are either colourless or yellow. A similar blue coloration of the spores of Saccharomyces mellacei with iodine has also been demonstrated by J. C. Holm. Photographs of micro-organisms are now frequently em- ployed. The preparation may be first stained, for instance, by Loffler 's method, to bring out the characteristics more sharply, and to remove foreign bodies. Whilst the usual sketches of microscopical preparations are always more or less diagrammatic, omitting everything except the special characters it is desired to emphasise, micro-photography provides a more correct representation of the object, and has a further incidental advantage. It is well known that s MICRO-ORGANISMS AND FERMENTATION. the photographic plate is sensitive to certain chemically active rays of light which cannot be detected by the human eye, so that photography can reproduce certain characters of the preparation which would be entirely missed by direct observation. It may be anticipated that the new method of photographing by ultra-violet light will bring about notable advances, for it will thus be possible to distinguish a greater number of fine characteristics and preparations otherwise colourless will appear to be composed of differently coloured parts. The ultra-microscope that has just been applied to technical purposes makes it possible to distinguish small details that are invisible with all earlier optical devices. Its design is based upon a special method of lighting, discovered by Sieden- topf and Zsigmondy, whereby the preparation is brightly illuminated with rays of light, falling in a direction at right angles to the axis of the microscope. The illumination is so arranged that a single layer of the preparation is lit up whilst the remainder is in shadow. With the help of this apparatus particles can be distinguished of a size of four- millionths of a millimetre. (The symbol fj. is used for mm. •a AyV/WV/ and ju/u for - mm.) The effect of such an illumination may be realised by recalling the well-known appearance of particles of dust floating in a beam of sunlight penetrating into a darkened room, when the particles are viewed in a direction approximately at right angles to the beam of light. A micro-chemical examination is of value for ascertaining the composition of the individual parts of a micro-organism. After treatment of the cells with reagents, the reaction brought about, and especially the colour reaction, may be studied under the microscope, with a view of determining the chemical composition of the part in question. As an example the proof may be cited that yeast cells, in the later stages of develop- ment, contain the reserve food stuff, glycogen (a carbo-hydrate). In order to detect this substance in the cell an iodine solution is added to the preparation (2 grammes iodine, 6 grammes potassium iodide, and 120 c.c. of water). The albuminoid MICROSCOPICAL PREPARATIONS. 9 portion of the cell contents is thereby coloured bright yellow, whilst the glycogen assumes a reddish-brown colour. A further proof that the coloured part of the cell contents really consists of glycogen is obtained by warming the preparation to 70° C., whereby the colour reaction of the glycogen dis- appears ; on cooling down the colour reappears. If the cells are cautiously pressed under the cover-glass, so that they burst, it may be observed that the brown coloured glycogen is liberated as a fluid mass, which quickly dissolves in the sur- rounding liquid. Oil or fat globules are often found amongst the granules that occur in the fully developed yeast cells. By the addition of 1 per cent, osmic acid they are coloured brown ; sometimes they assume a vermilion colour with alcanna tincture, and frequently they may be dissolved by treatment with alcohol- ether, benzene, or carbon bisulphide. The treatment must, however, be continued for some time ; the solution takes place more rapidly if the cells are crushed and the solvent brought into direct contact with the globules of oil. The albuminoids of the cells, as already stated, are coloured yellow, or yellowish-brown, with iodine ; with nitric acid and ammonia they are coloured lemon-yellow, and with Millon's reagent (mercuric nitrate), brick-red. In the examination of fermented liquids micro-chemical methods are used to determine the character of any suspended matter other than micro-organisms. To clearly recognise the reaction of the tiny particles floating in the liquid, it is some- times necessary to separate them by centrifugal action, and further to wash them free from the liquid. The starch or paste cloudiness which occurs in beer is produced by fine particles of starch or dextrin, which separate out when the liquor contains a given quantity of alcohol. They may be recognised by the addition of iodine, whereby they are coloured blue or brownish-red (dextrin). A cloudiness due to albumi- noids is often produced ; it is due to flocculent, membranous, and often very irregular agglomerates, and to minute granules, which are either isolated or enclosed in these secretions. Such a formation frequently occurs in pasteurised beer ; it may be distinguished under the microscope by the readiness with 10 MICRO-ORGANISMS AND FERMENTATION. which it absorbs the colouring matter from iodine or from aniline dyes ; it is coloured brick-red with Millon's reagent, and rose-red with RaspaiFs reagent (sugar and sulphuric acid). These bodies may also acquire a yellow colour (through the formation of xanthoproteic acid) by treatment with nitric acid and then with ammonia, or sometimes with the acid alone. Glutin cloudiness is a frequent form of albuminoid precipitation ; it takes the form of a fine network throughout the liquid. At 30° to 40° C. the liquid becomes absolutely clear ; on cooling, it again becomes cloudy. Under the microscope, fine flecks and small yellowish granules can be seen. According to Will, the latter consist of envelopes, more durable than the contents, which are easily attacked by water or dilute alcohol, acetic acid, or hydrochloric acid, and thus the sheaths become recognisable ; in 5 per cent, potash, the whole granule dissolves. On warming, the con- tents, but not the sheath, are dissolved ; hence the warm liquid is not always absolutely clear. Occasionally a cloudi- ness due to hop resin occurs in beer ; the yellowish-brown globules are recognised by the vermilion coloration given by an alcoholic alcanna solution. A cloudiness of beer due to a resin derived from pitch has been described by Will ; pre- cipitations of this kind are coloured violet by a mixture of acetic anhydride and concentrated sulphuric acid. This reaction is specially marked when the granules are separated from the liquid. 2. Biological Research by means of the Microscope ; Moist Chambers. The examination described in the previous section can give but a limited insight into the nature of micro-organisms. A more complete knowledge of their life characters can only be reached through a biological and physiological investigation. The methods adopted have gradually reached a high stage of development, and micro-biology now stands as an independent branch of natural science, which has given results valuable both to science and to industry. The subject of micro-biological research may be either a BIOLOGICAL RESEARCH. 11 growth or an individual, a single cell. In the first case, the certainty of the result is determined by the purity of the growth, and whilst the work is in progress this must be secured by the adoption of special precautions to be further described. In the second case, with which we now have to deal, the entire examination must be carried out under the microscope, special means being required to enable us to observe the series of changes that arise from the development and growth of the single cell. With this aim in view, Ranvier's moist chamber may be used (Fig. 3). This apparatus is made by grinding a slight hollow in the middle of a common object-glass ; round this hollow a groove is made of greater depth to carry water. The drop of nutritive solution, which must be very small, is Fig. 3. placed in the middle of the hollow and covered with a cover- glass, which extends beyond the groove. When the cover- glass is in place, it is cemented by means of vaseline, and the drop is thus enclosed between the cover-glass and the hollow of the object-glass, whilst the water in the groove prevents evaporation. If by suitable dilution, care has been taken that only one cell has been sown in the drop of water, the study of its development may be extended for any length of time, with the certainty that all forms that appear are derived from one and the same individual. It is obviously a condition of this and all similar investigations that the liquid and the closed part of the apparatus must be sterile. This chamber may be used again to decide whether fine 12 MICRO-ORGANISMS AND FERMENTATION. particles floating in a liquid are secretions or bacteria. Sub- stances are added to the liquid which favour the growth of bacteria, and by lengthened observation of the behaviour of the particles it may be determined whether they propagate or not. Amongst various kinds of moist chambers, that of Bottcher (Fig. 4), which now finds extensive use, may be mentioned. It consists of a glass ring fastened to a common object glass, and upon this a cover-glass is cemented with vaseline. The cover-glass carries on its under side a freely suspended drop containing the object to be examined. A few drops of water are placed on the floor of the chamber to prevent evaporation of the suspended drop. If the cover-glass has been completely freed from grease by cleaning with acid and ether, the drop will spread out to a thin layer, so that it may be observed under a strong power, and with a short focal distance. As the drop hangs freely, it is possible to lift the cover-glass Fig. 4. without disturbing the growth, if a sample is to be removed. If the cells are to be fixed, a little gelatine may be mixed with the liquid before sterilisation, as suggested by Brefeld. In his detailed researches on micro-organisms, Brefeld used this and other kinds of moist chambers, which are illustrated and described in Vol. IV. of his Botanischen Untersuchungen uber Schimmelpilze (Leipsic, 1881). In order to secure the presence of a single cell in the drop, he diluted the infected liquid until this proved to be the case. If the organism demands a full supply of air to reach complete development, a stream of moist air may be passed through two tubes fixed in the sides of the glass ring. By the help of such apparatus definite conclusions can be arrived at regarding the nature of the growth of micro- organisms, and this knowledge is essential for accurate work with mass cultures. To prepare the latter, the small pure STERILISATION. 13 culture is transferred with every precaution from the moist chamber to a flask containing sterile liquid (see detailed description in a later section). 3. Sterilisation. The principles of the whole technology of sterilisation, as well as the different kinds of apparatus required, were described in the early memoirs on spontaneous generation. The details of the development of this subject in its his- torical setting are given with the description of the theories of fermentation in Chap. v. Sterilisation of objects, whether a liquid or a piece of apparatus, means the riddance therefrom of all germs capable of development. This may be carried out either by removing all germs by mechanical means or by killing them by heat, or by the use of antiseptics. The choice of treatment is determined by the composition of the object to be sterilised, and obviously those means will be chosen that will render the adhering germs harmless, whilst producing the minimum of change in other directions. Sometimes, with this in view, the sterilisation can only be partial ; for instance, if the pro- perties of the liquid are changed to a great extent, by heating to the temperature at which the germs present would be killed, the lowest temperature must be found by experiment at which the organisms are so greatly enfeebled that they are no longer able either to develop or to affect the liquid. This is the object of pasteurisation, which will be discussed later on. One circumstance which often presents great difficulties to complete sterilisation is this — that the great majority of micro-organisms occur in two different forms of growth, vegetative cells and spores. Whilst the former are usually killed at a temperature below the boiling point, the latter, and especially the spores of bacteria, can withstand prolonged heating at the boiling point ; thus Fliigge has isolated a species of bacteria from milk, the spores of which withstood boiling for four hours. When such spores are encountered, it is necessary either to boil for several hours, or to adopt a 14 MICRO-ORGANISMS AND FERMENTATION. considerably higher temperature ; the latter alternative is specially necessary when dry heat is used. (a) Sterilisation of Glass and Metal Objects. — Sterilisation, properly so called, must always be preceded by a thorough mechanical, and often by a chemical, cleansing. Articles of daily use in the laboratory, such as spatulas, needles, wires, etc., are heated directly in a flame, and allowed to cool in a space free from germs. Heavy pieces of apparatus, however, do not admit of this treatment ; harm may be done by over- heating while ensuring that every part of the object has been sufficiently heated, or the number of objects to be sterilised may be so great that it would take too much time to treat each singly. The apparatus must in this case be placed in special sterilisation ovens, where it is exposed for some time to a temperature at which it is believed that all germs will be destroyed. Dry or moist heat may be used according to the. nature of the article. Dry heat is a much weaker disinfectant than moist heat at the same temperature. To make certain that all germs are killed when using a hot air steriliser, the air must be raised to a temperature of 150° to 160° C., and the articles must be subjected to this heat for one to two hours. Some objects are wrapped in paper, others (e.g., flasks) are closed with a cotton wool plug, which should be covered over with filter paper. If moist heat is required, the object can either be boiled in a water bath, or, better still, subjected to the action of steam. It is obviously of importance to see that the air is completely driven out, so that it cannot form a protecting layer, and prevent the steam from coming into contact with the object. Either a current of steam may be used, or steam under pressure. In the first case, the apparatus is placed in a vessel provided with a perforated false bottom, with a sufficient quantity of vigorously boiling water below it. The steam escapes slowly, as the lid of the vessel is not air-tight, and the apparatus is gradually raised to the boiling point. By boiling in steam at 100° C. all vegetative forms are probably killed, together with many spores of bacteria and other resistant forms if the treatment is continued for an hour. STERILISATION. 15 There are, however, spores which withstand this treatment ; thus, Sames and Christen have shown that the spores of certain species of potato bacilli which frequently occur in soil will withstand 10 to 16 hours' boiling. If, therefore, we have to do with material which has been in contact with the earth, higher temperatures must be employed, for it has been shown that the disinfecting power of stearn rapidly increases when its temperature rises above 100° C. Steam under pressure is, therefore, used in a Papin's digester or autoclave, constructed to stand a pressure of several atmospheres. This apparatus is specially useful for the sterilisation of several liquids used in the laboratory. If small quantities of liquids are to be sterilised, a pressure of one atmosphere, corresponding with a temperature of 120° C., is sufficient, if applied for half an hour. During the cooling of any variety of sterilising appar- atus, care must be taken that the incoming air is sterile, and this is secured by passing it through sterilised cotton wool. (l>) Sterilisation of Liquids and Solid Nutritive Substrata.— All germs can be removed from nutritive liquids by filtration, but this method of treatment, which is more troublesome than heating, is only used for liquids when their composition is affected by heat. Even in this respect it must be noted that filtration is not without eft'ect, for the investigations of Fliigge, Arloing, and others have shown that a filter retains or reacts upon certain of the soluble constituents, for instance, upon certain enzymes. As the filtering medium, either burnt clay, plastic charcoal, gypsum, asbestos, or kieselguhr may be used. The pores of these substances are very fine, and a thick layer must be used to ensure that even the smallest bacteria are retained. The pores are soon stopped up, and the filtration must then be hastened by pressure or suction. In labora- tories, and for the filtration of small quantities of water, the Pasteur-Chamberland filter, consisting of burnt porcelain clay, and Nordtmeyer's filter made of compressed diatomaceous earth are frequently employed. These filters take the form of a hollow candle, closed at one end ; the liquid flows into the hollow and out by a tube fixed at the other end. To test such a filter, it is immersed in water, and air is blown into the cavity. If bubbles of air rise through the water, the filter is 16 MICRO-ORGANISMS AND FERMENTATION. evidently perforated, and is, therefore, useless. The first runnings of a filter, even a perfect filter, are not always sterile, and after a filter has been in use for a short time germs always pass through it. This happens because the germs at length grow through the pores, since it is almost unavoidable that substances which supply nutriment to the bacteria should not penetrate into the filtering medium. The surface of the filter must, therefore, be frequently cleaned, and the filter sterilised, which is most easily done by boiling it in water. In breweries, the filtration of beer has been resorted to during the last few years, the filtering media commonly used being paper, cellulose, asbestos, etc. By such filtration brewers sometimes succeed, it is true, in freeing a beer of sound origin from deposits of various kinds, and in rendering it bright ; but, on the other hand, it has been directly proved by Thausing, Wichmann, Reinke, Lafar, and others that an indiscriminate employment of this method may prove ex- tremely dangerous. If the filters are not effective, it may happen that only yeast cells are retained and not bacteria, which can then react with much greater energy upon the liquid. Another great danger lies in the fact that a filter, when it is imperfectly cleansed, may harbour colonies of different kinds of germs, causing the contamination of all beer passing through it. If a single cask in a cellar has become infected, and the filter is not effectually sterilised after the filtration of its con- tents, the disease will be communicated to the whole of the beer. It is, of course, a great mistake to use a filter which has been allowed to stand for a day without previous steri- lisation ; the different species will have rapidly propagated in the favourable substratum, and will be swept off by the fol- lowing filtration. In this stage of development the cells of wild yeasts are much more vigorous than those of the cultured yeasts, so that the disease organisms will multiply rapidly, and cause serious infection. A warning must be given against treating the filter with water at a temperature below the boiling point ; a thorough cleansing can only take place by prolonged boiling. By careful handling of the excellent filters now manufactured by several makers a more stable product can be obtained than that before filtration, as the experience STERILISATION. 1 7 of the author has shown. It is certainly not allowable to lay it down as a general rule that beer must always be spoilt by filtration. The filtration of milk of any biological importance has proved, so far, impossible, as a fitter with pores large enough to allow the fat globules to pass will not retain bacteria, of which the vast majority are smaller than the globules. The filter is, therefore, only of use in removing the greater part of the dirt particles from the milk, and the micro-organisms that are attached to them. On a large scale sand and gravel filters are used. For instance, in the Danish system, as constructed by Busck, a vertical cylinder is used with per- forated diaphragms, between which are packed layers of sand, the grains of which are coarser at the bottom and finer at the top. The milk is run in from below. In Krohnke's construction the milk is passed through a cylinder partially filled with gravel, and carrying vertical diaphragms ; the cylinder is rotated round a horizontal axis. On a small scale, the fresh warm milk may be filtered through cotton wool, a layer of which is inserted between two sieves ; the filter requires renewing daily. A more complete biological puri- fication has been attempted by pasteurisation. The filtration of air is intended, not only to remove living germs, but also to remove all floating particles. It has already been stated that Schroder and Dusch accomplished this by means of a tube filled with closely packed cotton wool, and this still proves to be one of the best materials. In the labor- atory such filters are used to seal test tubes and flasks. When they take the form of glass tubes, as in the Freudenreich flasks, it is unnecessary to protect the surface at the open end of the tube, but it is otherwise in the case of test tubes, where a great part of the filter is exposed to the dust of the air. Germs may easily grow on the cotton wool when it absorbs moisture. Flaming the surface is not always sufficient, and in such a case it is desirable to keep the tubes in an atmo- sphere free from germs. By the diffusion of air, which goes on through such small filters, evaporation takes place, and as a consequence the liquid becomes more concentrated, or the gelatine hardens on the surface. Such evaporation can be 2 18 MICRO-ORGANISMS AND FERMENTATION. avoided by the use of the flask constructed by the author (Figs. 8 and 9). On the large scale in breweries, yeast factories, etc., cotton wool packed in suitable vessels is also used for filtering air, or else the air is led through a large number of layers of cotton wadding (Holler's filter). The complete sterilisation of the air on the large scale cannot always be attempted, and could not always be justified from an economic standpoint. In the filtration of water on the large scale, the conditions existing in nature are imitated, where water is allowed to sink through successive layers of soil, and the organic residues and micro-organisms are deposited on the finer layers, until, at a given depth, the water is sterile. Artificial filters con- structed of a number of layers of varying coarseness were first applied in London, and are now used in every country. Such a filter consists of a bed of large stones, covered with several layers of flints successively reduced in size so that the topmost layer is about the size of a pea, and on this is laid a layer of sharp sand about 5 feet in thickness, which has previously been washed. The water is first stored in a reservoir, where the larger particles settle out. When the filter is used for the first time water is led in slowly from below, so that all the air is driven out of the filter. It is then allowed to stand quietly for some hours before the true filtra- tion begins. This must be carried out slowly at first, and then more rapidly. It has been shown that the distance between the separate particles of sand is greater than the bacteria, and, therefore, the retention of the bacteria is not due to the sand filter. While the water is standing quietly over the filter, slimy matters in suspension settle down and form a fine skin of slime on its surface. This retains a few bacteria, and as it always contains organic residues, it supplies nutriment for the bacteria, and as a consequence they multiply. A few bacteria settle on the grains of sand in the upper part of the layer, and these become slimy, and so arrest the bacteria subsequently carried down with the current of water. In this way the upper part of the layer of sand gradually fills up, so that the pores between the slimy grains of sand are now smaller than the bacteria, and then, for the first time, it can act as a true filter. It is now " ripe," and the water ins the STERILISATION. 19 lower layers of sand will be found to contain few bacteria. Thus it is the matter contained in the water itself that converts the upper part of the bed of sand into a filter. The necessary condition for satisfactory working is that the water shall flow slowly, in order that the bacteria and other particles may have time to settle on the slimy grains of sand, and also to prevent the skin breaking or the formation of channels through the bed of sand. The rate of flow must depend on the nature of the supply. If rich in bacteria, it should not sink more rapidly than 2 to 3 inches per hour. For the same reason the water level must be retained within certain limits. During the slow passage of water, the bacteria embedded in the upper part of the sand are able to retain some of the dissolved organic matter in the water, so that when it leaves the filter it should be free from fermentative and putrefactive components. A stage is reached at last when the pores are so completely filled with bacteria that the capacity of the filter is greatly reduced, and it is then renewed by removing the top inch of sand. This process can be repeated until a layer of sand 16 to 20 inches thick is left; the layer must then be restored to its original thickness. / As many different factors condition the activity of the filter, such as the changing biological contents of the water, altera- tions in temperature, etc., its activity must undergo many variations from a biological point of view, and this necessitates a continuous bacteriological control. Completely sterilised water cannot, of course, be obtained from such a filter, but it has been shown that in a good filter only one out of every 1,000 or more germs is transmitted. Considerable use is also made of " rapid " filters. They may be constructed of sand, wood charcoal, etc., so as to allow of the passage of large quantities of water. The filtration is combined with the use of chemical precipitants, whereby the greater part of the slimy particles and organisms are separated from the water. The treatment of water with ozone is dealt with in a subsequent section. The exact method of sterilisation of liquid and solid sub- strata by means of heat is determined by their chemical and 20 MICRO-ORGANISMS AND FERMENTATION. biological nature. The methods employed include the use of a current of steam, steam under pressure, boiling in water or on the sand bath, and the treatment may be prolonged for a considerable period if it is desired to kill not only vegeta- tive cells, but also spores. In either case it is obviously of importance to take care that during the subsequent cooling only sterile air is admitted to the vessel. This is secured in the case of Pasteur flasks by the use of a tube with two bends, in which any germs that are sucked in are deposited ; in the case of Erlenmeyer and Freudenreich flasks, by sealing them with cotton wool filters. Whilst the hopped wort commonly used in zymophysiological laboratories will stand boiling on the sand bath, and after a comparatively short boiling can be preserved unchanged, wort gelatine and other gelatines cannot stand treatment on a sand bath or such prolonged boiling on a water bath or in steam, that will ensure the destruction of ah1 spores, because there is always a danger that after such treatment the gelatine will no longer solidify at a temperature of 25° C. The same difficulty is met with in the sterilisation of the mash in distilleries and of wort in the air yeast factories, owing to the great separation of albuminoid substances which takes place at the boiling point, causing a complete change in the character of the liquid. For this reason it is impossible to apply all the results of experiments obtained with properly sterilised liquids to the very different circumstances that obtain in practice. In all such cases use is made of the method of fractional or discontinuous sterilisation introduced by Tyndall. Its object is to bring about the germination of spores of bacteria and similar resistant organisms by main- taining the material at a gentle heat for some time, so that the cells may subsequently be killed at comparatively low temperatures. The material is first warmed, perhaps to a temperature of 70° C., or it may be heated for a quarter of an hour in a current of steam in order to kill the vegetative cells. It is then maintained at room temperature, or, better still, at the most favourable temperature for the development of spores (about 35° C.), and after the lapse of a day, or even of a shorter period, when it is assumed that germination is complete, the material is again heated. By repeated treat- STERILISATION. 21 merit of this kind it is possible to eliminate all spores and to kill all vegetative cells. This obviously depends, however, upon the regular germination of the spores. The treatment does not absolutely guarantee sterility, and before either liquids or gelatines are used they must be kept under obser- vation for a considerable time. In many cases filtration is to be preferred to discontinuous sterilisation. The liquids in daily use that are prepared with the help of micro-organisms, beer, wine, vinegar, etc., always contain a residue of these micro-organisms in a more or less vigorous condition. It is desirable, by heating them, to arrest the fermentation. The safest course is to sterilise the liquids, but as the temperatures required to effect sterilisation usually produce great changes in the liquids, it is necessary to limit the treatment to a temperature that will suffice to weaken the micro-organisms, so that under normal conditions they are extremely unlikely to propagate or to bring about further fermentation (" pasteurisation "). It is difficult to determine the best method when the nature of the liquid will not admit of a high temperature being used, while the result must depend upon the character and the activity of the different micro-organisms present, as well as upon the chemical composition of the liquid. It is, therefore, impossible to establish any general rule. It is essential in each case to determine experimentally both the temperature and the time of treatment, after forming a judgment as to what micro-organisms are present in the liquid. In the case of beer different temperatures are used — heating from 50° to 60° C. for two hours, or from 65° to 70° C. for half an hour or more — and for wine, two hours' heating at 45° to 50° C. (C. Schulze}. A slow cooling down after pasteurisation has often been experimentally proved to give better results than rapid cooling. The determination of the right temperature is obviously rendered more difficult if the liquid harbours different species of yeast, and still more so if at the same time the development of bacteria has taken place, especially those species that form spores. It has been proved that when the heating exceeds certain limits, the flavour either of beer or wine quickly deteri- orates, which is probably due in the first place to the decom- position of albuminoids. 22 MICRO-ORGANISMS AND FERMENTATION. If the liquid is particularly sensitive to high temperatures, it is necessary to fall back on the method of discontinuous treatment, whereby the liquid is heated to a moderate degree several times, with a suitable interval between each heating. Frequently the alteration in taste produced by pasteurisation can be partially removed by subjecting the liquid for a time to a low temperature. A special difficulty met with, par- ticularly in the case of beer, is that during storage or transport, particularly at low temperatures, the pasteurised liquid develops a turbidity, or forms a deposit, consisting usually of albuminoid substances separating in the form of small granules, or, in difficult cases, in flakes and skin formation. It has usually proved necessary to control the preparation of the malt if such a calamity is to be avoided. Care must be taken that a slow and sufficiently advanced development of the grain has taken place, accompanied by a full transformation of its contents. Further, it is obvious that the fermentation should have been vigorously carried out, and in this con- nection it is particularly necessary to adopt pure ferments. By cooling the beer to a low temperature before filtration and pasteurisation it is possible to avoid the subsequent separa- tion, as part of the material in question is separated in the cooling process. In dealing with milk, heat is applied in the same way. In this case the greatest possible difficulties are met with owing to the great range of micro-organisms present in milk (lactic acid bacteria, putrefactive bacteria, hay and potato bacilli, etc.), many of which are only killed at a high temperature, owing to their power of forming spores. Heating the milk further results in separating or modifying components, which may be of extreme value in nutrition (e.g., the enzymes), even at comparatively low temperatures. Pasteurisation at temperatures considerably below the boiling point may result in the milk bacteria being killed, whilst the putrefactive bacteria remain alive. As a consequence, the latter, freed from competition, multiply rapidly, and form putrefying matter in the milk, and this may occur to a considerable extent if the milk is not stored at a very low temperature. Actual sterilisation can only be secured if the milk is heated STERILISATION. 23 for an hour or more at a pressure of half an atmosphere, corresponding to 112° C. If the object is simply to destroy the pathogenic organisms that are present, especially the tubercular bacilli, it is only necessary, according to Bang and Weigmann, to heat for a few minutes at 85° C., or for a period of from a quarter of an hour to one hour at 65° C. The problem how to secure a product free from any organisms capable of development, and yet of good nutritive value, has not yet been solved, although long-continued treatment at 60° C. has proved of some value. (Absolute certainty cannot, of course, be obtained in this way.) The tendency is, however, to estab- lish a stringent control of the milch cows and of the milking operations, in order to make sure of a healthy product. Sterilisation of air can be best secured, as already stated, by means of cotton wool filters. Sulphuric acid or brine baths, cloth filters, etc., are less effective. In the laboratory, where it is often necessary to carry out work in sterile air, glass cupboards are used, the front of which can be sufficiently raised to admit the arms. Some time before using the cup- board the whole of the inner surface is washed over with either mercuric chloride solution or 60 per cent, alcohol, and the cupboard is then closed. Any particles and germs floating in the air will sink to the moist floor, and will be retained there. In breweries and other branches of the fermentation industry, the fermentable liquid is sterile at a particular stage in the manufacture, at the moment when the boiling is completed. After the zymotechnical analysis of air had shown that it may convey disease germs to the fermenting liquid, attempts were made to protect the wort during the cooling operation against such infection by the use of closed cooling and aerating apparatus, closed fermenting vats, and storage casks, and by the sterilisation of the incoming air through cotton wool filters. These precautions, together with the use of an absolutely pure yeast, should, theoretically, pro- duce an absolutely pure product. Incidentally, one important practical object was secured, for by blowing in a powerful stream of air during the fermentation, and by the removal of carbon dioxide, the rate of fermenting was greatly increased, and an earlier clearing of the liquor took place. The difficult 24 MICRO-ORGANISMS AND FERMENTATION. problem is to maintain such large vessels in a condition of absolute cleanliness. The experience of many years has, however, shown that in breweries with open refrigerators and cooling apparatus, open fermenting vats, and ordinary storage casks, a product can be obtained with such a small content of harmful germs that they have no practical influence on its quality, notwith- standing the fact that the wort, especially on the refrigerators, is exposed to a number of foreign germs. It has now been proved that the harmfulness of the atmospheric germs in the fermentation industry has been greatly exaggerated, for in competition with the enormous number of yeast cells which are established in the wort, the vast majority of these germs never come to development. If it happens that, notwith- standing the use of pure yeast, the product is strongly con taminated with disease organisms, the explanation is, in the great majority of cases, that these are developed in the plant itself. It is from the surface of the different vessels employed that the dangerous carriers of disease have developed, just because a rational method of cleansing has not been adopted. The chief importance must be attached to those stages in the process where the liquid is longest under treatment, in the fermenting vats and storage casks. In order to purify these vessels, as well as the connections, disinfectants are almost always used, and it may be remarked that a summary treat- ment with these is not sufficient. This, at any rate, holds good for wooden vats, in which it has often been proved that notwithstanding disinfection the disease germs retain their hold. A special investigation must, therefore, be made into the physical character of the vessels, and the necessary pre- cautions must be adopted. If in this way a rational method is worked out, it will be found that the atmospheric germs exercise no noticeable influence on the course of fermentation or on the character of the product, since no opportunity is given for them to establish themselves in the plant. Under special circumstances chemical reagents are used for disinfection, the antiseptics. The ground work of the technical application of antiseptics was laid by Schwann, who proved in 1839 that yeast cells die under the influence of certain STERILISATION. 25 chemicals. More recently the knowledge of antiseptics has been greatly extended by R. Koch. As in the case of the action of heat, so the individual species react differently towards the various antiseptics. Moreover, one and the same species of vegetation may react differently towards the same reagent, and that not only because the spores possess a greater power of resistance than the vegetative cells, but also because the activity of the latter plays a part. One practical problem is to determine how far the antiseptic can be diluted without ceasing to react. Whilst with a given concentration the antiseptic may prohibit life, with a greater dilution the action only restricts development, and with still greater dilution, if any further influence is felt, it may take the form of stimulating both the development and activity of the organism. Many organisms possess a special power of accommodating them- selves to strong doses of antiseptics. Disinfectants are placed on the market either in a solid or in a concentrated liquid condition. Their antiseptic power must first be determined by experimenting with the groups of micro-organisms which may be encountered in the fermentation industry. Once the limit of their activity is determined, it- is necessary to ascertain how rapidly a given dose operates. Should it prove that the action is too slow for practical appli- cation, other degrees of dilution must be tested until the minimum dose is found which will kill the micro-organisms in a short time (e.g., in thirty minutes). Flasks of 15 c.c. capacity, provided with ground glass stoppers, are used for the test. These are filled almost to the top with the disinfectant, and after a pure culture of each species has been placed in the flasks, they are thoroughly shaken. When the action is completed, every trace of the reagent must be removed from the vegetation by washing, and a sample of the growth is transferred to a suitable nutritive substratum, and exposed under the most favourable conditions. It must be maintained at a constant temperature, which should be higher than that of the room. Liquids are to be preferred to gelatine, because the nutritive value of the latter is generally smaller. Finally, the observation of such growths must 26 MICRO-ORGANISMS AND FERMENTATION. extend over a considerable period, as it often proves to be the case that the cells have not been killed, and after a considerable time they may germinate again. The degree of dilution at which an antiseptic operates restrictively on species is usually depend- ent on whether the action takes place in a nutritive fluid or not. In the first case, the chemical nature of the liquid obviously has considerable influence. Thus, for instance, liquids which are rich in albuminoids weaken the effect of many poisons. In a determination of this character for industrial purposes it is usual to limit the solvent to some particular fluid. As an example of the part that the solvent plays, the classical work of Koch in 1881 may be mentioned, which led to the proof that many antiseptics may wholly or partly lose their power according to whether they are dissolved in water, in ethyl or methyl alcohol, ether, or acetone. In this connec- tion an important fact may be noted. The addition of sodium chloride to certain antiseptics (e.g., to carbolic acid or mercuric nitrate solution) causes an extraordinary increase in their antiseptic power. Temperature, also, has an influence on their action ; the higher the temperature, the greater their activity. On the other hand, a dilute antiseptic exhibits the least restrictive power at that temperature which is most favourable to the organism. Numerous investigations regarding the influence of antisep- tics on different species of micro-organisms have shown that no general rule can be traced. One species may be much more resistant to one poison than many other species, whereas it may exhibit little resistance to another poison. The destruc tiveness of a given substance cannot, therefore, be defined in general, but only its behaviour towards a particular species. The application of antiseptics for the cleansing of vessels, etc., must always be preceded by a thorough mechanical cleaning. Antiseptic substances are partly inorganic, partly organic. Amongst the mineral substances, the first place must be given to mercuric chloride, on account of its extremely poisonous character. It is used in the laboratory in a solution of 1 gramme per litre of water, but it is impossible to use it in STERILISATION. 27 technical work. Like most of the other mercuric salts, mer- curic chloride belongs to that class of bodies which produce insoluble compounds with albuminoids, and thus do not react completely with bacteria. Attempts have been made to overcome this difficulty in such cases by the addition of a small percentage of sodium chloride. Hydrofluoric acid and its salts also belong to the most powerful antiseptics, especially as regards bacteria. Ammonium fluoride is generally used, and has a wide application. Chlorine is used in the fermenta- tion industry in the form of chloride of lime, but it is only applicable within certain narrow limits, owing to its strong and pungent odour. Another compound, sodium hypochlorite (antiformin), which has a weaker smell of chlorine, is more widely used. Chlorine is also used to disinfect water. For this purpose small quantities of chloride of lime are used, and after a short time the chlorine is fixed by the addition of calcium bisulphite. Sulphurous acid is used sometimes in the form of gas or of an aqueous solution, and sometimes as calcium bisulphite or sodium sulphite. It is used, not only as an antiseptic, but also as a means of restricting fermentative activity. This, as well as several of the above, usually appears to attack bacteria more strongly than yeasts in high dilutions. Soda is of value as a means of cleansing, as well as disinfecting, and this applies also to lime. Lastly, two gases must be mentioned which are now coming into use, ozone and hydrogen peroxide, the latter having an even greater disinfecting power than ozone. Amongst organic antiseptics, formaldehyde has found very extended application during the last few years, on account of its great antiseptic power. Thus, the spores of many resistant bacteria are killed by the application of a 0 1 per cent, solution for an hour. On the other hand, this reagent, when used in the form of vapour, has little action on man and the higher animals. Its vapours appear principally to attack the surface of articles, as its power of penetration is not great. A series of antiseptics which have proved of special im- portance in laboratory studies of fermentation includes ether, chloroform, and more especially acetone, toluol, and thymol, because they possess the valuable property that, while they 28 MICRO-ORGANISMS AND FERMENTATION. have the power of killing germs, they do not destroy enzymes. This fact has proved of importance in advancing recent studies of enzymes, etc., where it is necessary to inhibit the action of micro-organisms on the susceptible liquids employed. R. Koch first proved the antiseptic action of ether and alcohol, and recent research has brought to light the interesting fact that it is not absolute alcohol, but a 50 to 60 per cent, alcohol that exhibits the strongest disinfecting power. This may be explained by supposing that absolute alcohol absorbs moisture from the surface of the cells, and, therefore, makes them more resistant. The vapour of 75 per cent, alcohol appears to be equal in its action to a current of steam, and a still more powerful action is exhibited by a mixture of alcohol vapour of this strength with formaldehyde vapour. The mixture may be used to disinfect fabrics which would suffer by ex- posure to a temperature of 100° C. Carbolic acid (phenol), which plays an important part in medicine as a powerful antiseptic, cannot be applied in the fermentation industry, owing to its penetrating odour, but owes its interest to the fact that it does not attack enzymes. On the other hand, a whole series of compounds, of which carbolic acid is a component, are made use of in practice. The raw materials of the fermentation industry (rye, wheat, barley, etc.) contain peculiar compounds which, accord- ing to the researches of Jago, Delbriick, Lange, Henneberg, Hayduck, and Seyffert, act as poisons to yeast, and these are assumed to be of an albuminoid character. This action may be observed in the crushed grain or in an aqueous extract if the yeast is added in presence of sugar dissolved in distilled water. In their reaction to such influences, the yeast species do not behave uniformly. Thus, under certain conditions a stimulus may be given to some species, whilst under other conditions the poisonous substance may act destructively even in minute doses. Such is the case with the poisonous body present in rye bran, and also with that contained in a dilute hydrochloric acid (0-1 per cent.) extract of wheaten flour, in their action upon brewer's yeast, whilst with the addition of calcium carbonate, soda, gypsum, etc., the action of these reagents on the yeast is prevented. STERILISATION. 29 As stated above, it has been proved that very minute quantities of poisons may have such an influence on micro- organisms that they actually stimulate them to more rapid growth, often one-sided ; it may be the development of the vegetative organs at the expense of the organs of propagation, or again by bringing about an increase of the fermentative activity. In a few cases that have been closely examined it has been proved that the minute doses which can produce such an action have fairly well-defined limits ; the least excess brings about the opposite action — a weakening of the organisms in question. Thus a minute dose of a copper salt assists the development of the mould, Aspergillus niger, to a very great extent. In the same way Biernacki found that the addition of copper sulphate in the proportion of 1 : 600,000, added to the nutritive value of the liquid, and increased the activity of the ceUs. In larger quantities copper salts exercise a destructive action on yeast ; care must, therefore, be taken that when pure cultures of yeast are introduced into copper vats, these should be carefully tinned. Hayduck (1881) found that small quantities of lactic acid (about 0-5 per cent.) favour the propagation of yeast, and that anything up to 1 per cent, of lactic acid, under the usual technical conditions, is favourable to the production of alcohol. Heinzelmann proved in 1882 that salicylic acid in the proportion of 0-1 gramme per litre reacts favourably on yeast cells, and H. Schulze (1888), that minute traces of poisons, such as mer- curic chloride, iodine, chromic acid, and lactic acid, have the same action (e.g., mercuric chloride in a dilution of 1 : 500,000). Hirschfeld found that by the addition of 0-01 to 0-02 per cent, of hydrochloric acid the acetic fermentation is very consider- ably quickened. Richet proved a similar relation holds good with the lactic acid bacteria, while the addition of 0-5 mgm. of mercuric chloride, or of copper sulphate, per litre, intensifies their fermentative activity. In the same way Effront found that minute quantities of sulphuric acid and of fluorides have a stimulating action in nutritive liquids, both on the rate of propagation and the fermentative capacity of yeasts, but that this varies with the yeast species. The fungi have a curious power of accommodating them- 30 MICRO-ORGANISMS AND FERMENTATION. selves to poisons. By long-continued cultivation it has proved possible to introduce large quantities into nutritive substrata, and at the same time it has been noted in several cases that a marked change of character takes place. It has, however, proved impossible to fix these newly acquired characters ; they are of a purely transitory kind. As soon as the poison is removed the growth reverts to its original character. From the numerous examples, we select the following : — Galeotti accustomed Bacterium prodigiosum to grow on a 2 per cent, carbolic acid nutritive gelatine. Pulst accustomed Penicillium glaucum to withstand continually increasing quantities of poisonous copper salts, whilst its conidia germinated more rapidly than usual. The results obtained by accustoming yeasts to the presence of certain poisons are of special interest in the technology of fermentation. For example, the yeast in distilleries may work in a mash which by treatment with a disinfectant has been ren- dered more resistant to bacteria, a process which takes the place of the usual souring with lactic acid. For this object sulphuric acid, hydrochloric acid, and hydrofluoric acid have been made use of. Effront proved that much smaller quantities of hydro- fluoric acid were required than of the other two. In considera- tion of the different extent to which the yeasts are attacked by the hydrofluoric acid (or fluorides), Effront tried by special cultivation of yeasts to accustom them to work in a mash which contained so much of the reagent that the bacteria were suppressed. He found that the addition of 300 mgm. of hydrofluoric acid to 100 c.c. of liquid completely inhibited the growth of yeast, whilst its fermentative activity was only restricted. If, however, the yeast is gradually accustomed to the poison, beginning, for example, with 20 mgm. per 100 c.c., and rising by degrees to greater doses, a race of yeast will be formed that can multiply even in presence of the original dose. In presence of 200 mgm. per 100 c.c. the fermentative power of the yeast is increased, according to Effront, if it is introduced into a mash which also contains fluorides. In practice about 10 grammes of hydrofluoric acid are used for every hectolitre of mash. Even if this process succeeds in suppressing bacteria in the mash, which is not always the case, DISINFECTION IN PRACTICE. 31 other difficulties may arise when wild yeast are present, for these, according to Holm and Jorgensen, are stimulated in their development by the presence of hydrofluoric acid in the mash. 4. Disinfection in Practice. It has become clear, within the last few years, that the harmfulness of the germs in air and water has been greatly exaggerated, and that far and away the most important source of danger is to be sought in the growth of foreign organisms in the plant itself. The natural result is that increasing attention is being paid to a rational scheme for disinfecting the plant. As the raw stuffs used in breweries, distilleries, etc., form an admirable nutritive medium for micro-organisms, the distribution of these throughout the plant is much more widespread than usual, and it is often necessary, in addition to mechanical cleansing, to attack them by direct antiseptic means. By determination, on the one hand, of the maximum limit of such poisonous substances that can be allowed, and, on the other hand, of the necessary means to secure the desired object, the practical conditions are established. The con- centration must not exceed what is absolutely necessary. In the use of antiseptics it is essential to follow a recognised plan. A summary disinfection is insufficient if the individual parts present different possibilities for the development of foreign organisms. It is, therefore, necessary from time to time, and that frequently, to overhaul every single point in practice, before being able to say exactly where a particular infection has appeared. At certain points antiseptics must be discarded and mechanical means adopted. This is the case when the infection has penetrated so far into the material that the disinfectant is no longer able to attack it. This may occur in the great majority of wooden vessels as they are usually prepared. As many micro-organisms form slime, and may produce thick deposits when allowed an undisturbed development, it is often necessary to use a solvent of the slime before proceeding to actual disinfection, if the germicidal substance is not capable of completely dissolving the slime. 32 MICRO-ORGANISMS AND FERMENTATION. It is an established rule that two disinfectants should not be used simultaneously, or one immediately after the other, especially if their composition is unknown ; otherwise there is danger that they may neutralise each other's action. Thus, chloride of lime and calcium bisulphite should never be used at the same place. The literature of antiseptics used in the industry — Will, Lindner, Brand, Schonfeld, etc. — has grown to considerable proportions. A short resume of the methods of application of the respective substances follows. Ammonium fluoride, especially the acid salt, has a very wide application, owing to its great antiseptic power. It is a crystalline powder, readily soluble in water. In the pure condition it contains about 35 per cent, of hydrofluoric acid ; the commercial product, however, contains a less quantity, and sometimes not more than 20 per cent. It is used for the treatment of pipes, vats, etc. Pipes are filled with a solution containing about 0-5 per cent. In rinsing out vats a 3 to 5 per cent, solution must be used. Ammonium fluoride is not suitable for the treatment of metal, as it slowly attacks it. After treatment, a very thorough washing with water is necessary. Formalin has also been very largely applied in practice. It is an aqueous solution of formaldehyde (40 per cent, by volume or 37-2 per cent, by weight), and it forms one of the most powerful antiseptics. As it does not attack metal, it can be applied more generally than the fluorides. It may be used in the form of gas by soaking cotton wool or cloths in formalin, and hanging them up in the area to be disinfected, or it may be applied in specially constructed lamps, in which, by the imperfect combustion of methyl alcohol, formaldehyde is produced. The most frequent and most successful method of application is, however, to dilute formalin with water, and apply it as a spray to the walls of vats, etc. A solution of 0-5 per cent, of formaldehyde (about 1| litres of the commercial article to 100 litres of water) is most generally applicable. The vessel must then be well rinsed with water, and if the odour cannot be got rid of, ammonia may be applied. Chloride of lime has been used for many years, on account DISINFECTION IN PRACTICE. 33 of its powerful disinfectant properties. Its strong odour limits its application. It is especially used to disinfect racks and slimy walls in rooms where fermentation is going on. To disinfect filter bags in breweries, which often harbour large colonies of bacteria and wild yeast, Will recommends an application of chloride of lime in a solution containing about 1 per cent, of active chlorine (3 to 3| kilos, chloride of lime in 1 hectolitre of water). The mixture of water and chloride of lime is allowed to stand for some time, with occasional stirring ; the clear liquid is then decanted and applied to the filter bags, which are afterwards repeatedly rinsed with water. The dangerous development of micro-organisms on the filter bags may be avoided by cooling down the beer to the lowest possible temperature during filtration. Antiformin is a chlorine preparation which has found considerable application in recent times. It is a clear liquid with a strong alkaline reaction and a weak odour of chlorine. It consists of a crude sodium hypochlorite (cf. Eau de Javelle), and is prepared by decomposition of chloride of lime with soda. The solution is then separated from the precipitated chalk, and caustic soda is added. The liquor contains more than 4 per cent, of active chlorine, and not only possesses great antiseptic power, but also quickly softens organic sub- stances such as sediment, wort, crust, yeast, and slime, so that they can easily be removed. In other words, it acts both as a cleansing and as a disinfecting agent. Care must be taken, however, in applying it to infected wood ; for instance, to the staves of a fermenting vat, as the reagent, owing to its solvent power, can penetrate so far that it is difficult to remove it by rinsing with water. It may be applied in a dilution of 1 to 20. Antigermin appears to be specially adapted for washing down walls. It consists mainly of a copper salt of an organic acid, and the aqueous solution is without smell. It should be dissolved in boiling water, and mixed with lime before applying. Montanin, which is also free from smell, is equally appli- cable to walls and to connecting pipes, vats, etc., but the latter must always be well rinsed. It is a by-product of the glazed- 3 34 MICRO-ORGANISMS AND FERMENTATION. ware industry, and contains about 28 to 30 per cent, of hydro- fluosilicic acid (as aluminium fluosilicate) in a clear solution, pale green or yellow in colour, and feebly acid. The protection of walls by means of this preparation depends upon the pores being sealed by the formation of calcium fluoride, alumina, and silica, imparting to the wall a hard and smooth surface. Mikrosol appears in commerce as an acid green paste containing about 10 per cent, of copper phenolsulphonate, and smaller amounts of copper sulphate, free sulphuric acid and hydrofluoric acid. It is applied to walls in the form of a 2 to 4 per cent, solution. Antinonnin is largely used in order to coat moist walls, and is an excellent preventive of dry-rot, and protects woodwork from worms, etc. It forms a red viscous mass consisting of a potash compound of kresol mixed with glycerine, soap, etc. It is soluble to the extent of 5 per cent, in water. It does not attack either metals or organic substances, and, according to Aubry's investigations, may be applied to advan- tage throughout the brewery, where it cannot come in contact with beer. Pyricit, a new preparation, is a white powder soluble in water to form a colourless and odourless solution. According to Wichmann, a 2 per cent, solution forms a very powerful disinfectant, which does not etch or attack either metal, wood, or glass. It may be applied anywhere, inside or outside. It can be kept for a long time without losing its activity. Sulphurous acid is one of the oldest antiseptics, and is still frequently used for casks. A piece of linen which has been dipped in molten sulphur is set alight and introduced into the cask. The fumes do not, however, penetrate sufficiently to sterilise badly contaminated casks. Hops and occasionally malt are also treated with burning sulphur. In wine fer- mentation sulphurous acid is sometimes added to the must, to destroy the spontaneous germs before adding pure wine yeasts. Calcium bisulphite forms an energetic reagent, and usually contains about 7 per cent, of sulphurous acid. Diluted from three to six times with water, it forms an admirable agent for the treatment of vats and other apparatus, and is especially deadly to moulds. DISINFECTION IN PRACTICE. 35 Salicylic acid has also been applied to vats in the form of a dilute alcoholic solution, which is painted on to the surface, allowed to react for some time, and then washed off with an alkaline liquor, and finally with water. Amongst weaker antiseptics, lime and soda may be men- tioned. Milk of lime, freshly prepared, forms an excellent disinfectant for walls and ceilings, but as soon as the lime has absorbed carbon dioxide from the air it ceases to have any value. Soda, in the form of a 5 to 10 per cent, solution in warm water, is an excellent reagent for dissolving slime from connecting pipes, etc. It must, however, be very thoroughly removed by washing first with warm, and then with cold water. A very dilute soda solution (0-1 to 0-3 kilogramme per hectolitre of water) is of value in swilling new chips. Soda is not well adapted for disinfecting fermenting vats, as it imparts a rough surface to the wood. One of the most important disinfectants throughout the fermenting plant is steam, if care be taken that every part of the vessel to be treated is exposed to its action. Connecting pipes may be sterilised by steam if they do not exceed a certain length. In distilleries, sulphuric acid is used as a disinfectant in the mash to inhibit the growth of foreign bacteria, and to restrict that of yeast. Its application must, however, be kept within certain limits, as the yeast would otherwise be damaged. According to Hayduck, 0-024 per cent, can be used, and even 0-05 per cent, does not appear to prevent the development of yeast. Ozone has found application, in particular for disin- fecting water. In order to bring the gas into close contact with water, the latter is sprayed over a fine-grained material, where it comes in contact with a stream of ozone prepared by means of a high tension electric current, discharged from two electrodes of special construction. It has proved possible by this means to kill a very large proportion of the organisms in water (see the researches of Calmette, Schiider, and Pros- kauer, Ohlmiiller, etc.). Hydrogen peroxide has also been applied to disinfecting water, and preserving milk by Budde's process, which consists 36 MICRO-ORGANISMS AND FERMENTATION. in the application of 0-036 per cent., after which the milk is maintained for three hours at 50° to 52°C. 5. Flasks. All vessels in which cultures are made mustjsatisfy the condition that they are proof to every contamination from without. Pasteur flasks satisfy this demand in the highest degree. The illustration (Fig. 5) shows this flask in the improved form employed in the Carlsberg Physiological Laboratory. When the hopped wort (preferably filter-bag v ort) is boiled, the steam first escapes through the straight t ibe, attached to which is a short piece of rubber tubing ; Via. 5. — Pasteur's tin i^'. (i. -Chamberltnd flask. when this is closed (after boiling for about half an hour) the only outlet for steam is through the bent tube. About twenty minutes later, the flask is removed from the sand-bath. During cooling the germs are deposited in the lowest part of the bent tube, or are not carried beyond the enlargement of the tube, and, therefore, do not come into contact with the liquid. Hence, it is evident that the lower part of the bent tube must be heated whenever the flask is to be agitated or emptied through the straight tube. If the fiask is to be opened and connected with another flask, this must be effected either in a small sterile space, or else the opening and connecting must be carried out in a flame. A Bunsen burner is placed directly in front of the operator. FLASKS. sr the flask to be emptied to the left, and the one that is to receivd the liquid or culture to the right, alongside the burner. Then the tube of the left-hand flask is opened in the flame by quickly removing the rubber tube with its glass stopper ; while the open tube is in the flame, the glass stopper of the flask to the right is quickly withdrawn, and the hot tube of the first flask is connected with the rubber tube of the second flask after the tube has been cooled. The liquid is poured into the second flask, the bent tube of the first flask being at the same time heated. Then the side tube of the left flask is again introduced into the flame, while the stopper of the right flask is replaced directly after it has been passed through the flame ; finally, Fig. 7. the left flask is closed in the flame with its tube and stopper. When the operation is quickly performed the danger of con- tamination is almost excluded. Pasteur flasks will be found indispensable in certain opera- tions ; for instance, in physiological researches where large quantities of liquids are dealt with. The Chamberland flask (Fig. 6) is closed with a ground cap, which terminates in a short, open tube ; this tube is filled with tightly-packed sterilised cotton-wool. The Freudenreich flask is constructed on exactly the same principle (Fig. 7, centre) ; it has, however, a cylindrical shape. 38 MICRO-ORGANISMS AND FERMENTATION. These flasks must only be opened in a sterile cupboard. When gelatine is used the flask must be opened with the mouth downward. For special purposes the Hansen flask (Fig. 7, left) is employed. The ground cap is provided with a cotton-wool filter, and the flask has a small side-tube closed with an asbestos stopper. This flask is used in the author's laboratory for the dispatch of small cultures or of samples from the propagating apparatus.* For this purpose the lower part of the flask is filled with cotton-wool, and some cotton-wool is lightly packed into the cap. The asbestos stopper and the lower edge of the cap must be covered with sealing wax. A flask (Fig. 7, right, and Fig. 8) constructed by the author n carries a small, bent, and open tube in the cap, as a prolongation of the cotton-wool filter. By this means it has proved possible to prevent the evaporation of the contents of the flask for several years, provided that the lower edge of the cap and also , the lateral tube are well closed. This flask is used for pre- serving pure cultures of yeast in a 10 per cent, saccharose solution inoculated with a trace of the yeast. The flask is also suitable for a prolonged preservation of gelatines, if the surface is to be prevented from stiffening. Another flask constructed by the author, shown in section in Fig. 9, is also used for storing pure cultures of bottom and especially of top yeasts which will not stand vigorous shaking ; when further * This apparatus is described in Chapter vi. FLASKS. 39 development is required a drop of the yeast is transferred to a Pasteur flask containing wort. The small bent open tube on the right has its outer extremity packed with cotton-wool, to filter incoming air. The wide tube on the left, which is closed with an asbestos stopper, has its lowest bend on a level with the bottom of the flask. If this tube is connected with the side tube of a Pasteur flask in the flame, and then suction applied to the bent tube of the flask, a minute part of the yeast lying on the bottom of the small flask will be sucked into the Pasteur flask, without disturbing the ' Fig. 10 -Carlsberg flask— Old model. remainder of the yeast deposit. If it is necessary frequently to remove a small portion of a culture, this process may be recommended. Fig. 11.— Carlaberg flask Nt-w Connection between the flask and the bent tube. For the development of very large cultures the Carlsberg vessels (Figs. 10 and 11) are employed. They have a capacity 40 MICRO-ORGANISMS AND FERMENTATION'. of 10 litres, are made of tinned copper, are cylindrical in shape, and conical at the top ; at the apex of the cone a twice-bent tube (c d) with or without an enlargement (e) is either soldered into or screwed into the flask. At one side of the cone is the inoculating tube and glass stopper (a), and near the bottom of the vessel is another tube (6) for drawing off the fermented liquid and the yeast. This tube is provided with a pinch- cock. When the liquid is sterilised, the bent tube is closed with an asbestos or cotton-wool filter, which is tightly packed on to the end (d). In the new model (Fig. 11) the bent tube is ground into the upper part of the flask, and fastened by means of a screw, allowing the whole of this part to be detached, when the flask is to be cleaned ; the filter is screwed into the end of the bent tube. t 6. Nutritive Substrata. With regard to the nutritive substrata, the problem naturally consists in finding those which are best suited to the respective organisms. If they also possess the advantage of being less favourable for the development of competing forms, it is a great point gained. The fact must, of course, be borne in mind, when comparative investigations are made in different directions, that the nutritive liquid must always remain the same. For the investigation of yeasts, hopped - beer wort forms the best nutrient. It is best taken from the filter-bags, because these yield a smaller deposit on boiling in the flasks. It is also suitable for many bacteria and moulds, but for certain bacteria (e.g., lactic acid bacteria) the sweet wort, without hops, is used, and this is also adapted for use with moulds. Amongst the artificial nutritive fluids for yeast, Pasteur's (1860) has an historical interest. The renowned scientist used this to upset Liebig's theory with regard to the indispensability of albuminoids for fermentation. It consists of 100 c.c. of water, 0-075 gramme of the ash of yeast, 10 grammes of sugar, and 1 gramme of ammonium tartrate. A good nutritive fluid can also be prepared from yeast decoction with NUTRITIVE SUBSTRATA. 41 5 to 10 per cent, of sugar. Yeast decoction is an aqueous extract of yeast (about 1 litre of yeast to 2 litres of water, boiled under pressure), filtered, and either neutralised or rendered slightly alkaline with sodium carbonate or lime. For special research, compound liquids may be used containing sugar and the salts necessary for nutriment and normal growth of yeast, including potassium, magnesium, and calcium, phosphoric and sulphuric acids. An admirable means for preserving pure cultures of yeasts is the solution, first used by Pasteur, consisting of a 10 per cent, cane sugar solution. Special nutritive liquids are also used for bacteriological investigation. Cohn's solution has historical interest, and its composition is as follows : — 100 c.c. of water, -05 gramme mono-potassium phosphate, 0-05 gramme tri-potassium phos- phate, 0-5 gramme crystallised magnesium sulphate, and 1 gramme ammonium tartrate. To-day a nutritive broth is chiefly used, prepared by steeping finely chopped beef for a few hours in water, and then boiling and filtering the liquor. The liquid is generally neutralised with soda, or rendered slightly alkaline, and after adding 1 per cent, of peptone and 0-5 per cent, of sodium chloride, it is again boiled, filtered hot, and finally sterilised in flasks or test tubes. Such an extract must obviously vary in composition, and in special cases resort may be had to an artificial nutritive liquid, free from albumen. We may quote that prepared by Voges and Proskauer, con- sisting of 1 litre of water, 5 grammes sodium chloride, 2 grammes disodium phosphate, 6 grammes ammonium lactate, and 4 grammes of asparagin. A. Fischer's base consists of 0-1 per cent, of di-potassium phosphate, 0-02 per cent, magnesium sulphate, and 0-01 per cent, calcium chloride, dissolved in tap water. The solution is then added to peptone, or peptone and sugar, etc., according to the requirements of the particular species of bacteria. For the development of lactic acid bacteria (from milk) O. Jensen uses peptonised milk prepared by treating 1 litre of milk with 10 c.c. of hydrochloric acid and 2 grammes of pepsin. By keeping it in the thermostat and frequently shaking, the precipitated casein is redissolved ; the liquid is then neutralised, cleared with albumen, and sterilised at about 120° C. 42 MICRO-ORGANISMS AND FERMENTATION. For moulds, in addition to beer-wort, fruit decoctions and sugar solutions containing tartaric acid and tartrates are used, or, again, the complicated Raulin's liquid, which is also applicable to bacteria, and consists of — water 1,500 c.c., sugar 70 grammes, tartaric acid 4 grammes, ammonium nitrate 4 grammes, ammonium phosphate 0-6 gramme, potas- sium carbonate 0-6 gramme, magnesium carbonate 0-4 gramme, ammonium sulphate 0-25 gramme, zinc sulphate 0-07 gramme, ferrous sulphate 0-07 gramme, potassium silicate 0-07 gramme. If solid nutrients are required, 5 to 10 per cent, of gelatine is added, or, in the case of cultures which are to be developed at or above 30° C., about 1£ per cent, of agar-agar, a jelly derived' from salt water algae. For the cultivation of ther- mophilous bacteria at 60° to 70° C., Miquel uses carragheen moss instead of agar, in the proportion of 2 to 3| per cent. Slices of potato sterilised in an autoclave are often used as a solid nutrient. Black bread makes an excellent solid sub- stratum for moulds. For the cultivation of the nitrifying bacteria Winogradsky and Omelianski used gelatinous silicic acid. For plate cultures of acid-forming bacteria (lactic acid and acetic acid bacteria) some litmus or, preferably, according to Beijerinck, carbonate of lime (finely precipitated chalk) is added. The gelatine thus acquires a motley appearance, but the colonies of acid bacteria are surrounded by a clear zone, because the acid dissolves the chalk. By the use of zinc carbonate in plate-cultures, the acetic acid bacteria form colonies and display clear zones, whereas the lactic acid bacteria are relatively sensitive to this salt, and their growth is inhibited. Pasteur used liquids exclusively for his work on the ferments. Later, solid media became of great import- ance, and Koch laid the foundation for their application. Plate-cultures are prepared by introducing the growth into the liquefied gelatine, and then pouring the mixture into a Petri dish. When the gelatine solidifies the individuals are separated throughout the mass, and, on development, they appear as colonies, visible to the naked eye. Streak-cultures are those in which a minute portion of the growth is intro- PREPARATION OF PURE CULTURE. 43 duced on to the surface or into the upper layer of the solidified gelatine on a platinum spatula. Stab-cultures are those in which a fraction of the growth is introduced by an inoculating needle into a thick layer of solidified gelatine. Giant colonies are formed by pouring a drop of the inoculated liquid on to a stab in the solid gelatine. 7. Preparation of the Pure Culture. To prepare an absolutely pure culture, it is necessary to make sure by direct observation that the development begins with a single cell, and that this is so completely isolated that during the development no other cell can creep in and render the culture impure. If such a pure culture is required for experiments on a large scale or for actual fermentations, special rules must be observed in order that the absolutely pure growth at first developed shall be protected from every infection during its further growth in a succession of larger flasks. Care must, of course, be taken that the species is developed under the most favourable conditions to secure a vigorous and normal culture. The process in its later stages is described in another section. We are here concerned with the problem of securing the first absolutely pure culture as the point of departure for the mass culture. The desideratum of direct observation presents difficulties in the case of the smallest micro-organisms — bacteria. Whilst it has long proved possible to directly observe single cells of yeasts and moulds on account of the size of their cells, this has not been the case with the great majority of bacteria. In such cases we must be content with methods which give a certain probability for preparing a pure culture. It is only quite recently that the technique has been sufficiently de- veloped to allow of an approximately accurate solution of this problem. Long before there was any attempt to work experimentally with absolutely pure mass cultures, experiments in the culti- vation of micro-organisms had been undertaken with a purely botanical object, to discover what different forms a species may assume, and with this object the development of single cells was followed under the microscope. 44 MICRO-ORGANISMS AND FERMENTATION. As early as 1821, Ehrenberg observed the germination of the spores of certain fungi by careful observations of this kind. The propagation of yeast cells was observed by Mitscherlich (1843), Kiitzing (1851), and F. Schulze (1860), in the same way. A small quantity of top yeast was diluted with beer- wort until it contained only one or two yeast cells ; from a drop of this an ordinary preparation was made, the cover- glass was cemented on to the glass slide, to prevent the evapor- ation of the drop, and the development of the cell was watched under the microscope. Similar cultures were employed by Tulasne (1861) and de Bary (1866), in their famous researches on the germination of spores. A considerable improvement in the method was made by Brefeld during his detailed re- searches on mould, blight and mildew fungi, in which he followed the development of the mycelium until it, in its turn, again formed spores. The infection on the object glass was protected by means of a small shield of paper fastened on to the tube of the microscope, and this was afterwards converted into a moist chamber (1881), after Brefeld had recognised the danger of foreign germs penetrating into the cultures. He diluted the material with water, brought a drop containing a single germ on to the cover-glass, added some nutritive liquid containing gelatine, and placed the cover-glass, with the drop underneath, on to a glass ring (Bottcher's chamber), which was fastened to the object glass. As the apparatus and the nutritive liquid were sterilised, all the necessary conditions were fulfilled for carrying out a culture experiment without contamination. We may here see how improved methods of cultivation have led to the preparation of an absolutely pure culture. By the help of his cultures Brefeld made the interesting observation (1883) in quite a number of fungi — e.g., the smut of wheat, the boil-blight of maize, etc. — that the conidia are able to propagate by direct budding, like yeast, without throwing out new seed-carriers. A short survey follows of the different methods which have been applied for preparing pure cultures on the large scale. (a) Physiological Methods. — At the earliest stage, attempts were made to reach the goal by calculating the probabilities, PREPARATION OF PUKE CULTURE. 45 and treating the whole growth, without condescending to isolate single cells. The physiological methods — " the enriching process " employed by Pasteur, Cohn, and others start with the fundamental idea that the various species occurring in a mixture will multiply unequally according to their different natures, when they are cultivated in one and the same nutritive liquid and at the same temperature, so that those species for which the conditions are unfavourable will be gradually suppressed by the one or more species for which the conditions are favourable. When the growth has developed under the selected conditions for quite a short time, a minute fraction is inoculated into the same nutritive liquid in a fresh vessel at the same temperature, and this process is repeated many times. Different liquids have been employed for such cultures from time to time ; for instance. alkaline liquids for bacterial growths, acid liquids to free yeast growths from bacteria (lactic, tartaric, hydrofluoric acids, etc.). The weak point of all such methods is, that they start from an unknown material — namely, the impure mixture. It, is, therefore, impossible to know what results such a treatment will lead to, for we are not dealing with any true method, as contamination may take place at random. In fact, there is always the possibility that the weaker species are not destroyed, but merely checked and retarded, so that when the stronger species, after reaching their maximum development, become weaker, other species will have a chance of multiplying. This possibility also occurs when the growth is transferred to another substratum. Likewise, there is always the possi- bility that not one but two or more species thrive equally well in the liquid, and, consequently, develop to the same extent. Such, for instance, was the case with brewers' yeast before pure cultures were employed. This yeast often yielded several typically different speoies of " culture yeast," as they are termed, when examined by Hanson's method. Th^ method given by Pasteur for the puri.lcation of brewers' yeast may be mentioned as a marked illustration of the dangers connected with the physiological method of treatment. TL • impure yeast-mass is introduced into a can^-sugar solution. 46 MICRO-ORGANISMS AND FERMENTATION. to which a small amount of tartaric acid has been added. The object of the method is to free the yeast from any disease germs with which it may be infected. Hansen's investiga- tions have, however, proved that, even if the bacteria are suppressed or checked by this treatment, simultaneously the wild yeast, and among them those productive of diseases in beer, will develop abundantly, and in many cases the culture yeast, which it was intended to purify, is entirely suppressed. Even if there is primarily only a trace of the wild yeasts or " disease " yeasts, these are apt to develop to such an extent by Pasteur's treatment that they may eventually form the predominant part of the yeast-mass. The use of hydrofluoric acid or its compounds, such as ammonium fluoride, for the purpose of purifying an impure yeast — brewers' or distillers' yeast — as proposed by Eflront, is liable to lead to the same dangers as the use of tartaric acid. Methodical experiments made by Holm and the author have shown that by treating impure yeast according to Eff rent's process, the growth of wild yeast and Mycoderma species is forced more than that of the culture yeast ; they have also shown that such a dangerous species as Bacterium aceti is in many cases not suppressed at all by the treatment in question, but, on the contrary, multiplies more rapidly in presence of hydrofluoric acid or fluorides. If, now, we ask, whether it is advisable to employ any of the various methods mentioned above for the purification of an unknown and impure yeast-mass, the answer must be in the negative ; and this will be the case whether the culture is intended for purely scientific or for industrial purposes, for the danger will never be excluded that in prolonged cultiva- tion other species than the one desired will gain the supremacy. The starting point being uncertain, it necessarily follows that the result must be so too. In fact, all such methods must now be regarded as antiquated, and as complete failures. Nevertheless, they may possibly be used in isolated cases before proceeding to the preparation of a pure culture. In this way it is possible by suitable treatment of the impure material to secure a preponderance of the group of the desired species in the mixture, so that a pure cultivation PREPARATION OF PURE CULTURE. 47 is facilitated. Thus the treatment described with tartaric acid or hydrofluoric acid gradually converts the mixture into a growth of wild yeasts. If a mass of yeast is strongly con- taminated with bacteria, a cultivation at very low temperatures may possibly suppress the bacteria if the yeast are able to develop under these conditions. If it is desired to obtain a pure culture of the lactic acid bacteria from a mash, the material may be prepared by keeping it a short time at 50° to 55° C. At this high temperature many bacteria cannot thrive, whilst certain species of lactic acid bacteria can stand a high degree of heat, and thus spread throughout the material. In the same way in the cultivation of film-forming bacteria, such as acetic acid bacteria, the growth may undergo a preliminary purification by repeated inoculation of the film in fresh liquids. This process was used by Pasteur in his researches on acetic acid bacteria. To make an approximate separation of a large and small species of yeast in a mixture we may resort to decantation or filtration through a medium which will allow the small cells to pass. It is common to all these methods that with more or less luck it is possible to bring about the preponderance of one or more groups of micro-organisms in a mixture, but it is obviously impossible to obtain in this way the exclusive presence of one particular species. (6) Dilution Methods. — The second group of methods employed for physiological purposes embraces the dilution methods, or " fractional cultivation," the principle of which is to dilute the material to such a degree that it is ultimately possible to isolate a single cell. Brefeld used the dilution process for his botanical investigations of moulds, where he was able, owing to the size of the cells, to insure that only a single cell was present in a small drop of water in the moist chamber. He added sterile nutritive fluid, and observed the growth of the cell. Pasteur utilised air (Etudes aur la biere, 1876) as a diluting medium for preparing pure cultures. He started from the fact that if nutritive liquids are exposed to the action of air, fermentation takes place, excited by the germs which fall on to the surface. To isolate single germs from the yeast mass, he proceeded as follows : — A small 48 MICRO-ORGANISMS AND FERMENTATION. quantity of yeast was dried and ground with powdered gypsum. The fine dust was thrown into the air at as great a height as possible, and whilst the particles were floating down, a series of vacuum flasks were opened. Thus some of the yeast cells which were finely distributed throughout the dust cloud might penetrate singly into some of the flasks. The first application of the dilution method to bacteria was made by Lister (1878). To prepare pure cultures of lactic acid bacteria he first determined microscopically the number of bacteria in a minute drop of sour milk, counting them in several fields of the preparation, and thus calculating their whole number. He then estimated the amount of sterilised water it was necessary to add so that after dilution there would be on an average less than one bacterium in each drop. With five such drops he inoculated in one case five glasses containing boiled milk. The result was that the milk in one of these coagulated, showing that it contained Bacterium lactis, whilst the four other glasses remained unaltered, and did not show the presence of bacteria. The same method was subsequently employed by Nageli and Fitz (1882). In comparison with the physiological methods the dilution method now described is a distinct advance ; indeed we have thus approached much nearer to the goal. On the other hand, it is clear that, even if the dilution is carried as far as in the case mentioned, in which only one of several flasks shows development, it is not yet proved that this one flask has received only one germ. Thus, there is still great un- certainty, even in cases where the individuals with which we are working can be counted. Moreover, it is extremely difficult to count individual bacteria, and often, indeed, quite impossible. In all cases the accuracy of such calculations is very questionable. Thus, the problem remains to be solved : How are we to distinguish the flasks which have only received one cell from those which, notwithstanding calculation, have been infected with several cells ? For the bacteria, no means have as yet been found of solving this difficulty. In the case of yeast the process was further developed by Hansen (1881). He employed dilution with water, in the following manner : — The yeast developed in the flask is diluted PREPARATION OF PURE CULTURE. 49 to a given proportion with sterilised water, and after vigorous shaking, the number of cells in a small drop of the liquid is determined. The counting, in this case, is easily carried out by transferring a drop to a cover-glass, on the centre of which some small squares are engraved, which form a starting point for the eye, and this is then attached to a moist chamber (Fig. 4) ; the drop must not be allowed to extend beyond the limits of the squares. The cells present in the drop are then counted. Suppose, for instance, that 10 cells are found ; a drop of similar size is transferred from the liquid, which must first be shaken vigorously, to a flask containing a known volume — e.g., 20 c.c. of sterilised water. This flask, then, will in all likelihood contain about 10 cells. If it is now vigorously shaken for some time until the cells are equally distributed in the water, and 1 c.c. of the liquid introduced into each of 20 flasks containing nutritive liquid, then by calculation half of these 20 flasks should receive one cell each. If the infected flask is strongly shaken and then allowed to stand, the single cells sink and remain on the bottom. It is evident that if a flask contains three cells, they will, in the great majority of cases, be separated by the vigorous shaking, and be deposited in three distinct places on the bottom. After some days, if the flask is raised carefully, it will be observed that one or more white specks have formed on the bottom of the flask. If only one such speck is found, then in all probability the flask has only received a single cell. It was by this method that Hansen prepared all his earliest pure cultures, with which he carried out his fundamental researches on alcoholic ferments. Solid nutrient media have also been employed for the preparation of pure cultures by the dilution method. The foundation of such methods was laid by Schroeter (1872), who, in his researches on pigment-bacteria, employed slices of potato as a nutrient. He had observed that when such slices had been exposed for some time to the air, specks or drops of different form and colour made their appearance. Each of these specks usually contained one species of micro- organism. Koch considerably developed and improved this method. 4 50 MICRO-ORGANISMS AND FERMENTATION. He at first prepared his pure cultures by means of streak infections in nutrient gelatine. He afterwards devised a far better method, the plate-culture method (1883). The process is as follows : — A trace of the crude culture is transferred to a large proportion of sterilised water. From this a small quantity is transferred to a test-tube containing, for instance, a mixture of meat-broth and gelatine warmed to 30° C. The tube is shaken in order to distribute the germs, and the con- tents poured on to a large glass plate, which is then covered with a bell-jar. The gelatine quickly sets and the germs are enclosed in the solid mass. In a few days they develop to colonies — dots or specks which are visible to the naked eye. The purity of the bacterial growths in the gelatine is ascertained, according to Koch, partly by their appearance. An improvement in the method consists in the use of glass dishes with lids instead of glass plates, the Petri dishes (introduced by Salomonsen), into which the liquefied gelatine is poured : or the " roll-tubes " of Esmarch may be used, prepared by continuously rotating a test-tube round its longer axis until the inoculated gelatine has set in the tube, so that the whole of the inner surface is covered. When species are being developed which require a high temperature (at which gelatine would be liquefied), plates are made of agar, or of agar and gelatine. The growth can be mixed with the liquefied material, or else spread over the surface of the solid, either by strokes of a platinum pencil, or by stabs with an inoculated needle. After selecting colonies, which appear to be pure, from a plate prepared in any one of these ways, a new plate-culture may be prepared from one colony. If all the colonies that develop on this plate are pure, it is probable that we are dealing with a pure culture.* When regarded more closely it will be seen, however, that there is no essential difference between the distribution of * Great importance is ascribed to the appearance of colonies of bacteria on gelatine, to their colour, shape, the nature of the edge, etc., and whether they liquefy gelatine or not. Other characters of the gelatine cultures are taken from the streak- and stab-cultures. According to Hansen, colonies of many species of yeast on gelatine plates exhibit characters of great value. The giant cultures of Lindner are also used. PREPARATION OF PURE CULTURE. 51 the germs in liquefied gelatine, and Lister's method of dilution by means of liquids. The same uncertainty is always present ; neither the macroscopical observation of the appearance of the colony nor the microscopical examination of its contents gives any surety of its only containing one species. The only possibility of securing a really pure culture in the gelatine consists in the direct observation of one individual germ and its development. Hansen did this for yeast by using Bottcher's moist chamber. The lower side of the cover-glass is covered with a layer of wort-gelatine, in which the yeast cells are distributed. On account of the size of the latter, it is possible to see whether a single cell lies so wide apart from other cells that the colony developed from it will form a pure culture. The chamber is then either allowed to remain under the Fig. 12. — Jorgensen's moist chamber with etched squares and numbers. microscope, in order that the propagation of the germs may be directly followed, or the positions of well isolated germs are marked, either by dividing the glass-cover into small squares, or by means of the object marker, and the apparatus is placed in the incubator until the colonies are fully developed. The cover-glass is then lifted off and placed under a bell-jar, so that the gelatine layer is turned upwards, and the colonies are transferred into flasks. In the author's laboratory moist chambers like that represented in Fig. 12 are used, the cover- glass being etched with 16 squares and numbers. The situa- tion of the cells is then marked on a sketch plan, which shows all the etched numbers and squares. The author has altered the process by cementing the cover-glass on to the glass ring, and fastening the latter to the object glass with vaseline. To remove the chamber, the ring is lifted off, and this is a 52 MICRO-ORGANISMS AND FERMENTATION. more convenient and more certain process than lifting the cover-glass, for it is possible to transfer the colonies without inverting it. On one cover-glass there may be 50 to 60 well isolated germs. When the colonies are conveyed to the flasks by means of a small piece of platinum or copper wire, which has been previously ignited and cooled, the culture is moment- arily in the air, and is then exposed to contamination. But the danger of contamination at this, the single weak point, is reduced to an insignificant minimum, and disappears if the operation is performed in a small enclosed sterile space ; for instance, in a small cupboard with glass sides sufficiently large to admit the apparatus and the operator's hands. In this way the transference of the colonies is effected with all possible security. From the first flask the culture can be transferred without contamination to a continually increasing number of larger flasks. For the pure cultivation of brewery, distillery, and wine yeasts, vigorous cells must be conveyed to the gelatine in the moist chamber. According to J. C. Holm, on an average only about 4 per cent, of the inoculated cells do not develop, whilst from a growth of yeast that is taken at the end of the fermentation, in which the cells are weakened, about 25 per cent, do not develop. It is usually preferable to convey a small average sample of the yeast into wort or must, and then to use the very young growth, which is developed when the first trace of fermentation is observed. To decide whether any of the selected yeasts are of value for industrial purposes, a large number of cells must be isolated, as indicated by the author as early as 1885. After years of experience, it has proved impossible to speak of a preponderant species or race from which any individual can be chosen. The single type or species contains within itself so many varieties which have come to development under the conditions existing in practice, that a careful choice must be made from these. A thorough study of a type by means of comparative experi- ments will show which of the cultures is of the greatest value in practice. As early as 1883, Koch's method of plate-culture was tested by Hansen. He prepared a mixture of two species of PREPARATION OF PURE CULTURE. 53 yeast which can be distinguished from each other microscopi- cally— viz., Saccharomyces apiculatus, and a species of the group 8. cerevisice. This mixture was introduced into wort-gelatine, and after shaking was poured on to a glass plate. Of the specks formed, about one-half contained one species exclusively, the other half the other species, and in one of the specks both species were found. A similar control was carried out for bacteria by Miquel (1888), who introduced 100 colonies from a plate-culture obtained in an air analysis into 100 flasks containing meat- broth with peptone. The examination of the growths de- veloped in the flasks showed that they contained 134 different species of micro-organisms. This evidently depends upon the fact that it is very difficult, and often quite impossible, to separate all germs of bacteria and other organisms from each other by shaking the gelatine mixture. Holm has subjected the method to a thorough analysis (1891), in the case of a large number of yeast species, abso- lutely pure cultures of which were prepared by the Hansen method. The result of 23 series of experiments with different mixtures was that only in a single case were 100 colonies developed from 100 cells. In all the other series the method proved faulty. In the most unfavourable case 100 colonies were formed from 135 cells, and the average number obtained was 100 colonies from 108 cells. This proves the plate method to be defective also in the case of yeast. A modification of Brefeld's culture of a single cell in a hanging drop is that known as the drop culture, introduced by P. Lindner in 1893. It consists in conveying to a cover-glass a number of small drops of a diluted culture in a nutritive liquid by means of a mapping pen. The cover-glass is fastened by a ring of vaseline on to a hollow-ground object glass, and those drops are noted that contain only one cell. Care must, therefore, be taken that the drops do not flow together before the pure culture is conveyed to a flask. Burri attempted to solve the problem of preparing pure cultures of bacteria under direct observation of single cells by the help of his Indian ink point culture. He dilutes ordinary liquid Indian ink with water in the proportion of 1 to 10, and 54 MICRO-ORGANISMS AND FERMENTATION. after sterilisation infects with the bacteria, and then dilutes to such an extent that small drops of 0-1 to 0-2 mm. diameter contain on an average a single germ. Such drops are placed at suitable distances with a mapping pen on the surface of a layer of nutrient gelatine, where they immediately evaporate, and are then protected by flamed cover-glasses. Much smaller drops can be deposited on gelatine than on a cover-glass. The con- tents of these specks can then be controlled under a high power, and those noted which contain a single germ, a process that is rendered easier by the fact that the bacteria appear clear on a greyish-brown ground. If they can grow in gelatine the development is allowed to continue, but if they require high temperatures they may be conveyed to an agar plate by cautiously raising the cover-glass, and as the speck of ink is more firmly fastened to the glass than to the gelatine, the germ is carried with the glass, so that it can be conveyed along with this to the agar plate. After removing the germ from the gelatine plate a drop of nutritive liquid can be placed on the ink fleck of the cover-glass, and so the germ may be developed in a liquid. The process can also be used for the cultivation of anaerobic bacteria. Anaerobic bacteria demand special methods* of cultivation, in which the atmospheric oxygen must be removed both from the substratum and from the space in which the bacteria are growing. Pure cultivations may be carried out in nutrient gelatine or agar, in tubes filled almost to the top, the bacteria growing in the bottom layers. A still better process consists in removing the air from the test tubes by means of an air piimp. whilst the glass is immersed in water at 30° to 35° C., after which it is hermetically sealed. Another method is to remove the air with a current of hydrogen. This is conveniently carried out in the following way (Frdnkel) : — A wide test-tube is fitted with a stopper with two holes carrying two glass tubes, one of which reaches to the bottom of the test-tube ; the other terminates just below the stopper. When the vessel has been covered with nutrient gelatine or the like, and sterilised, it is inoculated with the growth, and a stream of hydrogen is passed through the long tube. The tube is sealed up as soon as the air is completely COUNTING YEAST CELLS. 55 driven out. The stopper is sealed with paraffin wax, and if gelatine or agar is being used the test-tube is rotated round its longer axis until the material has solidified. The bacteria develop slowly on the inner surface of the glass. The cultivation can also be carried out by utilising a sub- stance that will absorb oxygen — e.g., pyrogallic acid (1 gramme of the dry reagent in 10 c.c. of a one-tenth normal potash solution). To carry this out the open test-tube or plate- culture is placed in a larger air-tight test tube or vessel con- taining the reagent. The absorption of oxygen goes on slowly, and requires 24 hours or more. The culture may also be covered with paraffin, vaseline, oil, plates of mica, etc. Reducing substances like grape sugar, especially in an alkaline solution, or minute quantities of formic acid or sodium indigo sulphonate may be added to the nutritive substance, in order to favour the growth of anaerobes. If it is wished to ascertain with certainty, in using one of these processes, when all oxygen has disappeared, a con- centrated alcoholic solution of methylene blue may be used as an indicator. A few drops are added to the nutrient, and as soon as the last trace of oxygen is absorbed or removed the indicator will be entirely decolourised. The same applies to the addition of indigo carmine (neutral sodium indigo sulphonate). 8. Counting the Yeast Cells. The multiplying capacity of the yeast cells can be estimated by directly counting the cells that are present in a given volume of the liquid at different stages of the fermentation. Experiments on these lines have been undertaken especially by Delbriick, Durst, Hansen, Hayduck, and Pedersen, whilst Fitz has applied the method of counting to bacteria. The counting is performed by means of an apparatus constructed by Hayem and Nachet, and by C. Zeiss (Fig. 13), which was first employed for counting the corpuscles of blood (hence termed hcematimeter). Panum was the first to employ this apparatus for counting micro-organisms, in order to 56 MICRO-ORGANISMS AND FERMENTATION. determine their multiplying capacity. The hsematimeter consists, as shown in the diagram, of an object glass on which a cover-glass of known thickness (0-2 mm.) is cemented, and from the centre of which a disc has been cut out. A small drop of the liquid containing the cells is brought into the cavity thus formed, a second cover-glass is placed over the opening, and thus rests on the cemented and perforated cover-glass. The drop of liquid must not be so large that the pressure of the cover-glass causes it to flow out from the enclosed space, yet it must be high enough to be in contact with the cover-glass. The thickness of the layer of liquid is then known. In order to determine the other two dimensions, and thus be able to work with a given volume of liquid, one of the well-known forms of micrometer is introduced into the eye-piece of the microscope. It may consist of a thin piece Fig. 13. — Haematimeter — a, object-glass; b, cemented cover-glass with circular opening ; c, cover-glass. of glass on which 16 small squares are engraved. The actual value of each of these squares is known when a given system of lenses is employed, and thus, when the square is projected on the object, a small prism of known volume is defined. In certain cases it may be more expedient to make use of an appliance constructed by Zeiss, of Jena, from the instructions of Thoma, which consists of a fine system of squares of known size, engraved on the object-glass itself at the bottom of the cavity. This also improves the microscopical definition of the cells which are on the bottom of the chamber. When it is merely desired to determine the rapidity with which the cells multiply, or to make repeated observations of the number of cells in the same volume, it is quite superfluous to determine its size ; it is simply necessary to work always with the same volume. COUNTING YEAST CELLS. 57 The sample taken should always be a fair average. In most cases it must be diluted and thoroughly agitated for a long time, in order to obtain an equal distribution of the cells ; the specific gravity of the liquid must also be such that it will allow the cells to remain suspended in it for a short time. A small drop is then withdrawn in a capillary tube, transferred to the counting apparatus, and covered with the cover-glass. The apparatus is allowed to remain at rest for some time, in order that the cells may settle to the bottom of the enclosed space, and on this account the specific gravity of the liquid must not be greater than will allow this to take place in a convenient time. Both these require- ments are generally satisfied by the wort employed in breweries. If it is found that the determinate volume contains too many cells to be counted with certainty, the liquid must be diluted. This may be advisable for other reasons, partly to prevent the formation of froth, which may otherwise form abundantly from the violent agitation, and partly to isolate the single cells which frequently cluster as colonies in the wort, and are not always separated by shaking. Finally, it is necessary, whilst the counting is going on, to arrest the de- velopment of the yeast cells in the sample. Hansen found that dilute sulphuric acid (1 to 10) on the whole answers these requirements ; hydrochloric acid, ammonia and caustic soda may also be used, but they are not so good. If very great dilution is required, distilled water may be added, after the addition of one to two volumes of dilute sulphuric acid. When the different volumes of liquid are measured with accuracy, and particular care taken that the cells are thoroughly distributed by vigorous and prolonged shaking, the determin- ation can be made with great accuracy. Two similar dilutions must always be made, and samples taken from each for count- ing. As a matter of course, experiments must also be made to determine the number of the small squares, the cell contents of which must be counted to arrive at a true average. Such counting and determination of the average numbers is con- tinued until the number finally obtained is found to have no 58 MICRO-ORGANISMS AND FERMENTATION. further influence on the average value. The number of countings necessary, and the accuracy generally, depends on the experience and care of the observer. Hansen found that, as a general rule, it was sufficient to count the cells in 48 to 64 small squares. 59 CHAPTER II. BIOLOGICAL EXAMINATION OF AIR AND WATER. THE investigations into spontaneous generation already referred to naturally led to the study of the organisms in air, and after Pasteur, in particular, had demonstrated that air contained, not bacteria only, but also fungi giving rise to alcoholic fermentation, air analyses acquired an interest for the zymophysiologist, and for the fermentation industry. Such comprehensive researches are now available that it has been possible to arrive at an idea of the biological composition of the air on a large scale, and to form a judgment of these conditions in relation to the brewing industry. At first, when it became known that crowds of living germs, capable of development, could occur even in very small volumes of air, there was a natural inclination to exaggerate their effect in practice, and to attribute any excessive growth of disease germs in a fermentation to the direct influence of the air. An exhaustive study of the conditions occurring in practice, carried out in recent years under systematic biological control, has shown that this influence had been exaggerated, and that it is possible, even where an air analysis has shown the presence of numerous germs, capable of producing disease in a fer- menting liquid, to suppress the partly dried and weakened germs falling into the liquor by the addition of the excessive number of yeast cells contained in the pitching yeast. Large growths of disease-producing organisms were only found in practice if they had been allowed to develop on certain infected areas. The germs in the air are thus only indirectly the cause of disturbances in practice, and under normal conditions can seldom be of importance. 60 MICRO-ORGANISMS AND FERMENTATION. The majority of air analyses have been undertaken with a view of throwing light on the obscurity which surrounds most contagious diseases, nearly all of which are, as is well known, attributable to the agency of micro-organisms. With regard to the organisms of fermentation, these have been investigated by Pasteur, and, later, especially by Hansen. The French savant stated that, whilst these germs are always floating about in the air, they are present in much larger quan- tities in the dust which settles on the vessels and apparatus employed. The actual fungi giving rise to alcoholic fermentation are present in comparatively small numbers in the air, whilst the germs of moulds are more frequent ; he further showed, as was subsequently done by Tyndall, that the germ-contents of the air vary both with regard to quantity and species. These results were obtained by exposing beer-wort, wine- must, or yeast-water containing sugar, in open, shallow dishes, at different places, and examining their contents after some time for microscopical germs. Pasteur also employed for this purpose the so-called vacuum flasks, containing nutritive liquids and rarefied air. On opening the flask a sample of germ-laden air could be drawn in. The most important air analyses undertaken in recent years are, without doubt, those carried out by Miquel, the director of the laboratory specially arranged for this purpose at Montsouris, near Paris. His fellow-worker, Freudenreich, has also made valuable contributions to our knowledge of this subject. Miquel performed his first experiments with a so-called Aeroscope (Fig. 14), which is constructed in the following manner : — A bell-shaped vessel, A, is provided with a tube, C, through which air can be aspirated. A hollow cone, shown in the left-hand figure, is screwed into the bottom of A ; the mouth of the cone, B, points downwards ; in the apex, D, of this cone there is a very fine opening through which the air is aspirated, and immediately over this opening is fixed a thin glass plate covered with a mixture of glycerine and glucose. The particles carried in by the air settle to some extent on the viscous mix- ture. The intercepted micro-organisms are distributed as equally as possible on the glass plate, and counted under the AIR AND WATER. 61 microscope. This method is defective in so far as it gives no information on the most important point — namely, which and how many of the intercepted germs are actually capable of development. In order to determine the number of germs capable of development, and also their nature, Miquel employs the following apparatus (Fig. 15) : — The flask A has fused into it a tube, R, tapering below and nearly reaching to the bottom ; the upper end of this is fitted with a ground cap, H, provided with a narrow filter- tube containing sterilised cotton-wool, asbestos, or glass-wool, as. On one side of the flask is a tube, Asp, which is constricted in the middle, and is provided with two cotton-wool plugs, w and w. On the other side is another Fig. 14. — Acroscope. Fig. 15. — Miquel's apparatus for air analysis. glass tube connected by rubber, k, with the tube B, which is drawn out to a point, and closed by fusing the end. The flask is partly filled with distilled water, and the whole appar- atus sterilised. When the apparatus is to be used, the tube Asp is connected with an aspirator (e.g., a bottle filled with water and provided with an outlet cock) ; the cap H is taken off, and the air then passes, bubble by bubble, through the opening o, through the water g, and out through the cotton- wool plugs of the tube Asp. Since all the germs in the air are not retained by the water when the air-bubbles ascend through the latter, the cotton-wool plug w is intended to catch those which get past the water. When the experiment is finished, the cap H is replaced over the tube JR. By blowing through Asp, the liquid is made to ascend in E, in order that 62 MICRO-ORGANISMS AND FERMENTATION. any germs which may have settled on the walls of the tube may be washed down into the liquid. Then, by blowing with greater force, the inner cotton- wool plug w is driven down into the liquid, and its germs shaken off into the latter. After sterilising the thin tube B in a flame, the point is nipped off, and the liquid is now — by blowing through Asp — trans- ferred, drop by drop, into a large number of flasks containing sterilised broth. The main object then is, by means of preparatory experi- ments, to obtain such a dilution of the air-infected water that a considerable proportion of the small flasks (one-half for example) remain sterile after inoculation ; or rseveal samples of the water may be diluted to different degrees, and a series of flasks inoculated from each dilution (" fractional cultiva- tion "). If a large number of the flasks show no develop- ment of organisms, there is a certain probability that in each of the remaining flasks in which growths have developed, only one germ has been sown. A simple calculation will then show how many germs capable of development in the medium employed were present in the volume of air aspirated through the original flask. By these methods of investigation Miquel found that similar volumes of air in the same locality contained at different times a varying number of bacteria. Continued rain purifies the air from bacteria to a marked extent, and their number continually diminishes as long as the earth is moist ; but when the ground dries, it gradually increases again. Thus in the dry seasons of the year the number of bacteria is usually the greatest, whilst the moulds, which thrive best in moisture, and carry spore-bearing hyphae, which project upwards, are most abundant during the wet seasons. The purest air is found in the winter time ; the air of towns is less pure than that of the country ; germ-free, or nearly germ- free air is found at sea and on high mountains. In certain places — hospitals, for instance — the air has been found to be very rich in bacteria ; in one case even fifty times richer than the air in the garden at Montsouris. An entirely different method for determining the organisms contained in air is that employed in Koch's laboratory, and AIR AND WATER. 63 more completely developed by Hesse. A glass tube, about 1 meter long and 4 to 5 cm. wide, is closed at one end with a perforated india-rubber membrane, over which another non-perforated cap is bound. A little liquefied nutrient gelatine is then poured into the tube, after which the other end is closed with an india-rubber stopper, through which passes a glass tube plugged with cotton- wool. The whole apparatus is then heated sufficiently to render it sterile, after which the tube is placed in a horizontal position, so that the gelatine sets in a layer in its lower part. When the air is to be examined, the outer india-rubber cap is removed, and air slowly drawn through the tube. The germs contained in the air settle down on the gelatine, and after the aspiration is concluded the tube is again closed and placed in the incubator, where some of the germs produce visible colonies, which are easily counted. The results show that with a sufficiently slow current of air, the bacteria, which are often floating about in the air in larger or smaller aggregations, frequently clinging to dust-particles, small fibres, or splinters, settle sooner than the mould-spores ; so that the gelatine in the fore part of the tube generally showed a preponderance of the bacteria colonies, whilst the mould-spores developed further on. Miquel's method is to allow the air to pass through a hollow cylinder of solidified gelatine, in which the germs are retained. Hueppe, v. Schlen, and others use liquid gelatine for air analyses, the air being aspirated through the gelatine, after which the latter is poured on to glass plates. Frankland, Miquel, Petri, and Ficker use porous solid substances for the filtration of air for analytical purposes ; as, for example, powdered glass, glass-wool, sand, sugar, etc. The sand-filter employed by Petri is 3 cm. long and 1-8 cm. wide. It is packed with sand, previously ignited, the size of the grains being from 0-25 to 0-5 mm. Two such sand filters are placed one behind the other in a glass tube. In the first filter all the dust-particles containing germs should be retained, whilst the second filter serves as a control. The sand charged with germs is distributed in shallow glass dishes and covered with liquid gelatine. The germs accompanying the dust- particles will then form colonies in the gelatine. 64 MICRO-ORGANISMS AND FERMENTATION. When samples of the air contents are to be sent from one place to another, these air filters will answer the purpose. On receipt of a sample, the sand may be washed into gelatine or, preferably, into sterilised water. After vigorously agitating the water, it is added in drops to flasks containing nutritive liquid, or it may be used in plate-cultures. When samples of air are to be sent to the author's laboratory short cylindrical glasses are used, having india-rubber stoppers, which project well beyond the mouth of the glass. The latter are half-filled with sterilised water or with the nutritive liquid in actual use. When the glass is opened at its destination the stopper is placed by it with the wet end turned upwards, care being taken, of course, that this is not touched. A suitable time having elapsed, the stopper is replaced and tied down. Miquel has raised an objection to the employment of gelatine plates for this purpose, based upon numerous experi- ments. He asserts that many bacteria, when exposed to a temperature of 20° to 22° C., require a fortnight's incubation before developing distinct colonies in gelatine ; on the other hand, there are species which very soon liquefy the gelatine, thus rendering further observation impossible. The same is the case with the moulds, which often spread all over the plate in a few days. Thus, it becomes necessary to count the colonies at so early a stage that many of them are not yet visible. An additional drawback to the gelatine plates is, that the development cannot take place at a temperature higher than 23° to 24° C., otherwise the gelatine will liquefy, but many species of bacteria present a characteristic development only at considerably higher temperatures. Other species, moreover, do not develop in gelatine at all, but only in liquids. Finally, it is urged as a very material objection to the gelatine plates that many of the colonies consist of several species. Miquel proved this by introducing the colonies, one by one, into meat decoction with peptone, and then again preparing plates from these growths. This is in part due to the fact that the bacteria, as shown by Petri, often occur in aggregates in the air, and these will either fall directly on to the gelatine plate or become mixed in the liquid gelatine, where it would AIR AND WATER. 65 be very difficult to separate the individuals from each other by agitation. E. C. Hansen's investigations of the air were made between 1878 and 1882. His main object was to throw light on ques- tions affecting the fermentation industries. As is well known, his researches on Saccharomyces apiculatus (1880) were partly based on work of this nature. Since the question concerned the organisms which occur in brewing operations, the choice of a nutritive liquid was easily determined — namely, wort as ordinarily employed in breweries. The apparatus used consisted either of Erlenmeyer flasks closed with several layers of sterilised filter paper, the contents of which were boiled for a certain time, or of vessels similar to Pasteur's vacuum flasks, the necks of which were drawn out to a fine point, and closed with sealing-wax while the contents were boiling. A little below the point a scratch was made with a file, so that the point might be easily 'broken off when it was desired to admit air. When these flasks had been filled with the air of the locality to be examined, they were again closed with sealing- wax and thoroughly shaken in order to mix the contents of the infiltrated air with the liquid. The flasks were then put aside for a longer or shorter time, lasting in some cases for six weeks, and their contents examined under the microscope. In these investigations Hansen often found that the wort remained bright and apparently unchanged, even although a growth had taken place. Hence, the examination with the naked eye alone cannot be relied on. He names the following forms which, when present in a feeble state of growth, cannot be detected macroscopically : — Aspergillus, Mucor, Penicillium, Cladosporium, Bacterium aceti and Pasteur •ianum, and Myco- derma cerevisice. .Even when these micro-organisms have formed vigorous growths, the wort used has remained bright. It was further shown that pure cultures may often be obtained by the use of these flasks, when only one species gained access to the flask along with the air. It very seldom happened that three or four species were found in the same vessel. This arises from the fact that only a very small volume of air enters each flask. The advantages of this are evident : — 66 MICRO-ORGANISMS AND FERMENTATION. A true knowledge of these germs can only be obtained when they have developed ; in cases where several germs penetrate into the same flask, the strongest germ would by its growth, in all probability, prevent the development of the others, so that these would not be detected in a subsequent examination. At the same time this method necessitates the opening of a large number of flasks, which makes the operation cumbersome and costly. As the flasks only show what was present in the air at the moment of opening, Erlenmeyer flasks were also used to give supplementary information, for which purpose they were allowed to remain in the same locality for a long time, in some cases as long as 48 hours. After these preliminary remarks, we will give a brief summary of the results obtained by Hansen. He confirmed the statement made by Pasteur and Miquel that the air at adjacent places, and at the same time, may eon- tain different numbers and different species of organisms ; and he found that this holds good for adjacent parts of one garden. Hansen mentions, amongst other factors determining the distribution of micro-organisms, that those forms, for instance, which in the first half of July commonly occurred under the cherry trees in the garden, were in the latter half of the same month entirely absent from this locality ; further, that organ- isms which at one time could be found under the cherry trees, but not under the vines, were to be found later only under the latter. As a proof of the inequality of distribution of the organisms, he showed that flasks opened in the same place in the same series of experiments often had the most diverse contents. The experiments with vacuum flasks have further taught us that the micro-organisms of the air often occur in groups or clouds, with intermediate spaces, which are either germ-free or only contain a few isolated germs. As the organisms are not generated in the air, but on the earth and on fruit, it follows that their presence in the air must be dependent on the condition of the surface of the ground and of the fruit, which again depends to some extent on the weather. Hansen's numerous analyses have further proved that the Saccharomycetes comparatively seldom occur in the dust of AIR AND WATER. 67 the air. Their number in the open air increases from June to August to such an extent that flasks at the end of August and the beginning of September are frequently infected with these organisms, after which a decrease takes place. The Saccharomycetes which are found at other times of the year in the atmosphere may be regarded as unimportant in numbers and accidental in occurrence. As most species of the Sac- charomycetes have in all probability — like Saccharomyces apiculatus — their winter quarters in the earth, and their propagation areas on sweet succulent fruit, the latter must be considered as the most important source of contamination. During the same season bacteria are also found in the largest numbers. This constitutes a real danger in technical opera- tions, since wort, when spread in a thin layer on the open coolers, is exposed to a source of contamination from the atmospheric germs. Bacteria are found in the flasks in somewhat greater number than the Saccharomycetes, whilst the moulds occur in still greater numbers. Amongst the latter Cladosporium and Dematium are especially prevalent in gardens, and after these Penicillium ; whilst Botrytis, Mucor, and Oidium occur less frequently. After Hansen had thus demonstrated which of the micro- organisms existing in the open air are capable of developing in flasks with sterilised wort, he proceeded to communicate the results of his examination of different parts of the brewery. When grains (draff) are allowed to stand in the open air, they evolve, as is well known, acid vapours, and since they always harbour a rich growth of bacteria when they remain exposed for a short time, the question naturally presents itself, what is the condition of the air in the neighbourhood of heaps of grain ? It was found that only 30 per cent, of the flasks opened in these areas became infected, and of these 3-6 per cent, with Saccharomycetes and 2-4 per cent, with bacteria, whilst parallel experiments in the garden gave a contamination of about 44 per cent., of which 8-5 per cent, were bacteria. The air near the grains thus contained fewer bacteria than the air in the garden. The most abundant contamination was that of moulds, as in all other localities. 68 MICRO-ORGANISMS AND FERMENTATION. After a thorough examination Hansen came to the conclusion that, without doubt, scarcely a single organism which entered the flasks proceeded from the grains. The result tends to show that in this, as in other cases, air does not take up organisms from moist surfaces. This, however, must not be misunderstood to mean that grains may be allowed to accumulate, without risk, and that after removal, the residue may be exposed to the weather. It is clear that this would constitute a great danger. When the remains become dry and are blown about in the air as dust, masses of bacterial germs will be carried up at the same time, and will, without doubt, constitute a source of frequent bacterial contamination. For this reason, places where grains have remained for any length of time must be washed with lime- water or, preferably, with chloride of lime.* In a corridor which led to a room where the barley was turned, the flasks always received a greater contamination than anywhere else ; bacteria especially were found in them in great abundance. On the malt floors the condition of the air was also charac- teristic ; it always contained a very strong contingent of mould spores. In the case in question these consisted of Eurotium aspergillus, which was otherwise rare. On the malt itself, as usual, Penicillium glaucum occurred most frequently. The greatest interest, however, attaches to the examination of the different fermenting-rooms, partly in the " Old Carlsberg " brewery and partly in the brewery " N." In the former the air contained fewer organisms than in any of the rooms examined during the whole research ; in the fermenting- cellars of the brewery " N," on the contrary, a large number of flasks (55, 75 to 100 per cent.) were infected. The organisms which occurred in the air of these cellars were — Saccharo- myces cerevisice, Mycoderma cerevisice, S. Pastorianus, S. ettipsoideus, Torula, and other yeast-like cells ; further, Penicillium, Dematium, Cladosporium, and rod bacteria. * The germs are not killed during the treatment of the grain in drying machines. Such forms of apparatus, therefore, constitute a very great danger in the brewery, since dust-clouds of bacteria may be transported from the dried grains to the open coolers or into the fermentation vessels. AIR AND WATER. 69 Hansen was thus enabled, by a favourable chance, to contrast the state of the air in the most important part of these two breweries ; on the one hand an almost germ-free air, on the other hand an atmosphere teeming with germs. That the product of the latter place must have been affected by the atmospheric conditions then existing admits of no doubt, and this brings us face to face with one of the most important of all facts to the practical brewer — i.e., that the air in the fermenting room itself may contain a multitude of those germs which are productive of the most calamitous results. It is, however, possible to keep the air free from these invisible organisms, and there is no doubt that the recorded results are directly due, first, to the purification of the air entering the fermenting-room by passing it over brine, and, secondly, to the rigidly maintained order and cleanliness in the cellars of the Old Carlsberg brewery. Hansen's investigations, therefore, point a moral which cannot be too frequently emphasised. Saito carried out very comprehensive investigations on the distribution of moulds at different times of the year in many places in Tokio, both in the open and indoors, with the aid of Soja-gelatine (Soja, decoction of onions and cane sugar), confirming by this means the results of earlier investigations already cited. The zymotechnical analysis of water has been of greater value to the brewing industries than the analysis of air. The germs contained in water which give rise to disease in fer- mentations and fermentation products are not usually so enfeebled as those in air, and water on many occasions comes into closer touch with the different products during manufac- ture than does air. The examination of water in reservoirs, and the effect of filtration on the micro-organisms, is of especial practical value. A few details may be quoted here from the researches of Holm and of the author as examples of the results obtained from this form of investigation in the fermentation industry. Holm's researches showed that among the various micro- organisms in water the moulds are those which develop most quickly in flasks containing wort and beer, and generally also 70 MICRO-ORGANISMS AND FERMENTATION. those which occur in largest numbers in the flasks. Penicillium glaucum and Mucor stolonifer were found among them. Next to the moulds come the bacteria when wort is infected with water, whilst if sterilised beer is used, they develop only scantily. The following bacterial forms were found :— Bacterium aceti, Bact. Pastorianum, a third form which made the beer slimy and ropy ; and lastly, several species which imparted a disgusting smell to the wort. Yeast-like cells were of rare occurrence. Holm did not observe any growth of Saccharomycetes, although some Torula forms and Mycoderma occurred. The number of these germs varied at different times of the year, yet it did not seem to be dependent on the season — the rainfall, the condition of the surface water, and of the air had great influence. Of practical importance was the discovery of strong contaminations injurious to wort and beer, in reservoirs situated near granaries and malt-lofts insufficiently protected against dust. It was also shown that water which had been filtered through charcoal filters contained much larger numbers of wort bacteria than the unfiltered water. The water analyses made in the author's laboratory during a period of more than twenty years have given the following chief results : — The samples of water in only very few cases were found to contain Saccharomycetes (culture yeasts or wild yeasts). In one series of analyses S. anomalus and S. membranes faciens were met with. The bacteria observed by Holm which produced slime formation or imparted a putrid smell to the wort, occurred very frequently. If a pure yeast was infected with such species and used for pitching hopped wort, these bacteria did not usually develop further. Although, however, the bacteria did not develop during the fermentation, a difference was often observable between the condition of this beer and that of beer fermented with pure yeast. Acetic acid bacteria were not infrequently found in the analyses, and were usually able to assert themselves in the flasks, even in competition with rival species. In a few cases the experi- ments with wort showed a growth, and sometimes even an abundant one, of Sarcina forms, which did not occur in the AIR AND WATER. 71 parallel series of experiments with sterile beer. They rendered the wort turbid, and imparted a peculiar smell to it. Among the moulds the following were the most frequent : — Asper- gillus, Mucor stolonifer, M. mucedo, Oidium lactis and Dematium- like forms. In the water conduits of the breweries a coherent layer of Crenothrix was not infrequently found. In many cases it has been proved that water received a very considerable contingent of its wort and beer organisms in the reservoirs or conduits, and it may safely be asserted, as the result of many years' experience, that brewery water is most seriously contaminated in the brewery itself. Biological analyses of natural and artificial ice have shown that in both, organisms can exist capable of developing in wort and beer. Sarcina-like bacteria can also be introduced along with ice into these li quids, and may develop freely in them. In artificial ice, the inner snowy layer of the ice-block appears to be particularly rich in micro-organisms. If large quantities of water are to be analysed, it is of the utmost importance to take due care that real average samples are obtained. Hansen gives the following method for the zymotechnical analysis of air and water, a method based upon a long series of comparative trials. The principle underlying it is as follows : — For brewing purposes it is only necessary to know whether the water and the air contain germs capable of developing in wort and beer. This cannot, as was formerly assumed, be ascertained by means of the meat decoction peptone gelatine employed in hygienic air and water analysis. The zymotechnologist has this great advantage over the hygienist, that he is in a position to make direct experiments with the same kind of liquid as that employed in practice — namely, wort. All disease germs that have hitherto been shown with certainty to occur in beer are also capable of developing in wort. Hansen's comparative investigations have proved that the use of gelatines introduces great sources of error. Thus, for instance, in a series of comparative experiments with corresponding samples of water, the following numbers were obtained :— In Koch's nutrient gelatine— 100, 222, 1,000, 750, and 1,500 growths obtained from 1 c.c. of water ; in wort — 72 MICRO-ORGANISMS AND FERMENTATION. 0, 0, 6- 6, 3, and 9 growths ; whereas, in beer, none of these water samples gave any growth. In another series, Koch's gelatine gave for 1 c.c. of water 222 growths, wort gelatine 30 : but none of the flasks containing wort or beer, after infection with the water, showed any development of organisms. Thus, only very few of the great number of germs living in the water developed in wort or beer. Hansen has further shown that in zymotechnical analyses of water and air, it is a mistake to employ gelatine at the outset, and then to transfer the colonies that have been formed into wort. Thus, he demonstrated by experiment that several of the bacterial germs occurring in atmospheric dust and in water are capable of developing in nutrient gelatine, but not in wort ; whilst several of these species become invigorated to such a degree, after having formed a new growth in the gelatine, that they are then enabled to develop in the less favourable medium, wort. Another, and a still greater, objection to the gelatine method is that several organisms, and just those of importance, do not develop at all when transferred directly to the gelatine in the enfeebled condition in which they generally occur in atmospheric dust and in water. Temperature naturally plays a much more important part in the development of the germs on gelatine plates than in nutritive liquid ; at a less favourable temperature they will develop with greater difficulty in gelatine, owing to its defi- ciency in nutriment. Reference may also be made in this connection to the difficulties that are encountered in determining the number of germs which can develop on gelatine plates. Species of frequent occurrence in water, that tend to liquefy gelatine, generally develop very rapidly, and may encroach so exten- sively on the space that other species do not succeed in forming colonies at all ; others require so long an incubation before visible colonies are formed that the examination of the plates is often concluded before these growths appear.* * In comparative investigations, as, for instance, the examination of air and water before and after filtration, gelatine plates are usually employed ; the possible sources of error accompanying their use must, therefore, be borne in mind. AIR AND WATER. 73 Based upon these observations, Hansen devised the following method : — Small quantities of the water, either in its original state or diluted, are added to a series of Freudenreich flasks containing either sterilised wort or beer.* After in- cubation at 25° C. for fourteen days the contents of the culture flasks are submitted to examination. If only a part of them show any growth, the rest remaining sterile, it may be assumed with approximate certainty that each of the flasks belonging to the former set has received only a single germ. Information is thus gained concerning the number of germs capable of development existing in an ascertained volume, and the different germs are also under more favourable conditions for their free development. An exact examination will then show to what species these germs belong. Although wort - cultures give a very small number of growths in this method in comparison with plate-cultures, yet in many cases the number of wort-growths will still be too high, for these growths are able to develop in the flasks un- disturbed and without hindrance from other organisms, but when wort is mixed with good culture-yeast in the fermenting vessel, many of these germs will be checked. Further, the flasks which show a formation of mould will have no import- ance for the brewery itself, but only for the malt-house. In order that the conclusions based on the results should approxi- mate more closely to practical requirements, Hansen proposes the following method of procedure : — The flasks containing a development of yeasts and bacteria are divided into two groups —(1) those in which the growths appear rapidly, and (2) the remainder, in which they make their appearance later ; for instance, after five days. Among the latter are those species which develop less readily in wort. To these, then, less importance is attached in forming an opinion as to the nature of the water or air. The same principle is used by Wichmann, who endeavoured to give a numerical expression for the " harmfulness " of water — that is to say, to express to a definite degree the destructive property of water with regard to wort and beer. * In the analyses of air the germs are aspirated into sterilised water, or first into cotton-wool and then transferred to water. 74 MICRO-ORGANISMS AND FERMENTATION. He impregnated a series of Freudenreich flasks, contain- ing sterile, clear wort or beer, with different volumes of water, and noted the day on which a change in the contents of each flask (cloudiness, formation of a skin, fermentation) became visible. The more rapidly decomposition sets in, and the smaller the necessary quantity of water, the more noxious the character of the water. By giving numerical expressions to the time (setting in of decomposition), and to the quantity of water, the figure specifying the destructive capacity will be a product of these two factors. Beer, however, possesses a greater power of resistance than wort, so that in cultivations in the former, the coefficient must be correspondingly reduced. Lindner added sterile wort to the water, and distributed the mixture by means of a pipette drop by drop into a series of Petri propagating dishes. The growths which developed were counted, and from this the number of germs per c.c. was calculated. The result of many years' experimenting in the author's laboratory have led to conclusions which are at variance with those stated above. The first growths to appear in the wort flasks are almost always putrefactive bacteria, water bacteria, and the like ; just those forms which are of little interest in brewing operations, because they do not develop in the finished product. The special technical character of the analysis is lost if the time taken for the appearance of the bacteria is made the basis on which the character of the water is judged. Moreover, in forming an opinion as to the character of water from the technical standpoint, it is not essential that the quantity of wort-bacteria in a given volume should be esti- mated. On the contrary, when growth in the flasks has been allowed to continue for some time, it has been shown that in more advanced stages species appear which are known as disease germs, as, for instance, the wild yeast. Thus it is the last stage of the development in the flasks which is of real importance. In making the investigation both the wort and the beer flasks should be infected with small quantities of water. In some flasks the water is added in an undiluted condition ; in others it is more or less diluted. Samples are taken from time to time for microscopical, and eventually, AIR AND WATER. 75 for microbiological examination, in order that the micro- organisms which appear may be more closely investigated in regard to their action on wort and beer. For water analyses in connection with distilleries and allied industries, sweet wort is usually employed. This has been found to answer the purpose well, and is easier to obtain clear after sterilising than the mash — i.e., the worts from distilleries and yeast manu- factories. If special problems arise, as, for instance, the presence of certain species of micro-organisms, the analytical apparatus should be modified to suit the characteristics of the particular species. Thus, for yeast factories, the appearance of moulds and putrefactive bacteria will be of special importance. Provision for the former can be made by cultivation of the water on the surface of congealed gelatine which has been mixed with a decoction of fruit, and for the latter by employing ordinary plate - cultures consisting of neutral nutrient gelatine. For special demonstration of the Sarcina forms in beer a neutral decoction of yeast, with the addition of a small quantity of alcohol, will be suitable. For developing " wild yeast," wort may be used with an admixture of hydrofluoric or tartaric acid, and so on. Experiments may also be arranged in connection with other branches of the brewing industry by mixing the liquid which is to be fermented with a certain quantity of the sus- pected water at different stages of the fermentation, the addition of pure yeast having been previously made. The difficulty of this method when working with small quantities consists, as is well known, in approaching sufficiently near to practical conditions to make it possible to draw direct con- clusions. If, as is the case in our own laboratory, it is desired to ascertain what groups or species of organisms occur in the water, then it will be of no consequence if the sample has been delayed in transit. 76 CHAPTER III. BACTERIA. BACTERIA occur in every shape, from the smallest specks or spheres to green algae - like filaments ; and they are found in nearly all possible localities, under the most varied conditions. According to their action, a distinction is made between pathogenic, zymogenic, and chromogenic bacteria, or those that produce disease, fermentation, and coloration respectively. Our first knowledge of these living forms was obtained by placing small quantities of different substances under the microscope, and examining them with high powers. In putrefying meat minute spherical bodies were found, which clearly multiplied by division ; in sour milk short rods occurred, and in decomposing vegetable matter large spherical bodies and long fine threads ; in the mucus of teeth very fine spiral threads were found. Thus it was convenient provisionally to retain these forms, and to describe them as independent species. Credit is due to Cohn for the first systematic classi- fication of bacteria. Bacteria always consist of single cells. In their simplest form they occur as spherical bodies (coccus, Fig. 16, a) of varying size, often so small that they can only just be seen even with the strongest powers, and only give evidence of their existence as organisms during propagation by division. If the coccus divides in one plane, Diplo- and Streptococcus are produced (b) (c). By division in two planes, the Micrococcus (b) is ob- tained, and by division in three directions the Sarcina form.* If the cells assume a cylindrical form we have bacteria (e), * In the Sarcina forms that occur in beer, the division is incomplete in all three directions, but appears to vary, so that an irregular piling up of spherical bacteria takes place, or else a marked displacement of single cells is found. Two spherical bacteria are often found strung together. All such conglomerates of cells are surrounded by a gelatinous envelope, the development of which is de- pendent upon the nutritive conditions. BACTERIA. 77 which may be of very varied length. A distinction may be drawn between the motile (bacillus] and the motionless form (bacterium). When the rods are swollen in the middle, and thus form spindle shapes, we have the Clostridium form (/). If the bacilli are elongated, so as to become more or less thread- like, they are called Leptothrix (g), which may also occur as pseudo-filaments when several bacteria are grouped length- wise, or as Cladothrix, when they lie close to one another and appear as irregular branching threads. The bacilli and n****j K\f/.*SS& S&j&Vfiyl' Fig. 16.— Growth-forms of Bacteria (in part schematic).— a, Cocci; b, diplococci and micro- coccus ; c, streptococci ; (Leuconostoc) and similar species. Bacteria contain a number of enzymes of a more or less pronounced albuminoid character, and this is also the nature of the various poisonous substances which occur in several species. For nutrition, bacteria require carbonaceous and nitro- genous compounds, as well as the inorganic substances found in the ash. The majority of bacteria do not appear to possess the power of building up their organic constituents from inorganic material ; they are dependent upon those organic compounds that have already been built up in animals and plants. The nitrifying bacteria form an exception in that they can directly absorb carbon dioxide from the air, and the bacteria which occur on the nodules of the leguminosse in like manner absorb nitrogen from the air and assimilate it. It has been customary to distinguish between Saprophytes or organisms of putrescence and parasites which feed only upon living animals and plants. A corresponding classification of the bacteria has been attempted in a biological sense. Alfred Fischer divides them into prototrophic, metatrophic, and para- trophic (from the Greek, trophd, nourishment). By prototrophic bacteria are meant those, like the nitri- fying, the iron and sulphur bacteria, which can take up these substances in an inorganic form. The vast majority of bacteria are metatrophic ; they utilise organic compounds of the most varied kind, while they promote putrescence or fermentation. Lastly, the paratrophic are parasites ; they do not occur in . nature in a free state, but can only grow upon other forms of 6 82 MICRO-ORGANISMS AND FERMENTATION. life. Nevertheless, it is possible to cultivate them — e.g., in blood serum — at the temperature of the body. The metatrophic bacteria, which form the vast majority, and are of special interest to the fermentation physiologist, are not equally responsive to the different carbonaceous and nitrogenous food-stuffs. Peptone and amides are good sources of nitrogen. Many bacteria can also utilise ammonium salts and nitrates under given conditions, thus Bact. aceti can assimilate ammonia in presence of acetic acid. Similarly, according to Henneberg, certain species of acetic acid bacteria can utilise potassium nitrate and ammonium tartrate as sources of nitrogen, if the culture material contains sufficient dextrose. The carbohydrates constitute the most important source of carbon. Of the different varieties of sugar, grape sugar forms an excellent food for bacteria. According to the conditions of nourishment, bacteria may bring about varying decompositions of the substrata. The different nutritive fluids and gelatines that are used in the culture of bacteria are described in Chap, i., sec. 6. Pasteur made the important discovery that there are certain bacteria and other micro-organisms which do not require free oxygen, but are capable of effecting active decom- position of the fermenting material, even when oxygen is excluded. He, therefore, distinguished two classes of micro- organisms, aerobic and anaerobic. Whilst the aerobic bacteria breathe in a similar manner to all other organisms, and thereby convert organic substances (non-nitrogenous) into carbon dioxide and water, and bring about similar decompositions with the nitrogenous compounds, the anaerobic bacteria com- prise on the contrary those whose life activity is sustained without free oxygen. To this class belong some of the butyric bacteria, as well as the bacteria that ferment ceUulose. Since Pasteur's discovery (1861) numerous bacteria have been investigated in this respect, and it has been proved that there is every possible transitional stage between the obligatory aerobes (amongst which the hay bacillus, Bac. subtilis, must be classified) and the obligatory anaerobes. A number of facultative anaerobic species are now known which grow well with access of air, but also develop, to a degree varying with BACTERIA. 83 the species, either in diluted air or even in the absence of oxygen ; examples may be found among the lactic acid bacteria. It is a well-established fact that with one and the same species the demand for oxygen varies according to the other life con- ditions. The heat-loving (thermophilous) bacteria form a typical example, for they can grow at a high temperature in the presence of air, whilst at a low temperature they grow only in the absence of air. Of the obligatory aerobes,many, when well nourished, can develop in air containing only traces of oxygen. Whilst a few bacteria are motionless (e.g., lactic acid and, in part also, acetic-acid bacteria), the majority show a capacity for free movement ; such bacteria are commonly met with in decomposing fluids. This motion, which is not to be confounded with the Brownian molecular movement, usually consists of a forward swimming action, together with a rota- tion round the longer axis. The organs of motion, which only become visible on staining, consist of fine protoplasmic hairs — flagella or cilia — which are connected with the plasma of the cells through holes in the cell-wall. A few species have only a single cilium, attached to one end of the cell ; others have a bunch of cilia at one end, whilst cilia are distributed over the •entire surface of others — e.g., the hay bacillus, in certain stages of development, and some of the common putrefying bacteria. By means of these organs of motion, bacteria are enabled to penetrate to that part of the nutritive fluid which offers the most favourable conditions for existence. Thus, Engel- mann has proved that aiirobes move to the stratum of liquid which is richest in oxygen, whilst the anaerobes move in a contrary direction. Similarly, Pfeffer has shown that bacteria move to these parts of a fluid that contain nutriment of suitable concentration. The rate of motion is conditioned by tem- perature ; thus, Bac. subtilis moves more rapidly at 37° C. than at 20° C., whilst other bacteria cease to move at the former temperature. The propagation of bacteria takes place by division. It has been observed in detail in the larger species. The cells expand, fine transverse lines appear, which gradually increase in thickness and split into two leaflets ; after this the organism separates into smaller rods, which sometimes remain united, 84 MICRO-ORGANISMS AND FERMENTATION. sometimes become detached (Fig. 24, ^4). Long before a trace of these transverse walls can be observed, staining will show that the organism consists of a series of segments, each of which corresponds to a subsequent individual. The newly formed segment cells are all in the same plane. A division in either one, two or three planes has been observed in certain cocci. In the case of many bacteria, formation of spores takes place in the following manner (Figs. 16, I, m; 24, B). The plasma in the cell becomes darker, and often distinctly granu- lar ; a small body subsequently appears — frequently at one end of the cell — strongly refractive to light, which quickly increases in girth, and is surrounded by a membrane. Meanwhile, by far the greater portion of the remaining plasma of the cell disappears, being used up in the formation of the spore. This is seen enclosed in a clear liquid which gradually disappears f and finally the cell-wall shrivels up, and only remains as a withered appendage to the ripe, egg-shaped spore. In many cases a swelling takes place in the mother-cell during spore- formation (Figs. 16, /; 24, B). Before spore-formation begins, the cells of many, especially anaerobic species, are coloured blue with iodine like starch-granulose. Probably at this stage the cells store up reserve food material. Usually only a single spore is formed in a cell. One cause for spore - formation is that during vege- tative growth the products of its own activity — acidsr alkalies, etc. — accumulate in the nutritive substratum, and. as a consequence, further vegetative growth is checked. The exhaustion of the nutritive medium may produce the same effect. Spore-formation demands a suitable temperature, and a certain quantity of moisture. The membrane of the spore is very strongly developed, and is frequently surrounded by a gelatinous envelope. The contents are strongly refractive,, and contain but little moisture. Spores are of value in en- abling the species to survive when conditions occur that are unfavourable to vegetative life. They possess quite an extraordinary power of resistance to harmful influences. The membrane cannot be easily moistened or penetrated by water, and the great durability of spores is especially due to the fact that their plasma contains little or no moisture. Thus, BACTERIA. 85 according to Fliigge, species occur amongst the peptonising bacteria of milk, the spores of which will withstand boiling for four hours. Spores of hay bacillus will also withstand boiling for hours. Spores can usually stand dry heat better than boiling in steam or water. On the other hand, many spores show special resistance to heating in milk, and the same is true of neutral or feebly alkaline liquids, whereas acid liquids are unfavourable to their existence. Spores are generally difficult to stain. On the other hand, colouring matters once taken up by spores are retained better than by vegetative cells ; and after bleaching, therefore, coloured spores become visible in a colourless cell. As soon as favourable conditions of nutriment and tem- perature recur, spores germinate. They first swell up by absorption of water, and the contents lose their strong re- fractive power. A bacterium then grows out from the spore ; the wall of the latter is sometimes seen to burst or to unfold into two valves (Figs. 16, 24). The full-grown rod then multi- plies in the usual manner. Spores may maintain their germina- tive power through a long period, sometimes for many years. In addition to the endosporic bacteria just mentioned, *' arthrosporic " bacteria were formerly described which do not form spores in the interior of the cell, but in which it was believed that members split off from vegetative cells form the starting point of fresh vegetative generations. A micro- scopically discernible difference between the " arthrospores " and other cells, however, occurs only in a few cases, in that the walls of the latter thicken (Chlamydospores). Perhaps by continued investigation endogenous spores will be found in all such species. The gonidia which occur in Crenothrix constitute a special kind of cell ; in the case of some bacteria, as, for instance, Clado- thrix, they are motile. These cells are organs of propagation. Temperature plays an important part in the life processes of bacteria. We distinguish the minimum, optimum, and maximum temperature at which life can exist. These three cardinal points differ, not only for each species, but also for the individual functions of each, such as its rate of growth and its fermentative activity. 86 MICRO-ORGANISMS AND FERMENTATION. Many bacteria are very resistant to low temperatures. J. Forster, B. Fischer, Miquel, and others have shown that bacteria exist which multiply rapidly at the freezing point. Certain species are not killed by exposure to a temperature of - 70° C., — 110° C., or even to the extreme temperatures of - 213° C. and - 252° C. (Frisch, Pictet and Young, Macfadyen, and Rowland). In contrast to these, a number of thermophilous bacteria have been discovered. Miquel has described Bacillus thermophilus, which multiplies readily at 70° C., whilst its development is arrested at 42° C. Other species will only germinate above 60° C. In the excrement of animals many species of frequent occurrence continue to grow at 25° C., whilst their growth is inhibited at about 39° C. (L. Bahino- witsch). The lactic-acid bacteria and certain organisms occurring in molasses, in the fermentation of tobacco, and in the spontaneous heating of hay, belong to the thermophilous species. The bacteria occurring in hay have been examined in detail by Miehe. F. Cohn has also proved that the cause of the spontaneous heating of moist cotton waste is the presence of a micrococcus belonging to this group. It has already been stated that spores will stand a considerably higher temperature than vegetative cells. It is, therefore, obvious that only high temperatures can be used for disinfection. With regard to the germicidal action of light, Downes and Blunt found, as early as 1877-1878, that direct sunshine powerfully restricts their growth, and that the most active rays of light are the strongly refractive blue and violet rays, well known to possess powerful photo-chemical properties. On the other hand, red and orange rays are less active, and heat rays which accompany the light rays possess no activity. H. Buchner and S. Bang, amongst recent workers, have studied the action of light upon bacteria. Buchner records that sunlight plays a part in the spontaneous purification of rivers by bacteria. It is assumed that the effect of direct sunlight on bacteria is not entirely due to the action on the cells, but also to the alteration brought about in the sub- stratum, whereby it becomes less suited for nutrition. For example, the formation of hydrogen peroxide in nutritive agar by exposure to sunlight has been demonstrated. A few quite BACTERIA. 87 exceptional bacteria are known which appear to thrive in bright light. This is true of the purple bacteria, which, like green plants, assimilate carbon in presence of light. Bacteria are capable of living at considerable depths. Russel found bacteria alive at a depth of 1,100 metres. They are, therefore, capable of withstanding a pressure of over 100 atmospheres, and certain putrefactive bacteria have survived a still higher pressure. With regard to the action of antiseptics on bacteria, the rule has already been laid down, in the section on sterilisation, that the higher the temperature the more easily they are killed. But, with regard to the restrictive action of antiseptics, it is usually weakest at the optimum temperature, and stronger at both higher and lower temperatures. Very dilute solutions of an antiseptic may encourage the growth of bacteria. Various species react differently to the same concentration of a reagent, and the action depends to a great extent, with any given species, upon the state of nourishment of the cells. Spores are much more resistant than vegetative cells. It has proved possible to propagate bacteria in the presence of successively increased quantities of an antiseptic, but the charac- ters so obtained prove not to be fixed, but disappear as soon as the culture is prepared in a substratum free from poison. Bacteria and other micro-organisms when subjected to mechanical vibration behave very variously. Horvath proved that gentle vibration has no action on their growth, whereas violent shaking hinders or entirely inhibits it. Meltzer arrived at the conclusion, after prolonged experiments with liquid cultures, that gentle vibration promotes the multiplication of micro-organisms ; with a given degree of motion the rate of germination of the species is at the maximum, whilst any stronger vibration restricts it. The optimum and maximum differ for each species. According to Appel, cultures of bacteria on solid substrata behave the same whether they are shaken or not. Attempts have been made ever since the discovery of bacteria to define this large group of organisms, and to classify the various species in one system, like other sections of the vegetable kingdom. 88 MICRO-ORGANISMS AND FERMENTATION. With the exception of a small number of doubtful forms, amongst which Crenothrix may be named, the bacteria form a fairly uniform group, exhibiting the same simple structure and the same method of propagation throughout. It is an interesting fact that amongst the lowest green plants, the algae, there is a group which exhibits the same construction and the same method of propagation as the bacteria, so that the lowest green plants connect up with the lowest fungi. It has already been mentioned that green plants possess the power of absorbing and assimilating the carbon dioxide of the air, owing to a special constituent of the cells, chlorophyll. This power is not possessed by fungi, and the lowest group of algse has been classified, therefore, under the name of Schizo-algce, in contrast to the Schizo-fungi. It is not only a physiological difference that distinguishes the two groups — that could not be used as the basis for a systematic classification —but rather that the structure of the cell contents is entirely different, since the Schizo-algce contain grains of chlorophyll. On the other hand, the Schizo-algce have not the power possessed by bacteria of forming endogenous spores, but the method of division exhibited by Crenothrix (see Fig. 31) frequently occurs in the algae. To illustrate how difficult it is to define strict limits in nature, it may be stated that there are undoubted bacteria which contain green colouring matter, and yet others that can assimilate atmospheric carbon dioxide. Most people are agreed that bacteria are to be classed amongst the fungi, although, in their method of propagation, they stand nearer to the algae. There are, however, fungi which show the same kind of cell division as bacteria, and even the remarkable formation of endospores also occurs amongst many fungi, especially amongst those that will occupy us next, the Saccharomycetes. We have seen that bacteria have points of contact, both with the lowest forms of algae and also with the lowest forms of fungi. It must also be noted that in so far as certain bacteria move about with the help of cilia, a similar relation- ship exists between them and the lowest group of the animal kingdom (the flagellata), which again have other characters in common with bacteria. BACTERIA, 89 On account of their simple structure, the attempt to form a true system of bacteria is surrounded with great difficulties. It must, indeed, be based upon morphological characters. Thus it came about that for a long time it was generally held (Billroth, Nageli) that no single species can possibly endure, for it may pass freely and without limit into any other species, and it was further assumed that a so-called species can react in various ways upon its substratum, so that physio- logically no hard and fast lines can be drawn. In both direc- tions the hypothesis went far beyond the facts, and it must be considered to have been a great advance when Cohn, in 1875, first published his system of bacteria. His system must now be regarded as out-of-date, since Zopf, de Bary, van Tieghem, Hueppe, and many others have established new divisions, which correspond more closely to the natural boundaries. Nowadays general use is made of the schemes established by A. Fischer and Migula, and we must limit ourselves to describ- ing the main lines of Migula 's system. He recognises two orders, the Eubacteria and Thiobacteria, the latter distinguished by containing sulphur, and being either colourless or coloured pink, red, or violet by bacterio-purpurin. 1. Order — ETTBACTERIA. 1. Family — Coccacece (Zopf) Mig. Cells in free condition completely spherical, in state of division somewhat oval. (1) Genus Streptococcus, Billroth. Cells motionless, round, division only in one plane, occurring singly, in pairs, or in chains like strings of pearls. (2) Genus Micrococcus (Hall), Cohn. The cells divide in two planes, and, after subdividing, combine to form plate-like layers. No cilia. (3) Genus Sarcina, Goods. The cells divide in all three planes, and after subdivision mass together in the form of bound packets. No cilia. (4) Genus Planococcus, n.d. The cells divide in two planes like Micrococcus, but possess cilia. (5) Genus Planosarcina, n.d. The cells divide like Sarcina in three planes, but possess cilia. 2. Family — Bacteriacece. The cells are cylindrical and vary in length ; they are straight, and never twisted like a screw ; division takes place only in one plane after the rods have expanded in length. (J) Genus Bacterium. Cells without cilia, often forming endospores. 90 MICRO-ORGANISMS AND FERMENTATION. (2) Genus Bacillus. Cells covered entirely with cilia ; often forming endo- spores. (3) Genus Pseudomonas. Cells with polar cilia only ; seldom form endospores. 3. Family — Spirillacece. Cells twisted spirally, or forming part of a spiral-like curve. Division only in one plane after cells have expanded in length. (1) Genus Spirosoma, n.d. Cells without cilia ; rigid. (2) Genus Hicrospira. Cells rigid, with one, and, less frequently, with two or three polar and undulating cilia. (3) Genus Spirillum. Cells rigid, with a bushy formation of polar cilia, shaped in semi-circular curves. (4) Genus Spirochcste. Cells in serpentine curves ; cilia unknown. 4. Family — CJilamydobacteriacece. Cells cylindrically arranged in threads surrounded by a sheath. Propagation through motile or motionless gonidia, which project from the vegetative cells, and grow into new threads without undergoing any resting stage. (1) Genus dilamydothrix, n.d. Cells cylindrical, motionless, without branches. Grouped as threads surrounded by thick or thin sheaths, with nothing to dis- tinguish one end from the other. (2) Genus Crenothrix, Cohn. Thread-forming bacteria without branches, differing at the ends ; stationary. Thick sheaths often impregnated with ochre. Cells at first divide in one plane, and later in two or three planes. The products of division are rounded off, and grow into gonidia. (3) Genus Phragmidiothrix, Engler. Cells at first assembled in unbranched threads, which divide in three planes, and so produce a strand of cells. At a later stage the single cells penetrate through the fine close sheath, and give rise to branching. (4) Genus Sphcerotilus (incl. Cladoihrix). Cells cylindrically enclosed in sheaths, forming asymmetric branched threads without distinction between the ends. Propagation through gonidia, which swarm through sheath, and settle on surrounding objects, developing immediately into fresh threads. The gonidia possess bushy polar cilia. II. Order — THIOBACTEBIA. 1. Family — Beggiatoacece. Bacteria forming threads, containing no bacterio-purpurin. (1) Genus Thiothrix, Winogradsky. Motionless threads clustered together, and surrounded by a fine sheath. Division takes place in one plane. At the end of the thread rod-like gonidia form, having a crawling movement. (2) Genus Beggiatoa, Trevisan. Threads without sheath formed of flat cells, crawling like the Oscillaria, and rotating round their axis in a free condition. Gonidia unknown. BACTERIA. 91 2. Family — Rhodobacteriacece. Contents of the cell coloured pink, red, or violet by bacterio-purpurin ; cell containing granules of sulphur. ( 1 ) Sub-family — Thiocapsacece. Cells grouped in families ; division in two or three planes. (1) Genus Thiocystis, Winogradsky. Small families closely packed, surrounded by one or more gelatinous cysts ; motile at all stages of existence. (2) Genus Thiocapsa, Winogradsky. Flat spreading colonies on substratum, consisting of spherical cells loosely embedded in one mass of jelly ; motionless. (3) Genus Thiosarcina, Winogradsky. Family grouped in a packet form, incapable of movement. Corresponding with genus Sarcina in the Eubacteria. (2) Sub-family — Lamprocystaceoe. Cells combined in families. Division of cells first in three, and afterwards in two, planes. (1) Genus Lamprocystis, Schroter. Families at first solid, then forming hollow spherical mesh, and finally separating into small motile groups. (3) Sub-family — Thiopediacece. Cells grouped in families. Division in two planes. (1) Genus Thiopedia, Winogradsky. Families forming plates consisting of motile cells arranged rectangularly. (4) Sub-family — Amcebobacteracece. Cells grouped in families ; division in one plane. (1) Genus Amcebobacter, Winogradsky. Cells grouped in families; division in one plane. Families moving like Amoeba. Cells wrapped in threads of plasma. (2) Genus Thiotece, Winogradsky. Family with thick gelatinous cyst. Cells loosely enclosed in a common jelly ; always motile. (3) Genus Thiodictyon, Winogradsky. Families consisting of small rods, the ends of which are bound together to form a mesh. (4) Genus TJiiopolycoccus, Winogradsky. Families solid, motionless, con- sisting of small densely packed cells. (5) Sub-family — Chromatiacece. Cells free and motile in some stages. (1) Genus Chromatium, Perty. Cells ellipto-cylindrical or elliptical, com- paratively thick. (2) Genus Rhabdochromatium, Winogradsky. Cells free, spear or spindle- shaped. Motile at certain stages, with polar cilia. (3) Genus Thiospirillum. Cells free, motile, spiral-shaped. Always motile. This system provides a survey of the great groups of bacteria, classed together according to their form, structure, and the course of their development. 92 MICRO-ORGAN ISMS AND FERMENTATION. As soon, however, as a place has been found in the system for any pure species, it is necessary to distinguish it by a detailed and thorough investigation, and to give such an exact description that it can be clearly defined in relation to other species, and may be recognised again if new pure cultures were prepared from the impure material. After the far- reaching work of the last few years, and the thorough explor- ation to which the field has been subjected, it is possible to give such a description of many species of bacteria, notwith- standing the difficulties due to the great tendency of these plants to exhibit variations. In such a description of a species, it is necessary, owing to the poverty of these micro-organisms in definite shape, to take into consideration many other points. Cohn established the classification of bacteria already men- tioned by means of their physiological properties, dividing them into pathogenic, zymogenic, and ehromogenic bacteria. Of these, the second group is of importance to us, and the following examples embody typical species of those bacteria that are distinguished either by their special enzymes and fermentative products — acetic acid, lactic acid, etc. — or by their action upon fermenting liquids. 1. Acetic Acid Bacteria. The acetic fermentation is a process of oxidation. By the activity of the bacteria in presence of oxygen, the alcohol in the liquor is oxidised to aldehyde and water, and then the aldehyde is further oxidised to form acetic acid. It is a process differing greatly from those classed as fermentations, and has a certain similarity with the process of breathing. The entire content of alcohol undergoes change without the production of by-products ; the bacteria can, however, bring about the combustion of acetic acid to form carbon dioxide and water. It is, therefore, of importance to interrupt the process as soon as acetic acid is formed, if the full yield is to be obtained. Persoon, as early as 1822, was acquainted with the vegetable character of the film which forms on the surface of liquids under- going acetic fermentation, and he named the film Mycoderma. ACETIC ACID BACTERIA. 93 In 1837-38 the view was also expressed by Turpin and Kiitzing that the acetic acid fermentation is caused by a micro-organism, which Kiitzing described and delineated under the name of Ulvina aceti. Starting from this, Pasteur, first in his treatise of 1864 and subsequently in his work, Etudes sur le vinaigre, in 1868, furnished experimental proof of the correctness of this view. He sowed a trace of the film on a mixture of wine and wine vinegar, and thus obtained a stronger development of acetic acid than was possible by allowing the liquid to undergo spontaneous fermentation, and on this he based a process for manufacturing vinegar. He assumed that the acetic fermentation was caused by a single species of micro-organism, which he called Mycoderma aceti. His method consists in giving a large surface to the liquid employed — two parts of bright wine to one of wine- vinegar — and then sowing on the surface of the mixture a young film consisting of " mother of vinegar." When the temperature, the composition of the liquid, and all other conditions are favourable, the formation of acetic acid will proceed more quickly than in the older Orleans process. It is claimed that the installation is cheaper and the loss of alcohol scarcely greater than in the latter process. As early as 1879, E. C. Hansen discovered that at least two distinct species are concealed under the name of Mycoderma aceti, which now go by the names of Bacterium aceti and Bad. Pasteuri- anum ; and now a whole series of species are distinguished. To obtain the best results in this branch of industry, it is again necessary to start with an absolutely pure culture of a methodically selected species. The old Orleans process still prevails in France. In this method the wine which is to be converted into vinegar is placed in casks, half-filled, at about 20° C., to which air has moderately free access. The formation of acetic acid, as in Pasteur's process, takes place in consequence of the liquid being gradually covered with a film consisting of " mother of vinegar." In other countries the " quick vinegar process " is employed, in which " the goods " (diluted spirit mixed with vinegar) come into intimate contact with air. To allow free access of air, the liquid is broken up into small drops and distributed over a large surface 94 MICRO-ORGANISMS AND FERMENTATION. (beechwood shavings). The species occurring in this process have been the subject of a far-reaching investigation by Henneberg. Whilst Pasteur does not explicitly maintain in his memoir that the oxidation of alcohol to acetic acid brought about by bacteria is a purely physiological process, Adolf Mayer ex- pressed this opinion, the correctness of which he confirmed by proving that the vinegar film exercises its greatest activity at 35° C., and that it ceases to react at 40° C., and further that the film cannot react on more than 14 per cent, alcohol. The purely chemical action of platinum black on alcohol presents a contrast, for it is able to react at high temperatures and with higher concentrations. Pasteur showed that the acetic acid generated by the oxidation of alcohol is transformed, if the oxidation is con- tinued, into carbon dioxide and water. This has recently been confirmed by Adrian J. Brown. A few species are able to bring about other decompositions, owing to their strong oxidising power — e.g., the formation of butyric acid from butyl alcohol, gluconic acid from glucose — a few, again, have the power of inverting sugar (Bact. aceti and Bact. xylinum). An important advance was made in our knowledge of acetic bacteria when Buchner and Meisenheimer, as well as Herzog, proved that this remarkable fermentation is brought about by the activity of an enzyme. The cells may be killed with acetone, and then treated in the same way as the alcohol yeasts (see Chap, v.), and it can then be shown that, after evaporating the liquid, the residue can bring about the acetic fermentation, although it contains no living cells. By this discovery the real nature of the fermentation becomes clear. Like the alcoholic fermentation, it is caused by an enzyme, which may react independently of the living cell that brought it into existence. These bacteria grow vigorously in many nutritive fluids — e.g., in dextrose solution with peptone and salts. The presence of alcohol is not an essential condition of their existence, and, indeed, more than 4 per cent, of alcohol acts restrict! vely on their growth. ACETIC ACID BACTERIA. 95 Hansen's researches are among the first which proved that a definite fermentation is not induced by one species of bacterium only, but by several ; these researches also furnish some of the earliest experimental evidence of the fact that one and the same species can occur in very different shapes ; the correctness of his results was later confirmed by Zopf, de Bary, and A. J. Brown. By means of his staining experiments with Bacterium (Mycoderma) aceti (1879), he discovered that at least two distinct species are hidden under this name, of which the one, like most other bacteria, is stained yellow by iodine, whilst the other assumes a blue coloration with the same reagent. For the former he retained the old name Bact. aceti, whilst the one stained blue he named after Pasteur — Bact. Pasteur ianum. The film formations on wort and beer, and likewise the growths on wort-gelatine, give a fine blue colour with tincture of iodine, or iodine dis- solved in a solution of potassium iodide, whilst the growths which de- velop on yeast-water and on broth with peptone and gelatine are coloured yellow ; even very old films on beer show a yellow reaction. It is the slime formation secreted from the cell-wall that is coloured blue. At a later period Fis- v --Bacterium „ ,. , , . , . (after Hansen.) Hansen discovered a third species. These three species are characterised as follows : — Bacterium aceti (Hansen) (Fig. 17) forms a slimy smooth film on "double beer " (top-fermentation beer, rich in extract, containing 1 per cent, of alcohol), at a temperature of 34° C., and in the course of 24 hours. The slime is not coloured by iodine. The cells of this film consist of rod-bacteria, hour-glass-shaped, and arranged in chains ; occasionally longer rods and threads occur, with or without swellings. At 40°-40-5° C. long thin threads develop. In plate-cultures with wort-gelatine at 25° C. these bacteria form colonies with sharply defined edges, or, more rarely, stellate colonies, which appear grey by reflected light, bluish by transmitted light ; they mainly consist of single rod-bacteria. In peptone-gelatine broth the colonies are surrounded by milky zones, separated from them by 9(5 MICRO-ORGANISMS AND FERMENTATION. clear zones ; they may later become iridescent. On sowing drops on wort-gelatine, flat, spreading, rosette-shaped colonies are formed at 25° C. in the course of 18 days. In " double beer " the temperature maximum for growth is 42° C., the minimum 4°-5° C. This species is of common occurrence both in high- and low-fermentation beers. Bacterium Pasteurianum (Hansen) (Fig. 18) forms a dry film on " double beer " at 34° C., which soon becomes wrinkled and pleated. In young, vigorous films on beer or wort, at favourable temperatures, the slime surrounding the cells is coloured blue by iodine. The cells of the film form long chains, and are, on the average, larger, especially thicker, Fig. 18. — Bacterium Pasteurianum (after Hansen). Fig. 19. — Bacterium Kiitzingianum (after Hanson). than in the previous species. The thread-like form at 40°- 40-5° C. is also a little thicker than that of Bact. aceti. In plate-cultures, with wort-gelatine at 25° C., the colonies re- semble those of the previous species, but are a little smaller, and consist chiefly of chains. In peptone-gelatine broth the colonies are similar to the previous species. On sowing drops on wort-gelatine wrinkled colonies develop at 25° C. in the course of 18 days, which are slightly raised, and present a sharp outline or one slightly jagged. In " double beer " the maximum temperature for growth is 42° C., minimum 5°-6° C. This species is more frequently met with in high- than in low-fermentation breweries. Bacterium Kutzingianum (Hansen) (Fig. 19) forms a dry ACETIC ACID BACTERIA. 97 film, on " double beer " at 34° C., which creeps up the side of the flask. The slime is coloured blue under the same conditions as Bad. Pasteur "ianum. The film consists of small rod-bacteria, which are most frequently single or connected in pairs, and seldom form chains. The thread form at 40°-40-5° C. presents almost the same appearance as that of Bact. Pasteurianum. In plate-cultures with wort-gelatine at 25° C. the colonies are analogous to those of the previous species. They consist almost exclusively of small, single rod-bacteria. In peptone- gelatine broth the colonies resemble those of the two previous species. On sowing drops on wort-gelatine at 25° C., colonies develop in the course of 18 days resembling those of Bact. Pas- teurianum, but with a smooth surface without wrinkles. On " double beer " gelatine these colonies are slimy, whilst in the two previous species they have a dry surface. In " double beer " the temperature maximum of the growth is 42° C., minimum 6°-7° C. This species was discovered in " double beer." Hansen's thorough investigation of acetic acid bacteria has assumed great importance in the general biology and mor- phology of bacteria, owing to the light thrown on one of the factors causing multiplicity of bacterial forms. Each single species of the acetic bacteria examined by Hansen occurs in three essentially different forms dependent on temperature — chains, consisting of short rods, longs threads, and swollen forms. If sown on " double beer/' which is very favourable to their growth, the various species give a growth consisting of chains at all temperatures from 5°-34° C., which develops well, notably at 34° C. If a bit of this young film is transferred to fresh nutritive liquid at 40°-4O5° C., the cells grow into long threads in a few hours (Fig. 20). In some species these threads can attain a length of 500 /m* and more, whilst the links of the chain measure only 2 to 3 /JL. If this growth of long threads is then placed at a temperature of 34° C. a transformation into the chain form again takes place. Whilst developing at this temperature, the long threads increase, not only in length, but also in thickness, and * 1 ft - O'OOl millimetre. 98 MICRO-ORGANISMS AND FERMENTATION. that often very considerably. Thus an endless variety of polymorphous swollen forms are produced (Fig. 21). It is not till then that the threads are divided into small links, giving rise to typical chains. Only the thickest parts of the swollen threads remain undivided, and are at last dissolved. Thus the Fig. 20.— Bacterium Pewtewriant/iw.— The thread form after cultivation for 24 hours on "double beer" at 40°-40'5° C. (after Hausen). swollen forms play a regular part in the cycle of changes. This cycle furnishes a striking example of the effect of tem- perature in determining the form assumed by bacteria. The species Bacterium aceti and Bacterium Pasteurianum differ, according to Lafar, both chemically and chemico- ACETIC ACID BACTERIA. 99 physiologically. In sterilised beer they give different fermen- tation reactions. At higher temperatures Bact. Pasteurianum acquires a higher acidifying power than Bact. aceti ; on the other hand, Bact. aceti is able to carry on a vigorous fermentation at 4°-4-5° C., whilst at this temperature Bact. Pasteurianum Fig. 21.— Bacterium Pasteurianum.— Transformation of the thread-forms and chains after cultivation in " double beer" at about 34° C. (after Hansen). forms no appreciable amount of acetic acid. At 33°-34° C. Bact. Pasteurianum reaches the maximum of acetic acid formation — 3-3 per cent, by weight in seven days — after which the formation slowly diminishes, and finally ceases. After the maximum of acid formation has been reached, 100 MICRO-ORGANISMS AND FERMENTATION. irregularly swollen cells make their appearance in the growth, which under the existing nutritive conditions may probably be considered as diseased or degenerate forms (involution forms). More thorough investigation into this question is to be desired. The forms noted by Hansen in the cycle described have quite a different physiological significance, for they are then developing freely, and thus preparing the growth for the formation of new cells. Bacterium xylinum is essentially different from these three species. It was described by Adrian J. Brown in 1886, who examined it especially from a chemical point of view. It forms a film, the slime of which becomes cartilaginous and tough like leather. The growth, consisting of motionless rods resembling Bad. aceti, gradually fills up the whole liquid. This species is essentially different from the three first described in another respect — the slimy envelope shows the cellulose reaction, which is not the case with the slime of Hansen's three species. According to Emmerling the slimy sheath contains an albuminoid substance resembling chitin. According to an investigation undertaken in the author's laboratory, this species occurs in vinegar factories in many countries in a vigorous state of development. According to Henneberg, it may react unfavourably on the aroma of vinegar, and by forming slime may arrest the quick vinegar process. The sorbose bacteria investigated by Bertrand, which cause the conversion of sorbite, from the juice of mountain ash berries (Sorbus aucuparia), into sorbose, is identical with Bad. xylinum. Zeidler found an acetic acid organism in lager beer, Thermo- bacterium or Bacillus zeidleri, which occurs as short motile cells and involution forms. When a given quantity of acid has been formed in the liquid, the movement of the cells ceases. If the growth is sown on hopped wort, a cloudy turbidity forms on the surface ; the whole liquid gradually becomes turbid, and acquires a yellowish-brown colour. Small pro- tuberances form on the surface of the liquid, which soon sink, and thus a loose brownish deposit is produced. The species does not appear to be dangerous in the brewery. Bacterium oxydans, a species with motile cells, was de- ACETIC ACID BACTERIA. 101 scribed by Henneberg. The writer discovered it on low- fermentation beer which had been standing in vessels at a temperature of 25°-27° C. It forms roundish colonies on gelatine, which later assume irregular shapes with curious ramifications. On sterilised beer it forms a delicate film, consisting of separate prominences creeping up the sides of the vessel. In its younger stages the film consists of pairs of ceUs ; later, of long chains. Beer is rendered turbid by this species. In its younger stages, motile cells have been observed. At a temperature of 36° C. the growth on beer consists almost exclusively of long, uniform threads. This species also shows the irregular, swollen forms, as, for instance, on beer at 26° C. The cells are not coloured blue by iodine. The optimum temperature for the growth lies between 18° and 21° C. The upper limit of temperature for the formation of motile cells was found to be 37°-40° C. (or 44° when rapidly heated). The temperature at which the vegetation is destroyed lies between 55° and 60° C. for moist heat, and between 97° and 100° C. for dry heat. The oxidation of alcohol into acetic acid has its optimum between 27° and 23° C. This species oxidises many different kinds of carbon com- pounds. Henneberg has recently described the following species or varieties : — Bact. acetigenum, which occurs in the quick vinegar vats, forms small rounded swarming cells, which are not grouped in chains. At a later stage swollen cells may appear. The species forms a thin, matted, and very tough film, which finally sinks to the bottom in isolated patches, giving room for a new film formation. By treatment with iodine and sulphuric acid a blue coloration may take place. The acetic acid produced by this species is very aromatic, owing to the formation of acetic ether. The species has its optimum at 33° C. Bact. acetosum, which is found in high-fermentation beer, forms long chains, and, at the same time, irregular shapes. The film is solid, dry, and after a time wrinkled ; the liquid is clear. The optimum for this species is about 28° C. Bact. industrium occurs as short swarming cells without chain formation or irregular shapes. On gelatine it forms greyish- 102 MICRO-ORGANISMS AND FERMENTATION. white slimy colonies, and in liquids a thick slimy film and a solid ring attached to the side of the vessel. When shaken, the film separates in flecks. The liquid is rendered turbid. The optimum on wort-agar is 23° C. The upper limit of temperature for motile forms is about 45° C. The species oxidises a large number of compounds. The vinegar produced contains much aldehyde, Bact. ascendens, which is found in wine and wine-vinegar, likewise consists principally of single cells or pairs of cells r but also forms chains. On grape-sugar gelatine and grape- sugar agar the colonies are surrounded by a white halo. In liquids the species forms a very delicate uniform film, which creeps to an extraordinary height up the sides of the vessel. The film is easily broken up and forms a flocculent deposit, and the liquid is rendered turbid. On wort agar the optimum is 31° C. This species is only capable of oxidising a minute amount of material. The vinegar produced is distinguished by its odour of acetic ether. In old cultures the vinegar has a very pungent smell. Amongst sub-species, or varieties which occur in the quick vinegar process, Henneberg has isolated the following : — Bact. Schiitzenbachi, which occurs as round, oval, or longish cells, often also sickle-shaped or in irregularly bent and inflated forms, sometimes single and sometimes in chains. On wort gelatine it forms round, clear, glistening colonies with a yellowish-brown centre. On beer- gelatine the old colonies have a whitish, granulated surface. The very thin film that forms on liquids easily sinks to the bottom as a powder. The optimum appears to lie between 25° and 27-5° C. Bact. curvum has rounded, longish, oval, or elongated cells, with either rounded or pointed ends. The more or less bent cells are especially characteristic ; it also forms chains. On wort-gelatine the colonies are transparent and rounded, with a raised edge and projecting centre, and frequently have a whitish dry appearance. The film forming on liquids easily falls to the bottom. The optimum lies between 25° and 30° C. ACETIC ACID BACTERIA. 103 Bact. orleanense. The cells vary in shape from spheres to pronounced rod-like forms with all possible transitions. The rods are straight or bent, single or linked in chains, and swollen cells also occur. On wort-gelatine irregular whitish colonies form. On beer - gelatine the older colonies are reddish in colour with a moist glistening surface. The film on liquids adheres firmly, and the liquid, therefore, remains clear. The optimum is at 30° C., and later between 20° and 30° C. The species may be used either for the quick vinegar or the wine- vinegar process. The same author has described the following special wine- vinegar bacteria (Orleans process). Bact. xylinoides occurs both in the form of fully rounded and of short or long rods ; they may be straight, bent, or irregular, and sometimes swollen, single, or in chains. On wort-gelatine the colonies appear like drops of water, and often exhibit a light brownish nucleus. On beer-gelatine they have a moist glistening, pale brownish surface. The film formation on liquids differs greatly. On sugar-yeast-water and on beer it is thick and tough, like Bact. xylinum ; on other liquids it may exhibit every transition from a thin, dry, or smooth, to a thick tough covering. The thick films show the cellulose reaction on treatment with iodine and sulphuric acid, but the thin do not. The optimum for agar-cultures is at 28° C., and later at 20°-23° C. In wine-vinegar mash the optimum lies nearer to 24° than to 28°. The species is found widely distributed throughout wine-vinegar factories. It can be distinguished from Bact. xylinum by the multifarious forms of skin growth. Bact. vini acetati has rounded, oval, and seldom moderately elongated cells, single or linked two and three together. In- flated cells also occur. The colonies on wort-gelatine are rounded, clear, have a moist glistening surface, and a whitish precipitate in the middle. The films are not very coherent, and the culture liquid soon turns cloudy. On wort with 3 per cent, alcohol the film has a greasy appearance and pale yellow colour. The optimum is at 28°-33°. As a nutritive fluid for these different bacteria, dilute vinegar with diluted beer or mash may be used, or diluted vinegar mash. In the vinegar factories, where the useful bacteria are attacked by 104 MICRO-ORGANISMS AND FERMENTATION. many foreign micro-organisms, attempts are made to suppress the dangerous kinds (mycoderma, mould, etc.), by the addition of about 2 per cent, of acetic acid. Finally, an acetic acid bacterium may be mentioned, found by J. C. Holm on cocoa beans, which he has named Bact. aceticum rosaceum. It forms short, rounded, motionless rods 1'6 fjL in length, single or in pairs. On wort or beer it forms a very weak, pale-coloured film, whilst the colonies on wort- gelatine and agar are distinctly pink in colour. Acetic acid bacteria play an important part in the fer- mentation of beer, spirits, and wine. They do much harm, especially in wine, and if they once attain a strong develop- ment, the wine is irretrievably spoilt. In low-fermentation breweries they usually do less mischief, as their growth requires a high temperature and an abundant supply of air. Thus, they are readily suppressed in a well- arranged lager beer cellar. Hansen's experiments have shown that Bact. aceti and Bact. Pasteurianum are able to exist during the whole time of storage, whether the infection takes place at the beginning or end of the principal fermentation. In his experiments the contamination, however, did not manifest itself during the whole course of the fermentation either by the taste or by the smell of the beer. When the beer was bottled, and exposed to a higher temperature, the bacteria developed further ; yet, when the bottles were well corked, the beer did not turn sour. Just the same result was arrived at when the finished beer was infected. If, on the contrary, the bottles were badly corked, the growth turned the beer sour. In high-fermentation breweries, on the other hand, where fermentation is carried on at higher temperatures, these bacteria are capable of doing much mischief, even before the beer leaves the brewery. It is of practical interest to note that the species described by Hansen exert no influence on the colour or brightness of the beer, whilst most other bacteria cause turbidity. In distilleries, and more especially in air-yeast factories, acetic bacteria may occur in large quantities, as shown by numerous experiments made by the author. They are most ACETIC ACID BACTERIA. 105 frequently accompanied by mycoderma species. A careful control of the manufacturing process in this respect should never be omitted. While investigating the influence of acids, especially acetic acid, on wine yeasts, Lafar found that each of the different acids (malic, tartaric, lactic, acetic, etc.) exerts a peculiar influence on the yeast, and not only on the proportionate amounts of alcohol and carbon dioxide produced, but also of glycerine ; the acetic acid samples contained the smallest amount of glycerine and showed the weakest growth of yeast. Contrary to the previously accepted view that even small amounts of acetic acid prevent alcoholic fermentation, Lafar found that the presence of 0-27 per cent, had practically no influence on the rate of fermentation, the multiplying of the cells, or the yield of alcohol and glycerine. In must, before neutralisation, the yeast cells were not impaired by an addition of 0-74 per cent. ; and in neutralised must, after adding as much as 1 per cent, of acetic acid, 4-77 per cent, by volume of alcohol was formed — i.e., 60 per cent, of the maxi- mum yield. Yeasts differ considerably, however, in their sensitiveness to the action of acetic acid. Thus, a comparison of fifteen different wine yeasts showed that all were able to carry on fermentation in the presence of 0-8 per cent, of acetic acid in a must that had previously been neutralised, whereas with 1 per cent, of acid only three were active. With regard to the propagation of cells, yeasts behave differently with the same amount of acetic acid. Lafar also examined the influence of these acids on the chemical activity of wine yeasts — i.e., on the proportion between the amount of alcohol produced and the number of yeast cells formed. He found that in presence of 0-88 per cent, of acid the amount of work done by one cell was greater in the case of ten varieties, but smaller in two varieties, than in the presence of 0-78 per cent. Those yeasts, which are active in presence of 1 per cent, of acid, gave a smaller yield than in presence of 0-88 per cent. According to WT. Seifert, the nitric acid present in wines which have been diluted with water containing nitrates, is completely decomposed by the action of certain acetic acid bacteria. 106 MICRO-ORGANISMS AND FERMENTATION. 2. Lactic Acid Bacteria. If the micro-organisms of milk are subjected to spon- taneous development at a temperature of 30°-35° C., the lactic acid organisms soon begin to ferment the lactose present (about 4 per cent.), and the acid produced protects the milk from putrefaction. After a certain quantity of acid has been formed it checks the activity of these bacteria, and the milk mould (Oidium lactis) develops. This oxidises a portion of the lactic acid, and thus enables the bacteria to restart their action. The same effect is produced if the acid is neutralised, for instance with calcium carbonate, and thus the complete fermentation of the milk sugar may be carried out. Simul- taneously with the formation of lactic acid, casein, which forms the most important part of the albuminoid constituents of milk, separates out. Before souring, the casein occurs as a calcium salt (100 casein to 1-55 CaO), and is present in the colloidal form. When the milk is soured, the lime combines with lactic acid, liberating the casein, which is precipitated in a fine flocculent condition, causing the curdling of milk. In addition to this a greyish-yellow serum gradually separates out containing calcium lactate, lactose, albumen, etc. Other varieties of lactic acid may be developed by exposing a malt or other mash to a given temperature. If the mash is maintained at 40° C. a pediococcus form develops vigorously, if at about 50° C. a short rod form. If a fraction of the liquor is transferred to another mash at the same temperature, the respective forms each receive an impetus, and after a few inoculations only the two forms can be discovered in the respective mashes by an ordinary microscopical examination. It will be noticed that by the process described there can be no guarantee that a pure culture has been obtained, for in each case other bacteria survive, even if in an extremely weak condition, and, on the other hand, there is a possibility that more than one species, or variety, of lactic acid bacterium may develop at each temperature. Similarly in beer-wort and other liquids spontaneous lactic acid fermentation may occur. This is also the case in the souring of " sauerkraut," the preparation of leaven, ensilage, etc., and the bacteria which develop in LACTIC ACID BACTERIA. 107 these several fermentations doubtless represent many different species. The lactic acid developed in the fermentation of milk, an acid first definitely characterised by Scheele in 1780, corre- sponds approximately with the quantity of lactose that has disappeared. Only minute quantities of by-products are formed, as was proved by the detailed researches of Kayser. If the fermentation is continued for some time, many species will decompose part of the lactic acid originally formed. Kayser found that a pure cultivated species from cream grown in lactose-peptone-wort had lost 0-26 gramme of lactic acid per litre in eleven days. If volatile fatty acids are formed, they will tend to increase in quantity under these conditions at the expense of the lactic acid. According to 0. Jensen, lactic acid may itself be converted into volatile fatty acids. Lactic acid formed by the spontaneous fermentation of milk is usually optically inactive — i.e., it does not turn the plane of polarised light either to the right or to the left. If, however, the active bacteria are isolated in pure cultures, and inoculated into sterile milk, species are developed which produce a lactic acid turning the plane of polarised light to the right (dextro-rotatory bacteria), and others producing a laevo-rotatory acid. The dextro-rotatory species occur more frequently. Thus the species of bacteria determines whether one or the other sort of acid shaU be produced. It appears, however, that there are also species of lactic acid organisms in which the optical activity of the product . of fermentation depends upon the composition of the nutritive fluid, as shown especially by Kayser. The species reacts differently with dif- ferent sugars. Thus the common Bact. lactis acidi (Leichmann) ferments dextrose, lactose, maltose, mannite, and raffinose. Hueppe's Bac. acidi lactici ferments saccharose, dextrose, lactose, and mannite. A few species thrive best when they have access to atmospheric oxygen, whilst others carry on the fermentation equally well, or even much better, in the absence of air. They also exhibit differences in the rapidity with which acidification takes place at different temperatures. Thus, the Bact. lactis acidi has its optimum for the formation of acid at 32°-38° C., Hueppe's Bac. acidi lactici at 35°-42° C., 108 MICRO-ORGANISMS AND FERMENTATION. others at 20°-22° C. and 40°-48° C. In general the largest amount of acid is generated at temperatures somewhat below the given optimum. In 1903, Herzog proved that Bac. acidi lactici contains an enzyme which can be isolated from the living cell, and is capable of producing a lactic fermentation. He treated a pure culture mixed with kieselguhr with methyl alcohol, and afterwards with ether. The mass was then dried, and the resulting white powder, which contained no living cells, could convert minute quantities of lactose into lactic acid. Buchner and Meisenheimer subsequently proved that if a culture of one of the species growing in a distillery mash, Sac. acidificans longissimus (Bac. Delbrucki, Leichmann) is treated with acetone, whereby it is killed, and the mass is then dried, a powder is obtained which can bring about a lactic acid fermentation in a sugar solution. It may, therefore, be assumed that all bacteria of this group contain enzymes that can bring about fermentation independently of the living cell. To cultivate the lactic acid bacteria of milk, a preparation of peptonised milk made by 0. Jensen may be used : — To a litre of sterilised milk, 10 c.c. of pure concentrated hydro- chloric acid and 2 grammes of pepsin (P. granulatum) are added. The mixture is kept in an incubator, and frequently shaken. When the casein has dissolved, the acid is neutralised, and the liquid sterilised at 115°- 120° C. Gelatine or agar may be added before neutralisation. In addition to the proper lactic acid organisms, there are a large number of bacteria, and, amongst them, some patho- genic forms which develop this acid. We are indebted to Pasteur for the first important work on the subject of lactic acid bacteria. In 1858 he described the species which appears when milk spontaneously ferments. In his Etudes sur la biere he depicts certain bacteria growing in wort or beer in which lactic fermentation has begun (Fig. 22) ; he describes them as short rods slightly narrowed in the middle, and commonly occurring singly, rarely united in chains. In 1877 Lister prepared a pure culture of a lactic acid bacterium from sour milk, which he called Bacterium lactis. LACTIC ACID BACTERIA. 109 In 1884, Hueppe found a bacterium in a spontaneous lactic acid fermentation which converts lactose and other sugars into lactic acid with the simultaneous formation of carbon dioxide (Bacillus acidi lactici). It consists of short, plump, motionless cells, the length of which exceeds their breadth by at least one-half ; they are united chiefly in pairs, and seldom in groups of four. In gelatine plates they form whitish colonies ; those below the surface are stellate, uni- formly dark, and sharply outlined ; on the surface they appear as flat white glistening nodules, resembling porcelain surrounded by clear outer zones. Atmospheric oxygen is necessary for fermentation with this species. In recent times a large number of species of lactic acid bacteria have been found in milk. Marpmann, in 1886, described five species embracing both coccus and longer and shorter rod forms, and showed that the whole series was capable of producing a slight formation of alcohol. . O Fig. -22. —Lactic acid bacteria (after Pasteur). — In order to give an idea of the size of the bacteria, some yeast cells are figured among them. Hueppe and Grotenfelt have since described new species, of which Grotenfelt 's Streptococcus acidi lactici appears to be identical with the Bad. lactis acidi described by Leichmann. Adametz and Freudenreich have isolated species from Emmenthaler cheese (Bac. casei) which are for the most part facultative anaerobes. Leichmann has thrown new light on the conditions present during the spontaneous souring of milk. He found that a single species or type strongly preponderated, and named it Bact. lactis acidi (Streptococcus lacticus}. He described it as consisting of short motionless rods about one and a half times as long as they are broad, sometimes present in pairs, sometimes in chains (the latter particularly when cultivated in sugar- broth). On gelatine plates, the immersed colonies consist of round discs, white or pale yellowish-brown, at first transparent, 110 MICRO-ORGANISMS AND FERMENTATION. afterwards opaque. The surface colonies develop with extra- ordinary difficulty : they are transparent, and have a some- what irregular edge. This species excites fermentation even in complete absence of air ; in presence of a full supply of air the fermentation is restricted. Both Leichmann and Weig- mann consider this species to be the regular means of spon- taneously curdling milk, and explain the process of souring as follows : — Hueppe's Bac. acidi lactici, and other species of the aerogenic group, remain in the upper layers of the milk, to satisfy their great demand for air, and acidify these ; whilst the facultative anaerobe, Bact. lactis acidi, develops in the lower layers, and acidifies them. This species is easily grown in lactose- or grape-sugar-broth, and then forms long chains. As already stated, it ferments these sugars as well as lactose, mannite, and raffinose. It forms dextro-lactic acid, and produces no evolution of gas in sterilised milk. A number of so-called species described by different authors appear to be varieties of this species. In spontaneously soured milk, Leichmann discovered another species of frequent occurrence, which has great similarities with the above, but may be distinguished by its production of laevo-lactic acid, and by the evolution of gas (Micrococcus acidi Icevolactici). He also isolated a species which thrives best at 44°- 5 2° C., develops laevo-lactic acid, and forms thin rods of varied length. On agar it forms root-like, branching colonies. The great series of interesting varieties cultivated in a pure state by Weigmann deserves special attention. Their appearance at first resembles that of Leichmann's Bact. lactis acidi. Biologi- cally, however, they show important differences, and several have found widespread industrial application. Other species have been described by Marpmann, Conn, Kozai, Beijerinck, etc. In addition to these typical lactic acid bacteria, other species occur in the souring of milk which possess a curdling and a peptonising enzyme. The ripening of cheese, which consists in a conversion of casein (paracasein) into simpler albuminoids, and the breaking down of the latter, is brought about chiefly through the action of certain micro-organisms. As is well known, the curd may be separated from the milk by the addition of RIPENING OF CHEESE. Ill rennet,* whereby it remains sweet, or less frequently by the application of lactic acid (sour milk). The gradual decom- position of the curd is due to a slight extent to the action of pepsin contained in rennet, as well as to an enzyme (Galactase, Babcock and Russell), although, according to O. Jensen, this is precipitated from milk by bacteria at a very early stage. The action is, however, mainly due to the rich flora of micro- organisms embedded in the cheese. These are derived partly from the rennet, and partly, and, indeed chiefly, from the milk itself, and consist of lactic acid bacteria, peptonising bacteria, butyric acid bacteria, and moulds. The basis of bacteriological work on cheese was laid by Cohn, Duclaux, and Benecke, and their researches have been extended by Adametz, Freudenreich, Weigmann, 0. Jensen, and Harding. The main lines of this development must be regarded, according to these workers, as a modification of the albuminoids of the curd brought about during the first short period by peptonising bacteria — bacteria that had been active in the milk. Amongst these must be especially mentioned Micrococcus casei liquefaciens, which occur in great numbers. It multiplies at lower temperatures than the true lactic acid bacteria, and, therefore, is found in large numbers in the cooled milk, and is thus transferred to the cheese. It coagulates * Rennet is an enzyme which decomposes casein into paracasein and whey albumen. It is secreted in special glands of the stomach of various animals (e.g., the ruminants) ; calves' stomachs are used for the preparation of rennet. In the vegetable kingdom this enzyme is widely distributed. It is found, for example, in Pinguicula, Ficus carica, Galium vervm, in the calyx of the artichoke (Cynara scolymus), and in many bacteria. In 1892, Conn isolated an enzyme resembling rennet from bacteria which were isolated from cream, and completely lique- fied gelatine. They produced the enzyme most rapidly and freely at about 20° C. He isolated it from the filtrate of a ten-day-old milk culture. The filtrate was acidified with O'l per cent, of sulphuric acid, and then mixed with an excess of salt. A white foam separated out, which contained the comparatively pure enzyme. The dry foam formed a white powder. This enzyme also occurs in the ubiquitous putrefactive bacterium, Bacillus vulgaris (Prot. vulg.). It further occurs in Bad. prodigiosum (the bacterium of the Bleeding Host), and also in Bac. coli communia, which is always found in the intestines of men and animals, as well as in many of the " potato bacilli." Finally, it has been detected in torula species (Lactomyces), in different species of moulds (e.g., Aspergillus, Monilia), and in certain yeast species. 112 MICRO-ORGANISMS AND FERMENTATION. milk in 24 hours at 35° C., ferments lactose, and produces volatile acids, especially acetic acid. Its growth is, however, soon arrested by the true lactic acid bacteria, and, in particular, by the development of Bact. lactis acidi (Streptococcus lacticus), which causes a vigorous formation of acid. According to Weigmann, the lactic acid is then gradually displaced by acid-consuming fungi or by bacteria producing alkali, and the peptonising or casease bacteria come into activity ; in particular, the species producing cheesy aroma. The special characters of different kinds of cheese are due to special micro-organisms. The particular part played by the lactic acid bacteria in the process is to prevent the peptonising bacteria from getting too great a hold, and thus producing too quick a decomposition of the curd. The con- version process is thus regulated by means of the lactic acid organisms. According to 0. Jensen, they further stimulate the action of pepsin derived from rennet. Lastly, their importance in the ripening of cheese depends to a great extent upon the fact that certain species effect a further transforma- tion of the products of decomposition, especially of the albumoses and peptones produced in milk, and in the early stages of the ripening of cheese by peptonising bacteria. In particular, the production of volatile acids, such as propionic acid and acetic acid, detected by O. Jensen, is to be attributed to this cause. Quite recently Jensen has discovered a special propionic acid ferment in Emmenthaler cheese, which he believes to be a variety of Bact. lactis acidi. According to Jensen the carbon dioxide produced by this species forms the " eyes " in Emmenthaler cheese. Amongst the casein-digesting bacteria must be classed the aerobic Tyrothrix species, minutely described by Duclaux, thread-forming bacteria, which secrete an enzyme resembling trypsin, and belong to the group of hay bacilli. To this group belongs Bacillus nobilis, discovered by Adametz, and, lastly, Paraplectrum foetidum, detected by Weigmann in Limburger cheese, which occurs in milk as thick rods, and at the tempera- ture of the incubator (30°-40° C.) assumes mallet shapes, and quickly forms spores which are twice as long as they are wide. RIPENING OF CHEESE: ABNORMAL MILK. 113 Anaerobic bacteria also play a part in cheese fermentation, and, amongst them, butyric acid bacteria. Amongst moulds which are of special importance in deter- mining the character of different cheeses must be mentioned the Mucor, Penicillium and Dematium species observed by O. Johan-Olsen in Norwegian " Gammelost " (old cheese) ; a white Penicillium (P. candidum, Rodger) in Camembert cheese ; and a similar Penicillium album (Epstein) in Brie cheese. Weigmann, Conn, and others, on the contrary, attribute the special character of both these cheeses to the action of a particular Oidium. O. Jensen assumes that the peculiar flavour of Roquefort cheese is due to the symbiosis of Penicillium glaucum and Oidium lactis. All these moulds act partly by decomposing the acid contained in the cheese, and partly by neutralising the acid with ammonia formed by the breaking down of casein, and thus they prepare the ground for the peptonising bacteria, whilst also decomposing the milk fat, and liberating its volatile fatty acids. The abnormal characters of milk and milk products must in an equal degree be attributed to micro-organisms.* Thus, for instance, the bacteria introduced into milk from a diseased udder, and the consequent changes in the character of the milk, are accompanied, not only by a very great increase in the bacterial contents, but also by the presence of characteristic pus cells in the milk. " Soapy " milk, having a decided soapy taste and pro- ducing a strong lather, owes these properties to the presence of Bac. lactis saponacei, a short rod which forms slimy colonies on ordinary nutritive gelatine, turning to a rusty yellow on the surface. Other species may also produce this fault. Bitter milk may be the result of using certain food-stuffs, but may also be produced by certain bacteria, as was shown by Pasteur, Duclaux, Loftier, Weigmann, and others. In practice, micrococci which liquefy gelatine and certain vari- eties of aerobic, lactic acid bacteria appear to have this effect. * It is generally accepted that milk may act as a carrier of many dangerous disease germs ; typhoid epidemics in particular appear to spread in this way. Tubercular bacilli, capable of development, have frequently been found in raw milk. 114 MICRO-ORGANISMS AND FERMENTATION. Certain Torula yeasts may grow in milk and make it bitter (Calloghan and Harrison). " Ropy " milk has a marked slimy character, and can be drawn out into threads. This is due either to the enormously swollen membrane of certain bacteria, or to the formation of slimy albuminoid bodies. The active micro-organisms are either varieties of lactic acid bacteria, or more particularly the bacteria that digest albumen. A widely distributed species is Bac. lactis viscosus, described by Adametz, which gradually converts the viscid milk into a substance resembling honey. Another widely distributed and active species is Micrococcus Freudenreichii, described by Guillebeau, which liquefies gelatine. The lactic acid bacterium, Streptococcus hollandicus, Hueppe, described by Weigmann, Goethart, Boekhout, and others is of particular interest ; it occurs in Dutch " lange Wei " (a ropy cream used in the manufacture of Edam cheese). This organism is a facultative anaerobe, and has its optimum at 21°-22° C. Like many other varieties it easily loses its property of forming slime. A similar variety was found by G. Troili-Petersson in the Swedish " tatmjolk " (thick milk), and described under the name of Bact. lactis longi. It is believed that this species con- structs slime from lactose. It has its optimum below 20° C. Probably such bacteria are present on certain plants that are placed in the milk, such as Pinguicula and Drosera. By the action of micro-organisms milk may assume a blue, red, or yellow colour. The blue coloration of milk depends upon the growth of certain species of bacteria, the presence of which was proved by Fuchs as early as 1841. Hueppe was the first to prepare a pure culture of one of these species, and he described it under the name Bacillus cyanogenus (Bact. syncyaneurri), which occurs as a short motile rod. In the case of this, the most widely distributed species, the colour appears first on the surface of the raw milk, and afterwards penetrates to the lower layers. The production of colouring matter originates from the albuminoids, and may occur in the absence of sugar. A number of the water bacteria also have the power of imparting a blue colour to milk. The reddish colour which milk occasionally assumes is also due in certain cases to bacteria. Only a few examples are quoted in ABNORMAL BUTTER AND CHEESE. 115 the technical literature. Hueppe found a Bact. lactis eryihro- genes in red milk, which is described by Grotenfelt as a short rod coagulating milk, and producing a red colour on gelatine plates. Menge found a Sarcina rosea in red milk, which also forms red colonies on gelatine, and a few other species with similar properties have been detected. In yellow milk a Bact. synxanthum has been observed and described by Schroter. The taints observed in butter must also be chiefly attri- buted to micro-organisms, and in the technical literature it is strongly emphasised that a great development of such harmful species is frequently caused by a want of cleanliness, or by a wrong souring of the milk. The rancidity of butter, which is due to the presence of butyric acid and ethyl butyrate, is caused by the action of light and air. According to 0. Jensen this phenomenon is due to the presence of aerobic fungi, which cause the decomposition of fat, in particular Cladosporium butyri and Oidium lactis. This may further be due to the presence of two bacteria universally found in water, Bac. fluorescens liquefaciens and, occasionally, Bact. prodigiosum. The action of light may also produce the tallowy taste, but Storch has isolated a rod-shaped, lactic acid bacterium which can produce the same effect. A turnip flavour and a rotten- sweet flavour in butter, studied by C. 0. Jensen, proved to be derived from a special species, Bacillus joetidus lactis. Weigmann has subsequently observed similar bacteria. A whole series of other irregularities in the character of butter are also accompanied by the growth of different micro-organ- isms, and the assumption is reasonable that they must be the originators of these taints. One of the most pronounced faults with cheese is " blowing/' which is due to the presence of great masses of fermentation bacteria causing an excessive development of gas ; in particular, a species, Bac. Schafferi, belonging to the Bac. coli group, plays an important part in this respect. The same fault may be produced in cheese by the species originating from diseased udders. Yeasts may produce a strong evolution of gas and certain varieties of coli and aerogenes species may also bring about vigorous fermentation with production of gas. A 116 MICRO-ORGANISMS AND FERMENTATION. suitable degree of souring with lactic acid bacteria appears to be a certain means of preventing these mishaps. The blue flecks which appear in certain kinds of cheese may be produced, according to Beijerinck, in some cases by Bac. cyanofuscus, which is derived from water. Black flecks may be caused by growths of moulds, such as Cladosporium and Fumago. Rusty specks, according to Connell, Harding, and other American observers, are caused by a definite species, Bac. rudensis. In the same way a reddish colour is produced by red moulds, micrococci, etc. Lastly, a fault must be mentioned which may occur in all kinds of cheese, the bitter taste, which is caused by certain bacteria, as, for instance, by Micrococcus casei amari, described by Freudenreich, and also by a species occurring in bitter milk, and even by certain moulds and Torulce. Since 1890 methodically selected species of bacteria have been applied in dairies, to bring about a regular and certain souring of the cream used in the manufacture of butter, and to avoid any taint in butter. The progress made in this field is associated with the researches of Storch, Weigmann, Quist, in the author's laboratory, and others. The pure culture selected is added to skim milk, previously heated to about 90° C., and the culture is allowed to develop at about 15° C. After standing 24 hours, this "starter" is fit for use. In order to render the cream which is to receive the culture as free from germs as circumstances permit, it is pasteurised at about 85° C., and then quickly cooled. In the course of ten hours or so, the starter is allowed to develop in cream at about 16° C. It is then cooled below 10° C., and churning is begun. Among the forms isolated by Storch of Copenhagen (1890) from butter, sour cream, and butter-milk, the coccus form of the group Streptococcus lacticus seems to be most frequent and best suited to sour the cream. It occurs in a large number of varieties, which, according to their main characteristics, may be classed in two groups — one including those which give a specially pure and mildly sour taste and a fine aroma, and another embracing those which yield a product possessing great keeping powers. Morphologically the growths are dis- SOURING CREAM. 117 tinguished from each other by the fact that some are connected in chains, others are not (Fig. 23) ; the latter are of the most frequent occurrence, and are most widely distributed. These forms bear a certain resemblance to Pasteur's " ferment lactique." The species represented in Fig. 23, B, was isolated by Storch from a sample of butter having a pure and full aroma. It forms small globular colonies in gelatine of a pure white colour and smooth surface. In milk and whey it occurs in oval or globular forms. These lactic acid bacteria display fermentative activity, even at 20° C. At 28° C. milk is turned sour within eight to nine hours. Many species have been isolated by Weigmann and intro- Fig. 23.— Lactic acid bacteria (after Storch). duced into practice. A species which has been very success- fully applied at several places was prepared by Quist, and afterwards by J. C. Holm in the author's laboratory. It occurs both as micrococcus and in other forms, according to the different nutrient media in which it is cultivated. On gelatine it forms small, circular, slowly-growing colonies of a * whitish-yellow colour. In stab-cultures spherical colonies arise throughout the puncture-channel, and in streak-cultures this organism forms a continuous streak with wavy borders. It was prepared from a sample of butter of remarkable aroma and durability. Pure cultures of lactic acid bacteria have also been applied 118 MICRO-ORGANISMS AND FERMENTATION. in cheese factories to regulate the ripening of cheese. They are always added to raw milk, as it is of importance that other species of bacteria from the milk which play a part in the process should not be suppressed. Attempts have also been made to apply cultures of other bacteria and of moulds in the preparation of cheeses of pronounced character — e.g., Roque- fort, Camembert, etc. As the mash in distilleries is not allowed to exceed a temperature of 70° C., in order that the diastase may be preserved, many of the germs adhering to the raw materials are not killed, but are capable of developing during fermenta- tion, and thus they may not only utilise the nutritive sub- stances, but also disturb the desired alcoholic fermentation ; in the latter respect, butyric acid bacteria are specially dreaded. With the view of preventing too strong a development of germs injurious to the yeast, various acids have been added direct to the mash, or else a lactic acid fermentation has been previously carried out in a fraction of the mash. Thus a tenth part may be kept at a temperature of 50°- 5 5° C., till it shows about 2|° of acidity,* corresponding to about 1 per cent, of lactic acid. At this temperature the desired species of lactic acid bacteria develop, whereas it is too high for the majority of bacteria. An excellent means for maintaining the mash at this temperature is the acid chamber introduced by Kruis into distilleries, a small and well-isolated space in which the air maintains a constant temperature, and in which the mash to be soured is introduced and allowed to stand quietly as soon as it has cooled down to the same temperature. The mash is then heated up to 70°-75° C., whereby part of these bacteria are killed. After subsequently cooling down to about 20° C., the yeast is added. The yeast is not affected by this quantity of lactic acid. After it has developed sufficiently, the mixture is employed for pitching the principal mash. To devise a rational process for pitching the lactic acid mash with bacteria, a part of the mash must be placed on one side before yeast is added, and used to start the * i.e., 2i c.c. of normal caustic soda solution are required to neutralise 20 c.c. of mash. DISTILLERY MASH. 119 souring of the succeeding acid mash. About one-tenth of the fermented acid mash is used for pitching the following mash. The acid thus introduced into the principal mash, together with the surviving lactic acid bacteria; act as disinfectants, besides exerting an influence on the yeast cells, both directly and by reacting on the nutritive substances. The lactic acid bacteria occurring in the mash can be distinguished in many ways from those occurring in milk. Zopf was the first to prepare and investigate a culture of a species belonging to this class, from a mash obtained from dry malt and water at 50° C., according to Delbruck's process (1881), following up an observation of Delbruck's that at this temperature a vigorous lactic souring took place. A growth of threads, rods, and cocci was developed. Pediococcus acidi lactici, examined by Lindner, gives a strong acid reaction when cultivated in a neutral malt-extract solution at 41° C. Both in a solution of this kind and in a hay decoction, which has not been sterilised, this bacterium develops so vigorously that, according to Lindner, all other organisms are suppressed at this temperature. It has been proved chemically that the acid, which is abundantly produced, consists for the most part of lactic acid. When a malt mash or malt-rye mash is maintained at 41° C., the Pediococcus develops vigorously, and the rod-shaped lactic acid bacteria are suppressed. According to Henneberg the optimum for the formation of acid is 38° C. The optimum for growth on beer and wort agar lies between 36° and 40° C. In a neutral malt-extract solution the Pediococcus is killed after five minutes' exposure to 62° C. In gelatine it does not thrive well ; it is only in stab-cultures in neutral malt-extract gelatine that very vigorous white colonies are formed below the surface. This species appears, on the whole, to thrive better when air is excluded. In 1893, Kruis and Rayman isolated a vigorous lactic acid bacterium from yeast mash consisting of long and short rods, which produced 0-9 per cent, of lactic acid at 40° C. in a clear malt wort. It is of special interest to note that Kruis and Rayman in studying this species proved, for the first time, that lactic acid bacteria are capable of forming volatile fatty acids. 120 MICRO-ORGANISMS AND FERMENTATION. Lafar isolated from the sour yeast mash a species which he named Bac, acidificans longissimus, and since 1894 it has been applied in practice for souring the yeast " goods." It ferments saccharose, galactose, dextrose, laevulose, and maltose, but not lactose, and it occurs both in short rods and in very long threads. A short time afterwards, Leichmann described a bacterium occurring under similar conditions, Bac. Delbriicki, which is believed to be identical with Lafar 's species. It shows great resemblance to Leichmann's Bact. lactis acidi, and both species produce laevo-lactic acid. It cannot, however, like the latter, ferment lactose. In a lactose broth it produces no acid, and grows with difficulty, whereas in grape-sugar broth or maltose broth, as weh1 as in sweet wort, it grows vigorously. According to Henneberg this species has its optimum for acid production at 46°-47° C. In the mash it forms up to 1-79 per cent, of lactic acid. The amount of acid is reduced with free access of air. Its optimum for growth lies between 40°-48° C. In the mash,' it occurs with both short and long cells, single or grouped two and three together. On solid substrata it forms small, flat, clear colonies. Henneberg has isolated a number of other species of lactic acid organisms from mash and pressed yeast, which he has described as " wild," some of which may produce direct damage in the industry, if care is not taken to secure a vigorous yeast fermentation, for they not only carry on the production of acid throughout the fermentation, but form at the same time volatile acids, especially acetic acid, which damages the yeast and reduces the output of alcohol. Other members of this group appear to be harmless. All the species examined grow and produce acid in presence of yeast at 27'5°-30° C. Amongst the dangerous kinds may be named Bac. Hayducki, which occurs in mash in small short cells, mostly single, and forms round white colonies on gelatine (its optimum for acidification is first at 45°-46° C. : later at 33°-35° C.), and Bac. Buchneri, with similar cells in the mash and white or yeUowish colonies on gelatine (optimum for acidification first 39°-40°, and afterwards 23°-30° C.). There appears to be no doubt that the lactic organisms occurring in the mash have a tendency to variation, and that DISTILLERY AND BREWERY MASH. 121 not a few of the numerous species described must be regarded as sub-species of a certain parent form. Thus Beijerinck takes, as the parent form of Leichmann's species, Bac. DelbriicJd, a very vigorous acid producer Lacto- bacillus fermentum, a young culture of which prepared at 37° C. for 36 hours in an acid mash, and then transferred to wort jelly (gelatine agar), forms very small transparent colonies consisting of motionless bacilli of different lengths together with micrococci. In wort it forms threads of different lengths. The smaller the amount of oxygen present the more extended are the cells. Its optimum for the production of acid is 41°-42° C., and at 50° C. it ceases to develop acid. According to Beijerinck this parent form may be converted into Bac. Delbriicld through a continued series of cultivations with free access or air at 48° C. (well above its optimum for acid pro- duction), and then transferring to wort jelly at 37° C. In the distillery, Lafar, Leichmann and the author (in 1896), and more recently Kusserow and others, have introduced into practice pure cultures of predominant species occurring in normal sour mash (Bac. acidificans longissimus, Bac. Del- briicki), with the object of regulating the souring of the mash. By the proper application of such cultures in distilleries and yeast factories, and at the same time securing a uniform temperature of acidification throughout the whole mash, it is possible to absolutely prevent the development of foreign and harmful bacteria. In the brewing industry the lactic acid fermentation takes place in malting, and in particular in mashing. Accurate investigations were made by Prior. Some lactic acid is also formed during fermentation. Lactic acid is formed in large quantity in Belgian beers prepared by " spontaneous fer- mentation," imparting a sharp taste. " Weissbier " owes its refreshing taste to a vigorous lactic acid fermentation. In modern low-fermentation breweries attempts are made to get rid, not only of lactic acid bacteria, but also of bacteria of all kinds from the fermentation. The Saccharobacillus Pastorianus described by van Laer, which occurs in the form of rods of different lengths, produces a characteristic disease (" tourne ") in weakly hopped beer, 122 MICRO-ORGANISMS AND FERMENTATION. which manifests itself as follows : — The liquid gradually loses its brightness, and when it is agitated filaments of a silky lustre rise from the bottom, and the beer assumes a disagree- able odour and taste. According to Fellowes, this species is also found in English beer. It does not always react on the beer, probably on account of the larger amount of hops. In cultures, the bacillus develops either in the presence or absence of free oxygen. In nutrient liquids it ferments carbohydrates, and amongst them the saccharoses, without previously inverting them. Amongst its fermentation pro- ducts, lactic acid, acetic acid, and alcohol predominate. The acids produced cause the precipitation of nitrogenous com- pounds in the liquid, and these, mixed with the bacilli, produce a cloudiness, consisting of lustrous filaments. The nutritive mixture best suited to this bacterium is an extract of malt mixed with agar and a small quantity of alcohol, or, still better, neutral or slightly alkaline sweet wort. If this bacterium is exposed to a temperature of 55°-60° C. in beer, it is soon killed. Henneberg has closely investi- gated both this species and two other lactic acid bacteria occurring in beer which cause the same disease — Saccharo- bacillus Past., var. berolinensis and Bac. Lindneri. These all occur as thin and comparatively long bacilli, either straight or curved, and usually cluster together. In hanging drops they form very long threads. From a physiological point of view they behave quite differently. Saccharobacillus Past. gives the most vigorous formation of acid in arabinose and trehalose, weaker in saccharose, maltose, dextrose, laevulose, and galactose. Its optimum for acid formation lies between 24° and 33° C. The variety berolinensis gives a weaker yield of acid, and none at all in raffinose and trehalose. Its optimum for acid formation is at 20°-24° C. According to Henneberg it is this species in particular which grows in Berlin " Weiss- bier," and imparts to it its peculiar character. Other varieties of lactic acid bacteria occur, however, in this kind of beer. Bac. Lindneri produces acid in maltose, and to a small degree in dextrose. The optimum for acid formation is at 17°- 18° C. This species or variety frequently occurs in ordinary lager beer, and influences its flavour and aroma, LACTIC ACID BACTERIA IN WINE. 123 without, however, producing any considerable amount of acid. It may occur in the form of long cells in lager beer. Schonfeld observed a species in various high-fermentation beers, which looks like the bacterium of Berlin Weissbier, and gives to these beers a slight lactic acid flavour, and may make the beer membraneous. In the fermentation of wine lactic acid bacteria may also occur, and may produce great alterations in the consti- tution of the liquid. Amongst the better known phenomena are " vin tourne " and " pousse " (lactic acid, Zickendwerden), due to the presence of considerable quantities of lactic acid. Pasteur, as early as 1866, referred these diseases to the activity of bacteria, and at a later date Muller-Thurgau proved that a short bacillus regularly occurs in such wine, and is capable of converting many of the constituents of the wine into lactic acid, or of producing a lactic acid fermentation in wine must. The disease declares itself in this way : — The wine becomes turbid, and is at first pale, afterwards dark, and deposits a sediment. At the same time it assumes an unpleasant smell and taste. The disease appears in those seasons when the must is poor in acid, so that the bacteria find more favourable conditions for growth. A bacterium may be mentioned here that was isolated by Gayon and Dubourg, a rod-shaped motionless organism which occurs especially in red wine, and forms mannite (ferment mannitique), whereby the wine assumes a characteristic bitter-sweet taste. By the fermentation of different sugars it forms lactic acid, acetic acid, etc., and only forms mannite by fermentation of laevulose. It grows freely in dextrose and broth, and has its optimum at about 35° C. By heating the wine to 60° C. its development is restricted, and this is also the case if the fermentation is vigorously carried out below 30° C. Like the previous species its development is favoured by a wine with a low acid content. By the addition of tartaric acid its growth may be checked. Bact. mannitopceum, discovered by Muller- Thurgau, belongs to this group. It forms the bacterial bubbles described in an earlier section (zooglcea). It occurs in the form of motionless rods, both long and short, and threads, which may produce snow-white flecks in fruit wine. In 124 MICRO-ORGANISMS AND FERMENTATION. must-gelatine the colonies are round or sausage-shaped, white, and non-liquefying. The species is facultative anaerobic, and has its optimum for growth at 25°-30° C. It decom- poses laevulose and saccharose, and forms acetic acid, lactic acid, and mannite. According to Miiller-Thurgau's observa- tions, mannite fermentation occurs fairly frequently in fruit wines, especially those prepared from over-ripe fruit, lacking in acid. It is accompanied in fruit wines, as well as in grape wine, by the formation of larger quantities of lactic and acetic acid. In this connection a species must be mentioned which plays an important part in the reduction of acid in wine. According to Wortmann, Alfred Koch, and Seifert, certain bacteria bring about this result, and Seifert has isolated a species in pure culture. It is named MicrococciLS malolacticus, and destroys part of the malic acid, the most important acid of the wine, and forms lactic acid. As a consequence of the activity of this bacterium, the wine loses its acidity, and acquires a milder flavour, causing an improvement in its quality. Like other organisms that destroy acid, its activity is first displayed after the proper fermentation has ceased and the yeast is in a weak condition. The species has its optimum at 25°-34° C., and the limits for its growth are at 34°-37° C. It forms small milk-white transparent colonies on nutrient gelatine. Other bacteria forming lactic acid and producing diseases in wine are described by Maze and Pacottet, as well as by Laborde, who has also investigated the mannite ferment. In leaven, lactic acid bacteria also occur which, without doubt, play a part in the fermentation of bread. Peters, for instance, found a species which occurs in motile rods, and forms a slimy skin on neutral yeast-water-sugar at 30° C. Henneberg found Leichmann's Bact. lactis acidi by development in mash at 48° C., and at 38° C. a special species which he named Bac. panis fermentati, which occurs in mash in short and long rods, and forms small white colonies on wort-agar. The optimum for the production of acid is at first about 37°-42° C., and afterwards 34°-38° C. Henneberg also found different species of lactic acid organisms in pressed yeast along BUTYRIC ACID BACTERIA. 125 with the proper culture species, and amongst them Bac. Hayducki and Bac. Buchneri. Bac. Lister i should also be mentioned, which occurs in comparatively short rods and in chains. Its optimum for acid formation is at 34° C. Bac. Wortmanni has its optimum at 33°- 40° C., and later at 25°- 29° C. Lastly, three forms occur, Bac. Leichmanni (I., II., and III.). When cultivated in mash they appear chiefly as short rods linked in chains, with an optimum for acid formation of 35°-36-5° C. at first, and afterwards at consider- ably lower temperatures. When pressed yeast turns soft, the various kinds of lactic acid bacteria increase largely in numbers. The lactic acid fermentation plays a very important part in the means adopted in different countries for preserving vegetable foods for both man and beast. Vegetables are chopped up, in certain cases salt is added, and they are placed in vessels or in hollows protected from access of air. A fer- mentation sets in and lactic acid is produced as one of the products. This acid protects the material from the attack of other micro-organisms, and gives the peculiar character to the preserved vegetables. The temperature usually rises when fermentation begins, which allows of the partial develop- ment of special therm ophilous bacteria. As a consequence of the development of other micro-organisms, the amount of acid is always reduced with prolonged fermentation. The active species are described by Wehmer, Aderhold, Weiss, Henneberg, and others. A rich flora of species occurs, how- ever, in such ferments. In the same way such a souring is used for the preservation of different feeding stuffs. The heat evolved during the fermentation imparts a special character to the fermenting mass, which varies according to the extent to which the temperature rises. 3. Butyric Acid Bacteria. When stale milk in which lactic acid bacteria have de- veloped is neutralised by the addition of calcium carbonate, so that calcium lactate is formed, it will, as a rule, undergo a butyric fermentation. Pasteur showed in 1861 that this fermentation is brought about by particular micro-organisms 126 MICRO-ORGANISMS AND FERMENTATION. which are able to live without air (" vibrions butyriques "). This spontaneous butyric acid fermentation takes place most vigorously at 35°-40° C. Starch, glycerine, dextrin, cane- sugar, maltose, lactose, and dextrose are likewise decom- posed by the butyric acid ferments, and such fermentations a/re of frequent occurrence, as the bacteria belonging to this group are very widely distributed in nature. In order to induce a, butyric acid fermentation, Fitz recommends using a mixture of 2 litres of water, 100 grammes of potato-starch or dextrin, 1 gramme of ammonium chloride, the ordinary nutrient salts, and 50 grammes of chalk ; this mixture is to be maintained .at 40° C. Bourquelot recommends exposing slices of raw potatoes, standing in water for two or three days at a tem- perature of 25°-30° C. (a temperature of 39° C. is more favourable). A convenient process for assisting the growth of butyric acid organisms is given by Beijerinck as follows : — 5 per cent. of finely ground fibrin is added to a 5 per cent, solution of grape sugar. After vigorous boiling, it is inoculated with garden soil, and immediately placed in an incubator at 35° C. The fermentation will set in within a day or two. The liquor is then neutralised with soda solution. A growth is thus obtained of Beijerinck's Granulobacter saccharo-butyricum, the majority of other bacteria being destroyed by boiling, or else checked by the butyric acid fermentation. If, instead of grape-sugar, cane-sugar is used together with 3 per cent, of calcium carbonate, 0-05 per cent, of sodium phosphate, 0-05 per cent, of magnesium phosphate, and 0-05 per cent, of potassium chloride, the Clostridium form develops in the liquor. Botkin's process is also worthy of mention ; it consists simply in heating milk in closed flasks in a current of steam for half an hour, and then maintaining it at 35° C. The most important products of the butyric acid ferment- ation are butyric acid, carbon dioxide, and hydrogen. In the saccharine mashes of breweries, distilleries, and pressed-yeast factories, species of butyric acid bacteria always occur, and if the mashes are maintained for a lengthened period at certain temperatures, they develop very rapidly, and exercise a retarding influence on the alcoholic ferments. BUTYRIC ACID BACTERIA. 127 According to Pasteur's experiments, the butyric acid ferment can perform its functions without access to free atmospheric oxygen. The usual spontaneous butyric acid fermentations proceed most vigorously when oxygen is ex- cluded. It has, however, been shown by recent experiments that there are many butyric acid bacteria which behave otherwise regarding free oxygen, for some are incapable of growth in presence of oxygen — anaerobic species — whilst others multiply and induce butyric acid fermentation when they have access to oxygen — aerobic species. In the course of years a very large number of butyric acid bacteria have been described. By the study of this mass of material, it has been shown that they are divisible into two groups — first, the true butyric acid bacteria, being those that produce butyric acid as the chief product of fermentation by decom- position of carbohydrates or calcium lactate ; and, secondly, there remain many species which form butyric acid along with other products by the breaking down of albuminoids. This applies particularly to putrefactive bacteria, many of which only produce minute quantities of butyric acid. Thorough chemical investigations have been carried out by Fitz, and more recently by Perdrix, as well as by Schattenfroh and Grassberger, who investigated the action of a number of species upon starch, the sugars, glycerine, cellulose, and the albuminoids, and determined the products of fermentation. One of the first species to be minutely described is Praz- mowski's Clostridium butyricum (Bac. butyricus, Fig. 24). It occurs in the form of short and long threads and rods, which may be either straight or somewhat curved. The rods are in brisk movement, and under a strong magnifying power they are seen to be covered with a large number of cilia (Fig. 25). Before the formation of spores in the rods, the latter swell and form peculiar spindle and lemon-shaped, elliptical, or club-like forms, as shown in the diagram ; at the same time they are coloured blue by iodine. The spores can withstand boiling for five minutes. On germination the spores burst their outer envelope, and the germ filament grows in the same direction as the longitudinal axis of the spore. Clostridium butyricum grows most vigorously at a temperature of about 128 MICRO-ORGANISMS AND FERMENTATION. 40° C., and may then rapidly become predominant in sugar solutions if the lactic acid ferment has previously converted a portion of the sugar into lactic acid. This species is decidedly anaerobic. A t§ Fig. -24.—Clogtridium butyricum, Prazm. (after Prazmowski).— .4, Vegetative state; c, short rods ; d, long rods ; at a and b, rods and filaments curved like vibriones. B, Formation of "resting spores"; b, d, rods, previous to, c, e, during, f, g, h, after the formation of resting spores ; c, elliptical ; d and h, lemon-shaped ; e, g, spindle-shaped ; f, tadpole-form ; at a rods still in their vegetative state. C, Germination of resting spores ; the spore « expands into b ; c, shows the differentiation of the membrane into exo- and endosporium. The contents surrounded by the endosporium issue from the polar fissure of the spore in the form of a short rod (d), which appears prolonged at e. Fitz has described a species belonging to the aerobic organisms — a bacillus of a short cylindrical form, which is not coloured blue by iodine, is motile in a moderate degree, and forms no spores. It ferments all carbohydrates with the exception of starch and cellulose. BUTY-RIC ACID BACTERIA. 129 According to Fitz the spores of butyric acid bacteria can withstand the temperature of boiling water for a length of time, naturally dependent, as in all cases, on their condition and on the nature of the substratum ; Fitz gives three to twenty minutes as limits. They can, however, be killed at a lower temperature if maintained long enough ; thus they are killed by being heated for six hours at 90° C. in a solution of grape-sugar ; but in glycerine, at the same temperature, for a period varying from six to eleven hours. Hueppe has likewise described a species (Bac. butyricus) found in milk, and occurring in the same forms as the species discovered by Prazmowski, but it proved much less sensitive to oxygen, and must, therefore, be classed as an aerobe. This species does not, however, form butyric acid from carbo- hydrates, but from albuminoids. Another aerobic species, Bac. booco- pricus, was detected by Emmerling in cow dung. It forms short rods, and is characterised by not liquefying gelatine, and by converting glycerine into butyric acid. Gruber found three well-defined species associated under the name of Clostridium butyricum, two of which are exclusively anaerobic. One of the latter species consists of straight or. slightly- curved rods, which become spindle- or barrel-shaped during the formation of spores. In nutrient gelatine it forms colonies which, when seen in reflected light, appear brownish- black or black. The second species consists of strongly- curved vegetative rods, at the end of which spores appear ; it forms yellowish or yellowish-brown colonies. The third species is also capable of growth and of causing fermentation in the absence of oxygen ; its development is, however, decidedly assisted by the presence of oxygen, and it is only then able to produce spores. The vegetative rods are cylin- drical ; with the formation of spores the rods become spindle- shaped, and in the centre of the spindle the large spore is formed. The colonies in nutrient gelatine are yellowish. 9 130 MICRO-ORGANISMS AND FERMENTATION. All three species form butyric acid and butyl alcohol from carbohydrates. In the water supply of Paris, Perdrix found an anaerobic bacterium (Bacille amylozyme), which occurs in the form of motile threads, four to six times as long as they are broad. This converts saccharose into acetic and butyric acids, with evolution of hydrogen and carbon dioxide, and it also produces amyl and ethyl alcohols. The optimum for its growth is 35° C. On slices of potato it forms whitish round colonies, which gradually liquefy the substratum. This species is very sensitive to acids. The spores can withstand ten minutes' heating to 80° C. Bac. orthobutylicus was isolated by Grimbert from seeds of the leguminosse. It was separated by heating for one minute at 100° C. Its spores survive this treatment. The species is anaerobic, and forms motile rods two to four times as long as they are broad, with rounded ends. In these rods two and three spores may occur. Its fermentation products are chiefly butyric acid, butyl alcohol, and acetic acid, together with carbon dioxide and hydrogen. It ferments saccharose, maltose, lactose, and glycerine. It hydrolyses starch, and converts dextrose into maltose. Grimbert proved by detailed experiment that the length of fermentation, the concentration, the reaction of the liquid, and the conditions of growth all influence the amounts of the fermentation products. Thus with an acid reaction the amount of alcohol increased and the formation of acid simultaneously decreased. On the other hand, the quantity of alcohol diminished and that of acid increased when the liquid was neutralised with calcium carbonate. Amongst other workers in this field may be mentioned Botkin and Fliigge, who isolated a species from milk by heating for one and a-half hours in boiling water or in a current of steam, the spores surviving this treatment ; v. Klecki, who cultivated Bac. saccharobutyricus from cheese ; v. Hibler, who examined pathogenic varieties, and proved that one and the same species may occur in different shapes not only as indi- viduals, but also in colonies, according to the nature of the substratum. By cultivation in gelatine without sugar the different forms of colonies may be most clearly distinguished. BUTYRIC ACID BACTERIA. 131 Clostridium Pasteurianum, discovered by Winogradsky, is of particular interest. He isolated it from garden soil by heating for ten minutes at 75° C., and then cultivating in a stream of nitrogen in a substratum free from nitrogen. The species can, therefore, absorb free nitrogen from the air and assimilate it. It can, however, utilise nitrogen in combina- tion. It forms butyric acid, acetic acid, minute quantities of alcohol, carbon dioxide, and hydrogen, and occurs as short, thick motile rods, which at a later stage expand into spindle shapes, and during the production of spores gives a violet- brown colour with iodine. The free spores are surrounded by an irregular mass of jelly. Beijerinck has drawn special attention to the possibilities of butyric acid bacteria which display bodies resembling granulose in the swollen cells and may be coloured with iodine, and has formulated a group which he calls Granulo- bacter. It consists of a series of species, some of which are identical with those previously described. The true origin- ator of the butyric acid fermentation, the preparation of which has been described, he calls Granulobacter saccharobutyricum ; it forms varying quantities of butyl alcohol, carbon dioxide, and hydrogen from saccharose, better from glucose, and also from maltose, and it secretes diastase. Schattenfroh and Grassberger examined a long series of species, both pathogenic and non-pathogenic, and found that the latter consisted chiefly of two species, one of which is motionless, and is very widely distributed. It forms both short and long rods, particularly on alkaline substrata con- taining starch. It exhibits the granulose reaction in the Clostridium form (this usually disappears with the formation of spores), and it liquefies gelatine. The other species is motile, and forms thin rods with from six to twenty cilia on «ach. In the spore stage they are motile, and they do not liquefy gelatine. These two species include many of those previously described, which may be regarded as varieties. Neither of them attacks cellulose. We must here recall Paraplectrum foetidum (Weigmann), which is widely distributed in milk. It coagulates the milk, and then dissolves the coagu- lated mass, and develops a very objectionable smell of cheese. 132 MICRO-ORGANISMS AND FERMENTATION. There is no doubt that butyric acid fermentation may take place both in breweries, distilleries, and yeast factories, as well as in the fermentation of wine, which is probably caused by the activity of certain species. Thus butyric acid has been detected in potato fusel oil and in cognac, as well as in the yeast " goods " of the distillery, but more exact information is lacking. If the preparation of the mash and wort goes on under indifferent conditions, a good opportunity is afforded for the development of such bacteria, and this applies also to the higher temperatures at which top fermentation is carried on. A bacterium that produced butyric acid together with other substances is Bac. lupuliperda, described by Behrens, which occurs frequently on hops. The spontaneous heating of hops has been shown by Behrens to be due to the development of this and other organisms. It consists of motile cocci and short bacilli which liquefy gelatine. In nutrients free from saccharose it produces large quantities of ammonium com- pounds, and, in particular, trimethylamine (the smell of rotten herrings). In presence of saccharose the nutrient solution soon turns sour, and butyric acid is formed. The species appears to have its chief habitat in the earth, and bears much resemblance to Bac. fluorescens putidits, described by Fliigge. The sulphuring of hops appears to protect them particularly from moulds. To ensure that micro-organisms do not develop on soured hops the amount of moisture must not exceed 8 to- 10 per cent., and they must be stored in a cold place. 4. Bacteria Fermenting Cellulose. These bacteria are just as widely distributed in nature aa butyric acid bacteria. These are the organisms that ferment cellulose in plant residues present in the mud of rivers and ponds, and thus give rise to the evolution of marsh gas. It is only quite recently, however, that, thanks to the admirable researches of Omelianski, we have obtained a clear conception of what bacteria are responsible for this action. He sowed horse dung and river mud on Swedish filter paper (pure cellu- lose), with the addition of 1 gramme of chalk, 1 gramme of ALCOHOL-FORMING BACTERIA. 133 potassium phosphate, 0-5 gramme of magnesium sulphate, 1 gramme of ammonium sulphate or phosphate, and a trace of sodium chloride to 1 litre of water. The fermentation was carried on at 34°-35° C. in flasks adapted for the cultivation of anaerobic bacteria. He thus proved that two different fermentations of cellulose are set up, a hydrogen ferment and a methane ferment, and that these are produced by two different species of bacteria. Omelianski separated the two by heating the fermenting material for fifteen minutes to 75° C. The hydrogen fermentation then proceeded, whilst before warming the methane fermentation took place. The reason is that the spores of the methane bacteria develop more rapidly than those of the hydrogen bacteria. If the liquid is heated to 75° C. after the germination of the spores of the methane bacteria, the vegetative rods of these bacteria will be killed, and only the spores of the hydrogen bacteria will remain alive and germinate. By repeated infection a microscopically pure growth of one or other species may be obtained. The cause of the hydrogen fermentation is a thin bacillus, straight or slightly curved, which forms spherical spores at one swollen end. It is not coloured blue by iodine. The fermentation products consist of fatty acids, carbon dioxide, and hydrogen. The exciter of methane fermentation presents a similar microscopic appearance, but the threads are thinner and the spores smaller. It is not coloured blue by iodine. Its fermentation products consist about half of fatty acids (butyric and acetic acids) and half of carbon dioxide and methane. Other bacteria, and amongst them aerobic species, may ferment cellulose, and even moulds — e.g., Botrytis and Cladosporium . 5. Alcohol-forming Bacteria. Quite a number of bacteria produce alcohol amongst their products of fermentation. The first known species was dis- covered by Fitz in a cold extract of hay, and was afterwards more exactly investigated by H. Buchner, and described as Bac. Fitzianus (Fig. 26). It occurs both in coccus and bacillus forms. In a nutrient solution containing glycerine it ferments the 134 MICRO-ORGANISMS AND FERMENTATION. latter, forming principally ethyl alcohol. Bac. ethaceticus. discovered by P. Frankland in sheep manure, produces ethyl alcohol and acetic acid from glycerine, starch, saccharose, lactose, glucose, mannite, and arabinose. Bac. pneumonia. described by Friedlander, is not only a pathogenic organism, but also has the power of decomposing saccharine nutritive solutions, and forming ethyl alcohol and acetic acid. In this connection may be mentioned a lactic acid bacterium found by Kruis and Rayman in sour yeast " goods " which produced ethyl alcohol as a by-product. Duclaux's Amylobacter eihylicus has certain charac- CD O O O a CO CO* teristics in common with A. butylicus, and occurs along with the latter, but produces ethyl alco- hol and acetic acid. Fitz found a species (Bac. butyli- cus) in cow dung which produces con- siderable quantities of butyl alcohol by fermentation of glycerine. Fitz de- scribes it as occur- ring in the form of Jig. 26.— Bacittug Fitzianm, after H. Buchner.— a, b,f, g, Coccus motile rods 5 to 6 U. forms and short rods ; c, e, long rods ; d, spore-hearing rods. . , in length and 2 ju. wide. He developed it in a solution containing 1 part of potas- sium phosphate, 0-5 of magnesium phosphate, 2 of pepsin, and 100 of glycerine in 2,000 of water, to which must be added 20 parts of calcium carbonate, and he found about 8 percent, of butyl alcohol in the fermented liquid. Bac. orthobutylicus, Grimbert, already described, also has the power of producing a considerable quan- tity of butyl alcohol, especially from glucose, when the nutritive liquid has an acid reaction, or when for any other reason the bacteria are in a feeble state. In the same way Perdrix's Bacille amylozyme yields this alcohol on fermentation. KEPHIH. 135 Beijerinck's genus, Granulobacter, includes a series of bacteria producing butyl alcohol. We shall only attempt to describe a process used by him for the preparation of such species. He introduces coarsely ground meal of husked corn, in successive portions, into boiling water until the mass has the consistency of a thick paste. The last addition should not be subjected to a temperature of 100° for more than a few seconds. After rapid cooling, it is placed in an incubator at 35°-37° C. The pure cultivation may be carried out in sweet- wort gelatine under anaerobic conditions. The pre- dominant species form white non-liquefying colonies, with Clostridium forms and oval spores. Duclaux describes a facultative anaerobe, Amylobacter butylicus, obtained by infecting a potato mash with garden soil. It exhibits the usual swollen sporogenous cells and the granulose reaction. It ferments starch, and produces butyl alcohol, butyric acid, and acetic acid. A large amount of alcohol is readily produced when calcium carbonate is used to neutralise the acid formed during the fermentation of starch. The same alcohol is produced by fermentation of saccharose (which is not inverted), maltose, lactose, glycerine, mannite, and calcium carbonate. Bacteria also occur which produce amyl alcohol (fusel oil) ; to these belong Perdrix's Bacille amylozyme, producing minute quantities of this alcohol from potato starch. A similar species was discovered by Pereire and Guignard, and H. Pringsheim isolated another from potatoes. It is still an open question how far the amyl alcohol produced during an impure alcohol fermentation is due entirely to the action of such bacteria. According to Ehrlich's experiments, fusel oil may be obtained by the action of alcohol yeasts on two of the decomposition products of albumen ; leucin and isoleucin. 6. Kephir, Koumiss, Mazun, Leben, Yoghourt, Ginger-beer. Kephir, on which the investigations of Kern have thrown some light, is an effervescent, alcoholic sour milk, prepared by the inhabitants of the Caucasus from cows', goats', or sheep's milk. It is made by adding a peculiar ferment, 136 MICRO-ORGANISMS AND FERMENTATION. " kephir-grains," to milk. These are white or yellowish and irregularly-shaped grains, not larger than a walnut and of a tough gelatinous consistency, and when dried become cartila- ginous and brittle. The essential part of these grains consists of rod-like bacteria, connected in threads, and enveloped in gelatinous membranes. Kern calls this bacterium Dispora Caucasica. According to Beijerinck this species, which he calls Lactobacillus caucasicus, produces in lactose, saccharose, glucose, and maltose a direct lactic acid fermentation. It produces solid, nodular colonies on whey gelatine resembling the kephir- grains. Besides bacteria, various yeast fungi and, frequently, moulds occur in the kephir-grains. In the preparation of kephir a little milk is first poured on the grains and allowed to stand for twenty-four hours at about 17° C. ; the milk is then poured off, and the grains preserved for future use. This milk is mixed with fresh milk, and poured into closed bottles, or leather sacks ; the fer- mentation is completed in two or three days if the liquid is frequently shaken. It now contains about 2 per cent, of alcohol. This result is probably brought about by the simul- taneous action of Dispora and yeast cells in combination with lactic acid bacteria present in milk. These ferments convert a portion of the lactose into lactic acid ; the alcohol and a part of the carbon dioxide result from the action of yeast. As the fermented milk, according to some authorities, contains less coagulated casein than ordinary sour milk, it may be assumed that the Dispora is also able to partly liquefy (peptonise) the coagulated casein, perhaps with the help of the gelatinous mass secreted by the bacterium, found in the kephir-grains, but not present in the fermenting milk. According to recent investigations of Hammarsten, however, the amount of casein does not appear to decrease, but a part of it undergoes certain alterations, partly physical, in consequence of which it becomes more finely flocculent. The want of agreement in these results may possibly originate in the different biological composition of the selected kephir-grains. Freudenreich regularly found Dispora Caucasica (Bac. Caucasicus) in a number of kephir samples, which readily developed on milk-agar plates and in lactose broth at 35° C. ; KEPHIR. 137 the bacilli frequently have glistening points at both ends, and Freudenreich assumes that this phenomenon coincides with what Kern regarded as spores ; unmistakable spores, however, were never found. Two lactic acid coccus forms and a yeast species also occur in all samples. One of the cocci (Streptococcus a) forms diplococci and chains, and produces in lactose gelatine large, white colonies, with coarse granulation at the edge ; the best temperature for the growth of this species is about 22° C. ; it coagulates milk most rapidly at 35° C., and contributes essentially to the production of a sourish taste and fine floc- culent appearance. The other coccus (Streptococcus 6), like- wise forming diplococci and chains, occurs in smaller colonies than a, and, in contrast with the latter, grows well at higher temperatures, and forms more acid than a, but does not coagulate milk. If this species is transferred, together with the kephir-yeast, to lactose broth, the fermentation is more vigorous than if the bacteria alone are inoculated ; Freuden- reich, therefore, presumes that Streptococcus b splits up lactose, and that its fermentation is rendered possible by the kephir- yeast. The kephir-yeast (a Torula) discovered by him grows remarkably well, and gives a weak fermentation in beer- wort, but does not appear to produce any fermentation in milk or lactose broth. The growth consists of oval cells (3 to 5 /x long, 2 to 3 /u. broad) ; it forms neither film nor spores, and its optimum temperature lies at 22° C. In the course of his experiments, Freudenreich succeeded in producing a liquor resembling kephir, for which purpose he inoculated a mixture of the four species in milk, and, after a lapse of some days, introduced a small portion of this sour, coagulated milk, which had been repeatedly shaken, into sterilised milk ; he, therefore, concludes that these four species, through their symbiosis, are able to bring about the kephir-fermentation. He could not observe any synthesis of kephir-grains, and it is not yet clear what part Dispora Caucasica plays in the whole process ; moreover, it appears to be highly probable that species of bacteria, other than the two coccus forms described by Freudenreich, in addition to other budding fungi, are active in the process. It may be 138 MICRO-ORGANISMS AND FERMENTATION. deserving of notice that in the author's laboratory it has been proved that a genuine Saccharomyces (S. fragilis) occurs in Russian kephir-grains which ferments milk-sugar independ- ently, whereas all previous investigators only found budding fungi incapable of spore-formation. In some parts of North America a ferment resembling kephir-grains is used in the fermentation of saccharine liquids. According to Mix' researches it contains a yeast-species which coincides with the one described by Beijerinck, and also a Bacterium which resembles Kern's Dispora. If one of the kephir-grains is allowed to remain in milk, it grows very slowly, and only attains to double its size, according to de Bary, after the lapse of several weeks. He considers it probable that under such conditions single Dispora cells separate and give rise to new kephir-grains. According to A. Levy's published process, kephir can also be obtained without the addition of Kern's ferments. When milk, which is turning sour, is repeatedly and violently shaken, an effervescent alcoholic kephir-like drink is obtained, which does not perceptibly differ from kephir prepared with kephir-grains as regards taste. Koumiss is- a similar fermented milk, prepared chiefly from mare's milk by the nomadic tribes of Southern Russia and Siberia ; it has been applied in many countries as a cure for various diseases. The true Koumiss, as prepared by the nomads, is fermented in leathern bottles, fermentation being started by adding a little dried milk from a previous ferment- ation. The organisms present sour and coagulate the milk during their development, and an alcoholic fermentation sets in, with evolution of gas. The coagulated mass is so finely divided that the liquid only turns thick. An accurate exami- nation of the active organisms was undertaken by Schipin, who proved the constant presence of a yeast species, a lactic acid bacterium, and a special species of bacteria which occurred in large quantities, and appears to be characteristic of the Koumiss fermentation. It is a facultative anaerobe which forms whitish colonies in gelatine, consisting of a central nucleus with streamers in all directions. It thrives best on sour-milk gelatine, and does not liquefy the gelatine. By MAZUN AND LEBEN. 139 the addition of cow's milk at 37° C. it coagulates to a thick paste without noticeable separation of whey. Its optimum lies between 20° and 30° C. Ten minutes' heating at 60° C. is sufficient to kill it. In experiments with mare's milk in presence of these three organisms, Schipin arrived at the conclusion that this species plays the most important part in the formation of Koumiss, and that it produces a lactic acid, as well as an alcoholic fermentation. It only displays its activity when the yeast and lactic acid bacteria have prepared the way for its development. At certain health resorts cow's milk is used instead of mare's milk, with the addition of sugar and alcohol yeast ; in other words, a pre- paration which has nothing at all to do with Koumiss. Mazun is, like kephir, a fermented milk (buffalo, goat, or cow's milk), which is prepared in Armenia, and is used both as a beverage and for butter-making. According to Kalanthar, Emmerling, and Lindner it contains a number of organisms, and amongst them yeasts fermenting lactose, an Anomalus form, Bac. subtilis, and lactic acid bacteria. In a similar way in Egypt, a sourish aromatic product resembling kephir is prepared from buffalo's, goat's, and cow's milk named Leben. It contains less alcohol than kephir, and coagulates in an alkaline mass. As in previous cases, boiled milk is brought into fermentation by the addition of dried milk from a previous fermentation. According to Rist and Khoury five different species are active in this fermentation ; a Streptobacillus which coagulates milk and produces lactic acid from lactose ; a very thin Bacillus which also yields lactic acid ; a Diplococcus which strongly coagulates milk : a yeast species which ferments glucose, saccharose, and maltose, but not lactose, but which, along with the Streptococcus, may give a vigorous fermentation in milk, as the bacterium hydro- lyses lactose ; and, finally, a Mycoderma species which can ferment glucose and maltose, but not lactose. The Strepto- coccus and the Diplococcus also possess a special coagulating enzyme. According to Rist and Khoury, by the use of these five species, Leben can be prepared from milk, and, best of all, if the two budding fungi are added first and the bacteria later. 140 MICRO-ORGANISMS AND FERMENTATION. Yoghourt is a species of sour milk or thick milk prepared in Turkey and Bulgaria. Sheep's or cow's milk is used, which is boiled and reduced by evaporation to half its volume, then cooled to 45° C., and the ferment — " Maya " or " Podkoassa "• is added. This consists of milk residues from previous pre- parations, dried under special conditions and ground, and contains many species of bacteria. After a fermentation lasting for nine to sixteen hours at a temperature of 40° C., the Yoghourt is ready for consumption. It is more or less solid, according to the degree of concentration, and possesses a sourish aromatic taste. It is eaten cold, either alone or with the addition of rice, bread, sugar, or fruit syrup. We owe the first bacteriological investigation to Grigoroff. who found three different lactic acid bacteria. The most important is Bacillus A. (Bac. Bulgarus or Bulgaricus}. It forms long motionless rods, often linked in chains, grows well on saccharine substrata, has an optimum temperature of 45° C., and does not multiply at room temperature. It produces alcohol, and attacks lactose, mannite, dextrose, maltose, and Isevulose, but not rhamnose, dulcite, and sorbite. Micrococcus B. occurs as single cocci or diplococci. In addition to the above varieties of sugar, it attacks rhamnose and glycerine. Streptobac. C. forms short rods linked in chains. It attacks lactose, saccha- rose, laevulose, and glycerine, but not maltose, mannite, rhamnose, dulcite, or sorbite. The optimum temperature for the last two species is 45° C., and they produce alcohol. Other investigators (Maze) have only found two species of bacteria. Luerssen and Kiihn, as well as Kunze, mention a " granule bacillus," perhaps a variety of Bac, Bulgaricus. Others have found yeast species to which they attach more or less importance. Metschnikoff, Piorkowski, and Henneberg have published further work regarding Yoghourt. The Ginger-beer Plant, which presents morphological resemblances to the kephir ferment, has been examined both botanically and biologically by Professor Marshall Ward. If this ferment is introduced into saccharine solutions containing ginger, it transforms them into an acid, effervescing beverage, ginger-beer. When fresh, it forms solid, white, translucent lumps, of irregular shape, brittle like dried jelly, varying in GINGER-BEER PLANT. 141 size from that of a pin's head to that of a large plum. It induces an alcoholic fermentation in the sugar solution, which at the same time becomes viscous. Marshall Ward isolated the numerous micro-organisms existing in these lumps, and described a series of yeast-fungi, bacteria, and moulds, and of these, two organisms proved to be essentially concerned in the fermentation of ginger-beer. One is a Saccharomyces (bottom yeast), belonging to the ellipsoidal group of this genus, and probably originating from the ginger and brown sugar com- monly used ; Ward named it Saccharomyces pyriformis. It inverts cane-sugar, actively ferments the products, and forms a pasty white deposit at the bottom of the vessel. It yields spores on gypsum blocks in 40 to 50 hours at 25° C. ; it also forms spores on gelatine. In hopped wort it induces a feeble fermentation, and forms a film on the surface containing many pear- and sausage-shaped cells. The other essential organism, which is always present, is a Schizomycete, Bacterium vermiforme, which, according to Pro- fessor Ward, emanates from ginger, and is active in the lactic acid fermentation. It is a peculiarly vermiform organism, enclosed in clear, swollen, gelatinous sheaths, and imprisoning the yeast cells in brain-like masses formed by its convolutions. It is the swollen sheaths of this organism which constitute the jelly-like matrix of the " plant." It also appears without sheaths, and in a great variety of shapes. The gelatinous sheaths are only developed when the saccharine liquid is acid, and free from oxygen. A Mycoderma and a Bacterium aceti were also found. Marshall Ward has proved experimentally that Saccharo- myces pyriformis and Bacterium vermiforme are the only two essential species in the ginger-beer fermentation, since it was only by inducing a fermentation with these two species that he was able to produce an effect similar to that obtained when the ordinary ginger-beer plant is employed. But it is only when both species develop together in the liquid that they bring about this result, and his experiments indicate that the relations between the yeast and the bacterium are those of true symbiosis, because the yeast ferments more vigorously in presence of the bacterium than it does alone. 142 MICRO-ORGANISMS AND FERMENTATION. 7. Slime-forming Bacteria. Among the various species of slime-forming bacteria there are several which are of peculiar interest in the fermentation industries, as they occur in wine, milk, beet juice, and fer- menting wort, causing morbid changes. By analogy, this slime formation, which usually consists of substances re- sembling gum, may be regarded as a phenomenon closely related to the commonly occurring zooglcea formation of certain bacteria. In his Etudes sur la Here (Plate 1, Fig. 4) Pasteur described bead-like chains of spherical organisms, which render wine, beer, and wort so viscous that they can be drawn out into threads ; this is caused by the formation of gum and mannite. Kramer has described Bacillus viscosus sacchari, which in a short time converts neutral or slightly alkaline cane-sugar solution into a tough mass of a gummy nature. He isolated a Sac. viscosus vini (2 to 6 /x long), which was cultivated in sterile wine, air being excluded. Sound wines infected with this growth thickened in the course of six to eight weeks. It grows best at 15°- 18° C., and apparently cannot exist at such a comparatively low temperature as 30° C. A mannite fermentation is sometimes associated with the formation of slime in wine. The motionless bacterium isolated by Gayon and Dubourg grows on the bottom as large zoogloea, and thrives only in saccharine solutions. On the other hand, a mannite fermentation investigated by Peglion in wine is never accompanied by the formation of slime. A very comprehensive memoir regarding slime-forming bacteria has been published by Kayser and Manceau. The disease occurs especially in wines protected from the action of the atmosphere. The formation of slime begins in the lowest stratum of the liquid, and increases by degrees without reaching the top. The disease occurs in those districts where it is customary to remove the skins, stones, and stalks, before fer- menting the juice. Such bright wines are usually poor in tannic acid, nevertheless other wines comparatively rich in tannin may be attacked. The percentage of alcohol and of free acid appears to be of greater importance than the content of tannic acid. SLIME-FORMING BACTERIA. 143 Bottled wines suffer more than wine in the cask ; white wines more than red, because the former contain more sugar, especially laevulose, which forms an excellent food-stuff for these ferments. Slime-forming bacteria have been isolated from eight different wines, some white and some red, and six of the species were subjected to an exact investigation. They all occur as short rods, varying in length and breadth (1-1 to 4- 2 //. long, 0-7 to 1-7 /j. wide). They seldom occur singly, usually linked in chains, which are short and straight in some cases, and in others long and spiral. They are non-motile, anaerobic, neither form spores nor liquefy gelatine, and all give the Gram coloration. They are surrounded by a slimy growth at every stage of development. The optimum tem- perature for propagation lies between 25° and 30° C. Bacteria have been found in bread which produce a strong formation of slime, and in particular the "potato bacilli" appear to be active — i.e., varieties of Bac. mesentericus wdgatus (Bac. panis viscosi), described by Kratschmer and Niemitowicz, and Toy Uffelmann, Thomann, Vogel, and others. As a consequence of the action of these bacteria the bread can be drawn out into long thin glutinous strings. They occur in rye meal and multiply in presence of moisture. They develop in bread if the spores survive the baking temperature, and the bread is stored in a warm place. According to Migula, Bac. panis (Vogel) occurs in long slender rods (4 to 7 //), forming chains, which have a rapid movement, and possess a polar cilium. They form oval spores, which survive the action of a current of steam at 100° C. for fifteen minutes (in a potato culture). On gelatine plates the colonies form flat liquefied depressions. With a magnification of 70, it appears as a colony having a yellowish-brown nucleus coarsely granulated, and delicate streamers in the gelatine. On agar also the colonies form a nucleus with streamers. The optimum is at 40°- 4 2° C. In plant infusion (digitalis leaves), Ritsert proved experi- mentally the presence of a Bad. gummosum which brings about a mucilaginous formation of slime. Its activity depends upon the sugar content of the liquid, and is greatly favoured by the presence of potassium and sodium acetate and yeast ash. 144 MICRO-ORGANISMS AND FERMENTATION. There is a rich formation of slime in 10 to 30 per cent, nutrient sugar solution, whereas none occurs in similar grape- sugar and milk-sugar solutions. The species has a pronounced demand for oxygen, and the cells exhibit movement at certain stages. It appears to occur both as rods and coccus forms, according to the composition and reaction of the substratum. It liquefies alkaline gelatine. In a stab-culture on agar it grows as a moist glistening whitish deposit, which forms two zones, the inner wrinkled and the outer smooth. Brautigam isolated a Micrococcus from an infusion of digitalis leaves, which converted a nutrient sugar solution into a complete jelly, and made apple juice viscous. In a similar infusion Happ found a slime-forming rod bacterium (Bac. gummosus) 5 to 7-5 /x long, 0-6 to 2 /u. wide. It assumes spindle shapes in old cultures, and is sometimes motile. On neutral gelatine it forms colonies with streamers ; the gelatine is liquefied. On potatoes it forms coccus-like involution forms. Saccharose solution is absolutely necessary for the production of slime. The optimum lies at 25°-30° C. He also found a Micro- coccus gummosus, which may be distinguished from Brautigam 's- species by its fermentation products. It forms yellowish colonies on gelatine, but a colourless deposit on agar. The optimum is at 15°- 20° C. This species may produce slime in saccharose and maltose solution. Schardinger has under- taken a detailed enquiry into the products formed by a slime bacterium, one species of which was isolated from impure drinking water. It is a very short, motionless, non-sporogenous bacterium which forms on gelatine a tough slimy film consisting of cells linked in long chains. On saccharose or grape-sugar gelatine it forms slimy and " ropy " colonies of a greyish- white appearance, which when removed leave a depression in the gelatine. In broth it forms slimy flakes, especially on the surface, and it also makes milk viscous. In nutritive liquids containing saccharose, maltose, lactose, etc., it causes fer- mentation with evolution of hydrogen, and by fermentation of an 8 per cent, saccharose solution, with inorganic salts and calcium carbonate to neutralise the acid, it forms lactic acid, acetic acid, ethyl alcohol, and succinic acid. The optimum for slime formation is 20°-30° C. It does not liquefy SLIME-FORMING BACTERIA. 145 gelatine. According to Schardinger, the species is related to Loffler's Bac. lactis pituitosi. A chemical examination of the slime formed by mass cultures from saccharose solutions, containing nutritive salts and calcium carbonate, shows that it chiefly consists of a carbohydrate which by oxidation with nitric acid forms mucic acid, and by boiling with hydrochloric acid produces optically active sugar. As a slime can also be formed by bacteria in the absence of sugar, it should pro- bably be regarded as a product of the swelling of bacterial membrane. As an example of one of the species producing a vigorous formation of slime in milk may be mentioned Bac. lactis viscosus, found in water, and described by Adametz. It forms a short, feebly-motile rod with a thick refractive capsule. Its average dimensions (in milk cultures) are 1-5/z long and 1-25/x thick. On glycerine-peptone-gelatine it forms whitish non-liquefying colonies with irregular jagged edges, which shows a bright opalescence in reflected light. By inoculation in sterilised milk, the milk becomes viscid like honey in four to six weeks, and may be drawn out into long threads. At the same time the fat globules of the milk disappear. Lactose is only attacked to a very slight extent by this species, whereas casein is greatly modified. Slime is also formed in nutritive liquids free from carbohydrates. It is believed to be a zooglcea formation. Other related species have been described by Duclaux, Leichmann, Schmidt-Muhlheim, Loffler (Bac. lact. pituitosi, motionless rods which quickly divide into coccus-like cells, and on gelatine give white colonies with sharp or slightly dented edges), Weigmann (the coccus of " lange Wei " with nitrogenous slime), Emmerling, etc. Emmerling has proved that Bac. lactis aerogenes forms a mucilage in lactose solutions possessing the properties of galactan, for by oxidation it may be transformed into mucic acid. In beer also slime-forming bacteria occur. Thus H. Schroder (1885) found a Micrococcus in " ropy " Berlin " Weiss- bier/' which was afterwards cultivated in a pure state by P. Lindner, who named it Pediococcus viscosus. The disease 10 146 MICRO-ORGANISMS AND FERMENTATION. could be produced by adding pure cultures to sterilised " Weiss- bier " wort. On the other hand, this organism had no action on hopped beer- wort or low-fermentation beers. By the addi- tion of tartaric acid the beer becomes normal. Schonfeld distinguished many species in long " Weissbier," and, in par- ticular, found two typical kinds (P. major and minor}. The optimum for the formation of slime lies between 20° and 26° C. These species form a considerable amount of acid, and impart to the beer a pleasant, acid- wine bouquet. In presence of larger quantities of alcohol the beer does not easily turn viscid, and the lactic acid present protects such beer from the disease. These organisms grow well, according to Schonfeld, in ammoniacal yeast decoction. Schonfeld has proved that such species occur in horse urine. In ropy Belgian beer, Van Laer found the cause of this disease to be small and very thin, sporogenous rods (1-6 to 2*4 //. long), which were partly isolated and partly united in pairs by means of a zooglcea-like substance. When added to beer-wort, this first becomes turbid, and afterwards ropy. Milk also turns slimy, and its lactose ferments. On beef- broth gelatine these rods give concave colonies with concentric rings of different colours ; streak cultures give broad, white bands, with a sinuous border; stab -cultures give a white stripe soon extending to the bottom of the glass ; the gelatine forms fissures which become filled with the growth, while at the same time a speck is formed on the surface. Experiments carried out with pure cultures of this bacterium in beer-wort have shown that one and the same form includes many vari- eties, which have a somewhat different action on wort. They are all included under the name Bacillus viscosus (I. and II.). If sterilised wort is infected with this bacterium, and alcoholic yeast added after the lapse of some hours, the liquid becomes viscous. If the wort is infected with a mixture of absolutely pure yeast and bacteria, the disease will develop in a varying degree, according to the proportion of bacteria. If, however, these are only added after the completion of the primary fermentation, the disease will not appear at all. The greater the proportion of nitrogenous matter in the liquid, the sooner it will become viscous ; even liquids which do not contain sugar SLIME-FORMING BACTERIA. 147 can be made ropy by these species. When the nutritive liquid contains much sugar, the fungus develops very feebly, and in pure sugar solutions the phenomenon does not occur. A high con- tent of acid greatly restricts the development of these bacteria. Van Laer has since isolated a Bac. viscosus bmxellensis which produces, in addition to slime, a peculiar disease called *' biere a double face." It occurs in " spontaneously " fer- mented Belgian beers, Lambic, Faro, and Mars, and can be recognised by the fact that the beer looks clear in transmitted light, and milky in reflected light. It forms a long rod making a white tough film on beer-wort, which grows down into the liquid. Subsequently the slime disappears, and the rods are then surrounded by a slimy envelope. On Avort gelatine large, round, slimy, transparent colonies are formed, with a yellow centre and with many zones. The species restricts the activity of alcohol yeasts, and beer attacked by it is consequently poor in alcohol, and richer in extract than sound beer. It forms lactic, acetic, and butyric acids. Vandam found in English beers an aerobic Bac. viscosus (III.), which occurs as small rods, single or in chains, consisting of two, three, or more links, with spore-formations in the centre of the rods. This bacillus develops best at about 30° C., and produces a slimy mass in brewers' wort, which under the microscope proves to consist of zooglcea formation. After the lapse of some time the liquid has the consistency of albumen. No gas is evolved, but the liquid acquires a peculiar odour. On meat-juice gelatine and on wort-gelatine the growth develops freely. The viscosity of the liquid does not seem to depend on the quantity of nitrogenous matter present, but on the other hand, the bacillus grows feebly in the absence of sugar. This species is incapable of producing •disease in beer unless it is thriving well, and is introduced in large quantities into the wort before or during pitching. Like the form discovered by van Laer, it ferments milk- sugar ; and, according to Vandam, it is easy to detect it in yeast, even in traces, simply by introducing a sample of the latter into nutritive liquid containing milk-sugar, a growth of this species soon making its appearance in the upper part of the liquid. Brown and Morris mention a Coccus form which also seems 148 MICRO-ORGANISMS AND FERMENTATION. to produce ropiness in English beer. This species occurs as diplococci and tetrads, and gives yellow wax-like colonies on meat- juice gelatine. The disease made its appearance in the beer after a lapse of six to eight weeks, but it was not usually possible to produce it by inoculation with pure cultures of the species in sterile beer. Close to the fermentation room there was a pork-butcher's premises, in which putrefying matter had accumulated ; after this had been removed and the soil dug and cleaned, the disease disappeared. Fellowes also examined several English beers affected by this disease, and prepared pure cultures of the bacteria present, but by inoculation of the cultures in beer he did not succeed in preparing a beer containing these organisms and showing a viscosity corresponding to that of the sample from which they came. Heron undertook a thorough study of a slime-ferment which occurred in English beers, a very small coccus, which gradually elongates, and by contracting in the middle assumes the form of a dumb-bell. The two ends may also expand in a direction at right angles to the first growth, and assume a similar shape. At a later stage the species takes on the form of rosaries (zooglcea). The beer attacked loses its acid simul- taneously with the formation of mucilage, and acquires an unpleasant taste. This species can only produce slime in presence of yeast. Beer may be protected against its action by increasing its acidity and adding more hops. The species originates in malt dust, according to Heron. The so-called frog-spawn fungus Leuconostoc (Streptococcus) mesenterioides was investigated by Cienkowski and van Tieghem and subsequently by Zopf and Liesenberg (Fig. 27). Both the European form and the variety found by Winter in Java occur spontaneously in beet-juice and in the molasses of the sugar factory, and in molasses distilleries, in which they form large slimy masses (" frog-spawn ") and multiply vigorously. The fungus forms chains of cocci, alternate pairs of which are always more closely united. In contrast to the observations of earlier workers, who thought that certain of these cocci enclosed spores, Zopf found that they present no differences morphologically or physiologically ; spore-formation could in SLIME-FORMING BACTERIA. 149 no case be proved. Consequently, the analogy formerly assumed to exist between this fungus and the algae genus Nostoc (implied in the name Leuconostoc) falls through. Under certain conditions the cells are surrounded by a strong gelatinous sheath with a sharp outline (Bb, Be, C), which in many of the above consists of a mucilaginous carbo- hydrate, dextran. This formation only takes place in the Ba. *t ! Fig. %7.—Leuconogtoc megenterioideg, Cienkowski (after Zopf).— A, Cell cluster of the sheath- less variety, taken from a potato cultivation ; B, series showing the development of a culture, grown in gelatine, free from sugar ; Ba, sheathless ; Bb, the same after 24 hours' growth in a solution of molasses, sheaths already seen but not strongly developed ; Be, after 48 hours' growth in molasses, the sheaths strongly developed and partly encased in each other; C, a small gelatinous mass from which the cells have been expelled. presence of cane and grape-sugar, and not in solutions of milk- sugar, maltose, or dextrin. Under the latter conditions, and in potato cultures, the species develop distinctive forms, in which the gelatinous sheath is completely absent (A, Ba). The formation of jelly is a phenomenon depending also upon certain conditions of nutriment. Leuconostoc ferments grape-sugar, cane-sugar (after previous 150 MICRO-ORGANISMS AND FERMENTATION. inversion), milk-sugar, maltose, and dextrin, with production of acid and gas. The acid proved to be lactic acid. Especially characteristic of this fungus is its power of resisting high temperatures, the younger growths possessing this power in a* higher degree than older cultures. It withstands gradual heating to 86°-87°C. for a few minutes. The optimum temperature for development lies between 30° and 35° C. ; the maximum at 40°-43° C. It is also remarkable that both the growth and the fermentative activity of the fungus are favourably affected by the presence of considerable quantities of calcium chloride. Cohn's Ascococcus (Micrococcus) Billroihi, the cells of which are enveloped in a jelly, under certain conditions of nourish- ment, forms mucilaginous slime from sugar, according to Zopf . The three following species may be classed along with the above : — Glaser described a Bact. gelatinosum betce which pro- duces slime in beet-juice and evolves gas. It forms short motile rods, giving white liquefying colonies on beet-juice gelatine. At its optimum of 40°-45° C., it rapidly forms a gelatinous film on beet- juice ; it does not, however, develop on molasses. It inverts saccharose, and produces alcohol during ferment- ation. The slime is of the same character as in Leuconostoc. Clostridium gelatinosum, described by Laxa and Schone, is- found in sugar factories, and forms a slime like that in Leuconostoc. It appears as rods of varying length, which are motile in their earlier stages, and form spores in the middle of the swollen cells. The optimum is at 40° C. The species inverts saccharose, and thrives best with free access of air. In soil where sugar-beet is cultivated it grows in great numbers. Other species, both coccus and rod forms, are described by Schone. Maassen has described a number of similar species under the general name of Semiclostridium, by which he wishes to express- that the rods, especially when the quantity of oxygen is restricted, swell at one end and in the middle. The ellipsoidal spores do not, however, develop in this swelling, but at the thin end of the cell ; the young rods are motile. The optimum for vegetative growth is about 45° C. The spores- are extraordinarily resistant, both to boiling and to anti- septics, and the organisms are widely distributed in the soil. S. commune, isolated from filter press residues, forms a BACTERIA WITH INVERTING ENZYMES. 151 jelly only from saccharose, which is inverted by this species, and fermented with evolution of carbon dioxide. It may be distinguished from Leuconostoc by the fact that the slime yields laevulose on hydrolysis, whilst Leuconostoc slime forms dextrose. Cobb describes a gum disease on the sugar cane, causing the production of a slimy yellowish mass in the vascular bundles of the stem, filled with bacteria of a single species, Bac. vasculorum, which, according to Cobb, produces the mucilage. In the gummy runnings of the sugar cane, a short rod with cilia always occurs, according to Smith, who named it Bad. Sacchari, The " gummosis " of turnips and sugar beets, recognisable by drops of gum appearing on the cross sections, which acquire a black colour, is accompanied by a strong development of bacteria. These gradually multiply, and entirely alter the character of the mass. Busse (experiments on the inoculation of pure cultures into sound beets) proved that the short motile rod which forms slimy colonies both on gelatine and on slices of beet was the cause of the disease. It inverts saccharose. 8. Bacteria with Inverting, Diastatic and Proteolytic Enzymes. We have already mentioned a number of bacteria that owe their importance in the fermentation industry to enzymes. Some further examples are given in this section which possess other enzymes. Invertase is present in the following, amongst others :— Bac. (Proteus) vulgaris, one of the commonest putrefactive bacteria, forming short motile rods often grouped in rows, and also forming long filaments with spiral and spirulina forms. Bac. fluorescens liquefaciens, which occurs frequently in water, as well as in decomposing substances, and derives its name from a greenish fluorescent colour which it imparts to gelatine. The gelatine is liquefied. It forms straight and curved rods of medium size, consisting of two or more members. Bac. Megatherium, found by de Bary on boiled cabbage leaves, is distinguished by its extraordinary size. The rods may be 2-5 /j. thick; they sub-divide into short cells. It forms whitish, liquefying colonies on gelatine. 152 MICRO-ORGANISMS AND FERMENTATION. Fermi and Montesano found that Bac. Megatherium, Proteus vulgaris, and Bac, fluorescens liquefaciens in neutral broth, invert a 4 per cent, solution of saccharose. Many of these bacteria, however, loss their power of inversion if the broth Pig. 28. — Bacillus subtilis. — A, Cells with cilia ; B, C, segmented threads; D, thread with spores ; E, spores in swollen mother-cell ; F, germination of spores ; G, film on hay infusion. is ^rendered alkaline, whilst most of them are uninjured in slightly acid broth. In broth without sugar, and in media containing no albumen, such bacteria pro- duce invertase ; thus almost all the species that were examined formed invertase in a nutritive salt solution containing glycerine. The invertase produced by these bacteria proves to be a soluble enzyme, which is destroyed at temperatures differing ac- cording to the species, but it is always J more resistant during its action on sac- charose than in a dissolved state ; it is very sensitive to acids and alkalies, and especially to organic acids and potash. According to A. J. Brown, the well-known Bac. subtilis (hay bacillus, Figs. 28 and 29), belongs to this group. Brown found that it cannot grow either in beer or wort of normal acidity. According to Hansen, many species of bacteria of common occurrence in beer secrete inverting ferments. Amongst these Fig. 29.- Bacillus (after A. Fischer). staining, x 1500. Cilia SARCINA. 153 there is a group which exhibits an inverting action on a pure saccharose solution, but loses this property when yeast- water is added. Wortmann in 1882 began some experiments on the diastatic action of bacteria, and used for this purpose drops of bacterial liquids on rotten beans or potatoes. He proved that in this mixture species were present which can bring about the same changes in starch paste and in soluble starch as the diastase of the higher plants can do. The bacteria only exert action on starch when no other available carbohydrate is present (e.g., sugar or tartaric acid). This was established by means of experiments with inorganic nutritive solutions. Krabbe showed that the presence of peptone increased the formation of diastase. Fermi proved that this enzyme was present in different Streptoihrix species, and found that the formation of diastase was prevented when the bacteria were cultivated on substrata free from albuminoids. Pfeffer and Katz observed a rich formation of diastase in Bac. Megatherium ; by the addi- tion of saccharose or maltose to the nutrient the diastatic activity was considerably reduced. Garbowski observed the enzyme in his detailed researches on Bac. luteus. The action is brought out most strongly by inoculating on an inorganic nutritive liquid, together with starch solution. Bac. vulgar is and Bac. prodigiosus are amongst the organisms containing proteolytic or peptonising enzymes. The latter, which belongs to the group of colour-forming bacteria (the " Bleeding Host ") forms very short motile rods in weakly alkaline substratum, but longer rods and filaments in weak tartaric solutions. These albuminoid-digesting bacteria play an important part in nature, in the decomposition and suc- cessive building up of organic substances. The anaerobic species appear to be particularly active ; for example, Bac. putrificus, which forms long motile rods, with spore-formation at their swollen ends. 9. Sarcina. The name Sarcina is given to the spherical bacteria (Coccus], which are motionless, and divide in all three planes. Under favourable conditions of growth, and especially in liquids, 154 MICRO-ORGANISMS AND FERMENTATION. the cells formed by division may remain clumped together, caught in the slime secreted by the cells, and thus more or less cubical groups are constituted, which sometimes have a certain resemblance to corded bales of goods. On solid substrata, on the contrary, the growth of many of these species breaks down rapidly into single cells, or else remains grouped in clusters of two and four. The harmful kinds occurring in beer, which belong to this group of bacteria, are really only known with division in two planes, and the pro- duction of regular shapes (diplo- and tetracoccus), whilst the larger clusters are composed of irregularly massed cells. Until something further is known about them, these species must, therefore, be counted in the group which shows division in two planes (Pediococcus, Micrococcus, and Merismopedia). A similar behaviour is shown by Sarcina rubra, discovered in red milk by Menge, which in milk only displays the micro- coccus form. The sarcina form is known in broth-cultures. The many species of Sarcina that have been described can give differently coloured colonies on gelatine ; white or greyish colonies are formed by S. alutacea, isolated by Gruber from leaven, which liquefies gelatine. This is also the case with Lindner's S. Candida, found in the water reservoir of a brewery. Yellow colonies are formed by the widely distributed S. flava which has been found in leaven, beer, and elsewhere. It forms both regular packets and irregular masses of cells. On gelatine it gives small round colonies, which gradually liquefy the gelatine, and on hay infusion it forms a film with a strong development of packet shapes. S. aurantiaca forms on gelatine orange-yellow, liquefying colonies, but develops the typical sarcina form only in hay infusion and plant decoctions. It gives a dark bluish-green colouration with sulphuric acid. S. casei, discovered by Adametz in cheese, forms pale yellow, liquefying colonies with concentric rings, and coagulates milk. Adametz also found S. butyrica in cheese ; it forms a yellowish- white colony on the surface of stab-cultures in potato gelatine, but dark liquefying colonies in plate-cultures. S. lutea always forms regular packet shapes, and gives on gelatine lemon- yellow, non- or only feebly liquefying colonies. Brown colonies on gelatine are given by 8. acidificans discovered by SARCINA. 155 Adametz in cheese. It develops a yellow colony in stab- cultures on agar. It precipitates casein from milk. A dark brownish-yellow colour is developed by S. fusca, discovered by Gruber in flour. Among the varieties exhibiting a red growth are S. rubra, giving glistening red colonies on the surface of gelatine, and slowly liquefying it. The colouring matter is insoluble in alcohol. 8. rosacea, occurring in air and water, usually forms irregular masses of cells in malt extract, but develops the typical shapes in a hay decoction, which are surrounded by a brownish envelope. On a neutral malt extract gelatine it forms a reddish deposit with a dry surface. The colouring matter is soluble in warm alcohol. S. maxima, found by Lindner, which develops in a malt mash at 40° to 45° C., has cells of 3 to 4 n diameter. 8. mobilis, isolated by Wolff from milk, is distinguished from each of the above by having motile cells. It shows the typical form both in liquids and on solid substrata, liquefies gelatine, and forms yellow colonies on whey gelatine and agar. In the fermentation industry, sarcina-like organisms occur in addition to those already mentioned in section 8, especially in low-fermentation lager beer, where they may develop during the secondary fermentation. Pasteur described and depicted the diplococcus and tetracoccus forms, and he noted that beer contaminated with such bacteria assumes a dis- agreeable flavour and smell. At a later date they were depicted by E. C. Hansen under & «, the name Sarcina (Fig. 30). He found them in ®y, <$ & many parts of the brewery plant, and estab- 8 $ fs lished by direct experiments the nature of their Fig. so.— sarciua. influence on beer. Balcke, who afterwards investigated such beer, regarded it as established that the sarcina-like organisms were responsible for the disease, and as he only found the diplo- and tetracoccus, and not the packet form, he gave it the name Pediococcus cerevisice. Other workers have since failed to isolate the typical sarcina form from diseased beer, and have only detected irregularly massed cells, so grouped that it was impossible to determine how they were produced. The possibility is not, however, excluded that we may be able, as in other cases, to detect typical sarcina forms in these? 156 MICRO-ORGANISMS AND FERMENTATION. •diseases. The name " Sarcina disease " is best retained, as •certain conceptions are associated with it.* Lindner has described a number of Sarcina species in pure cultures, and amongst them one which occurs in diseased lager beer, which he named after Balcke, Pediococcus cerevisicR. It grows best with access of air, and forms yellowish or 3rello wish-brown colonies on meat- juice gelatine, which is not liquefied. In a stab-culture it gives a flat white colony on the surface. In the streak culture it forms a greyish-white moist line, which appears iridescent in thin layers. On meat- juice gelatine it is killed by eight minutes' exposure to 60° C., but not by twelve minutes' exposure at 50°-55° C. It proved impossible by inoculating pure cultures to reproduce the unpleasant flavour and odour of the beer ; only turbidity ensued, and the isolated species doubtless cannot have been .a disease Sarcina. In later experiments Lindner succeeded in a few cases in reproducing the characteristic appearance in beer by the application of yeast which had been inoculated with a Sarcina isolated from the diseased beer. On the other hand, A. Petersen observed a case where a growth of these organisms had •developed in beer without affecting either its flavour or its smell. A. Reichard isolated from low-fermentation beer a Pedio- •coccus sarcinceformis, which developed freely in sweet wort and sterile beer, but not in pasteurised beer. This species developed best when access of air was limited. In ferment- ation experiments turbidity or peculiar changes of taste occurred in certain cases, but not in the majority. After many •experiments, he arrived at the conclusion that these contrary results were due partly to the condition of the various growths of this Sarcina form, partly also to the manner in which the fermentation took place. In cases of quiet fermentation in * As examples of the various typical Pediococcus = Micrococcus species, may be instanced M. candidus and concentricus growing in water ; M. urece, which converts urine into ammonium carbonate, and M. lacticus, which Marpmann found in fresh milk, giving white and non -liquefying colonies ; M. amarifi.ca.ns, which, according to Conn, makes milk bitter, and M. casei amari, which, according to Freudenreich, makes both milk and cheese bitter: these have white liquefying colonies ; M. luteu-s with yellow non-liquefying, and M. ftavus with yellow lique- fying colonies ; M. cinnabarium and carneus, isolated from water, with red colonies. SARCINA. 157 a lager cask the growth remained at the bottom, and th& bacteria did not exert any appreciable influence on the liquidr whereas in the case of a vigorous secondary fermentation they were carried upwards in the liquid, along with the carbon dioxide bubbles, after which the disease manifested itself. Rousing the beer might, therefore, be injurious in such cases, An addition of hops to lager beer exerts a retarding influence on these bacteria, as on the majority of bacteria occurring in beer. vSchonfeld inoculated cultures (from diseased beer cultivated on yeast- water gelatine) into pasteurised beer, and produced, not only a turbidity, but also an acid — sweet, disagreeable flavour. Two species have been described by N. H. Claussen, which were isolated after he had checked the growth of other organ- isms occurring in beer by a slight addition of acid ammonium fluoride. The beer-cultures were allowed to develop in hopped wort and in pasteurised beer, and when inoculated in beer brought about the characteristic disease phenomena. Both species grow in the usual nutritive liquids either neutral or slightly acid, whereas a minute quantity of free alkali restricts their growth. The most favourable temperature for their growth is 23°-24° C. Neither liquefies gelatine. They grow in wort both when oxygen is fully excluded, and also in presence of the full quantity in the atmosphere. The- one, P. damnosus, usually imparts an unpleasant odour and flavour to beer, but only forms a slight deposit in the liquid ; the other, P. perniciosus, causes, in addition to the deterioration of flavour and odour, a turbidity in the liquid. Schonfeld has isolated species from diseased beers with the help of sweet wort gelatine, and especially on dry-yeast gelatine. He found that species that are dangerous for lager beer, imparting the Sarcina odour and objectionable flavour, as well as turbidity, only produce a comparatively minute quantity of acid in sweet wort, and give a peculiar odour slightly resembling honey : for this reason he gave the group the common name of P. odoris mellisimilis (he assumes that this group is identical with Claussen's P. perniciosus). In contrast with this a group of species exists which occur in lager beer and " Weissbier " producing large quantities of acid in sweet wort, and, according to Schonfeld's observations, much turbidity, but not 158 MICRO-ORGANISMS AND FERMENTATION. the pronounced honey odour. In sweet wort they give a pleasant sourish odour and flavour. They are grouped together under the name P. acedulefaciens. Other varieties have been described by Schonfeld giving a red colour to lager beer. By inoculating a pure culture of a species producing strong turbidity into pasteurised beer which is allowed to stand, he showed that in most cases only a sedimentary growth developed, but if, on the contrary, carbon dioxide is passed through the beer, freely swimming bacteria develop which produce turbidity. This observation agrees with those made by Reichard in practice. The fact that cases do occur in which lager beer contains bacteria appearing to possess the characters •of true beer Sarcina, but having no recognisable influence either on the clearness, the odour, or flavour of the liquid, has .since been confirmed in the author's laboratory. Experi- ments have proved that in isolated cases growths of one and the same species have caused diseases in one beer and not in another. From a number of individual observations of the .associated conditions, the following conclusions may be drawn : — Species may be isolated from yeast and from lager beer capable of development, which appear to be incapable of •exciting any disease whatsoever in the latter. The extent to which such organisms occur in beer is not yet known with certainty. Amongst the true disease species, a given organism appears to be unable to produce the special disease under all circumstances, even when the conditions are favourable for the reproduction of cells. According to our present experience, the most likely assumption appears to be that this is caused by the condition of the liquid at the time when the contamination with bacteria took place. According to the published experiments, the possibility cannot, therefore, be excluded that these organisms are capable of variation like other bacteria, and the question arises how far they are able to retain their newly- won properties. No proof has yet been given that foreign species can be so completely acclimatised that they may act as disease bacteria in lager beer. Both the typical Sarcina and the Micrococci (Pediococci) .are widely distributed in nature, and may easily be recognised SARCINA. . 159 by the use of the usual liquids and gelatines. Certain materials, for example, horse urine and dung, appear to be particularly rich in pronounced Sarcina species. Their presence can easily be verified also in malt and malt dust. It has not proved possible as yet to determine the natural haunts for the beer Sarcince. One reason for this is that such species cannot be distinguished from others that do not attack the liquor, in an ordinary micro-biological analysis. The only accepted con- clusions are that all true beer Sarcince that have been exactly investigated cannot thrive in alkaline substrata (ammoniacal liquids or gelatine) ; that they form whitish masses in streak cultures, and on the surface of stab-cultures ; that they belong to the facultative anaerobes ; and further, that specially favourable conditions for their development are to be found in badly saccharified wort, and to some extent, according to Miskowsky (as in the case of many other bacteria), in malt extract with a high content of dissolved albuminoids (especially albumoses and peptones) ; in such a liquid they may remain for a long time unaltered ; and, lastly, that, like many other bacteria, they appear to be checked by a large amount of hop constituents. It follows that it is impossible to distinguish by any general test whether Sarcina-\ike bacteria in yeast or beer are able to produce a disease in beer. To answer this question we must proceed experimentally — a difficult investigation taking up much time. It would, however, be obviously foolish to neglect the usual test for Sarcina-like bacteria in yeast and beer, for if they are observed there is always a possibility that dangerous species may be present. It appears to be thoroughly established by experience that, in the early stages of ferment- ation, a weak growth of such bacteria may be concealed, and at present the problem is to provide means whereby the analyst may be able to detect minute quantities of these organisms. Amongst such means may be adduced Claussen's method for the treatment of yeast with minute quantities of acid ammonium fluoride which checks the yeast cells, so that a subsequent infection in wort-gelatine mainly gives a growth of Sarcina colonies. The liquid prepared by Bettges and Heller may also be used ; it consists of sweet wort completely 160 MICRO-ORGANISMS AND FERMENTATION. fermented by the addition of yeast ; starch is then added, and after clearing it is neutralised with ammonia, and reduced to- an alcohol content of 4 per cent. The sample is mixed with this liquid, and the development observed in the sealed pre- paration. It will be found that of bacteria, only Sarcince come to development. In the author's laboratory, for many years past, an addition of neutral yeast water (preserved in flasks along with an excess of calcium carbonate) is made to the sample taken at the end of the principal fermentation. After two days' standing the Sarcince present have multiplied sufficiently to be easily recognisable under the microscope. Until investigations have gone far enough to enable us to prove whether the haunts of these disease germs lie outside the plant, efforts must be directed in practice to finding their haunts within the plant. We must bear in mind the limits of our present knowledge, and we must not forget that without- pure culture experiments direct observation of Sarcina-lilie germs in the plant itself (for example in the vats) suggests a much greater probability that these growths are dangerous in practice, than if a large number of germs of similar microscopic appearance had been observed, with the help of plate-cultures, in the air surrounding the brewery or in the water used in the plant. By means of a properly organised system of disinfection, and often without the application of antiseptics, such growths may be entirely suppressed. It is an entirely false impression that the beer Sarcince described in this section cannot be fully excluded, a mistake that has arisen because it has proved impossible to distinguish between many of the organisms of this large group of bacteria occurring in air and the true disease species of beer. Investigations carried out in the author's laboratory on bright wines have frequently brought to light vigorous growths of Sarcina forms, and at the same time the wine has assumed a peculiar odour, which resembles to a remarkable extent the odour and taste of beers in which such growths occur. 10. The Fermentation of Tobacco. During the fermentation which dried tobacco leaves undergo, a number of organisms are present, and it is a natural FERMENTATION OF TOBACCO. 161 assumption that these play a part in producing the successive alterations in the leaf material. During fermentation the temperature gradually rises, and it is attempted in various ways to limit the temperature to about 50° C. The effect of fermentation is that aromatic bodies are produced in the leaves, and simultaneously part of the nicotine, according to Behrens, disappears. Suchsland was the first to investigate the micro-organisms present in fermenting tobacco. He attempted to improve its quality by introducing pure cultures of selected species of bacteria. Nothing further is known with regard to these species. More recently, Behrens, Vern- hout, Koning, and others have described some of the vast number of species that are present, and Koning found by parallel experiments that an inoculation with certain pure cultures selected from fermenting tobacco, partly aerobes, but chiefly facultative anaerobes, exercised a favourable influence on the aroma and flavour of the tobacco. In the same way the after-fermentation, which takes place when the leaves are packed together, appears to be due to the action of micro-organisms. A contrary view has been expressed by 0. Loew, who attaches no importance to micro-organisms in the fermentation, but seeks the active causes in the oxidising enzymes, which he proves to be present in the leaves. H. Jensen, as well as Splendore, found that leaves which had been heated in a current of steam (90°- 100° C.) showed every sign of a good fermentation, and that this was not prevented by treatment of the leaves with mercuric chloride, formol, and chloroform, which certainly appears to confirm Loew's conclusion. Behrens bases upon his observations the belief that micro-organisms play a part in the fermentation, a view the correctness of which is rendered more probable by the results of Schloesing's experiments on the fermentation of snuff tobacco. The experiments undertaken in the author's laboratory with parallel fermentations carried out with both American and African tobacco lead to the conclusion, especially when facultative anaerobes are employed, that certain species do play a part in determining the aroma and flavour of tobacco. 11 162 MICRO-ORGANISMS AND FERMENTATION. 11. Iron and Sulphur Bacteria : Nitrifying Bacteria. The bacteria described in this section are of particular interest, because they possess the property of oxidising in- organic substances. In microscopical examinations of water we often meet the characteristic forms of Crenothrix Kiihniana (Fig. 31), or spring pest, described by Cohn and Zopf. This ferment occurs in all water containing organic matter, and sometimes it multiplies to such an extent that it may render the water unfit for use. Thus, according to Zopf, great calamities have been caused by this fungus in the water supplies of Berlin, Lille, and certain Russian towns. In consequence of its power of storing iron compounds within its walls, it forms red or brown flakes in water. Its forms are very beautiful ; it occurs in the form of motionless cocci or gonidia (a-f), which by division and formation of viscous matter form zoogloaa (g) ; these cocci frequently grow to articulated filaments, which are provided with distinct sheaths (h, i-r) ; they then increase in thickness towards their free end, and when they reach a certain age, they divide within the sheath into smaller fractions, which become round and issue either as rods, macro- or micrococci. Leptothrix ochracea is a widely distributed iron bacterium with colourless cylindrical cells connected in threads and surrounded by a sheath, which at first is thin and colour- less, but afterwards, by accretion of hydrated oxide of iron, assumes a yellow or brown colour. Oval and motionless gonidia develop in the threads. The empty sheaths may form large yellowish-brown deposits in water containing iron. Cladoihrix (Sphcerotilus] dichotoma is also of frequent occurrence. Its cells are surrounded by a similar thin sheath. By displacement of single rods in a filament, false branching takes place. The rods are finally set free, and are then pro- vided with cilia, with which they swim about until they settle down, and grow into new threads. These iron bacteria are commonly met with in water containing the soluble basic ferrous carbonate. According to Winogradsky this salt is oxidised by the bacteria and ferric oxide is deposited on the IRON AND SULPHUR BACTERIA. 163 sheath. The great deposits of iron ochre found in nature are probably f or " spherical yeast." The carbon dioxide formed by the fungus is only of value in the formation of the yeast stage by excluding oxygen. Whilst the absence of oxygen is a general condition governing the formation of yeast cells from mucor, there are a few species which demand the presence of sugar. Mucor racemosus requires sugar, whilst Mucor alpinus (dis- covered by Hansen) does not require it. By cultivating M. racemosus in a flask completely filled with wort, through which a stream of carbon dioxide is passed, a growth consisting: exclusively of mucor yeast can be obtained. Mucor erectus, with greyish-yellow transparent sporangia^ which may be found, for instance, on decaying potatoes, has the same microscopic appearance as Mucor racemosus ; physio- logically, however, it differs from this species. Mucor circinelloides (Fig. 37) has a very characteristic appearance. The mycelium (1) shows the remarkable branch- ing which occurs in some of the species of Mucor. The main branches (6) send out short, root-like branches (c) with frequent forking ; at the base of these come new mycelial branches (r), which grow erect, and are able to form sporangia (2 to 5) ; the sporangiophore is sympodially branched. During its development it curls up, and to this fact the species owes its name of circinelloides. In this form, as well as Mucor spinosus, the mycelium, when submerged in a saccharine liquid, produces gemmae, similar in formation to those of Mucor racemosus and Mucor erectus. Mucor spinosus has a greyish- blue mycelium with spherical spores and brownish-black sporangia, which is distinguished by the uppermost part of the columella being studded with pointed, thorn-like protuberances. Finally, M. alternans belongs to this group, and bears a similarity to M. circinelloides. This fungus has the distinction of being the first of the Mucor species which was shown by Gayon and Dubourg in 1887 to possess the property of fer- menting dextrin. MUCOK. 203 The most interesting of all the species of Mucor is M~ (Amylomyces} Rouxii, on account of the great use which is made of it for the conversion of starch into sugar on a com- mercial scale. It was isolated by Calmette in 1892 from " Chinese yeast/' small greyish-white cakes, which consist of rice grains kneaded together with different kinds of spice. Calmette, however, only described the characteristic mycelium which exhibited gemmae (chlamydospores), and called the Fig. 37.— Mucor circinelloiden (after van Tieghem and Gayon).— 1, Mycelium ; b, main branch ; e, root-like branches ; r, axillary branches ; 2-4, development of sporangia ; 5, opened sporangia ; to different mycelia. Rh. nigricans occurs on a great variety of juicy fruits, causing them to decay, and thus working considerable havoc. Behrens has shown that the damage is caused through the secretion by the fungus of a poisonous substance, which kills the fruit cells. It is also of frequent occurrence on malt. Fig. 38. — Rhizopus nigrican» (after Brefeld). — a. End of a runner or stolon ; t, sporangiophore ; *, sporangium ; e, columella ; A, root-like hyphw or rhizoids. A similar species, Rh. Oryzce (Chlamydomucor Oryzce) wasr discovered on Javan rice cakes by Went and Prinsen Geerligs. On account of its power of dissolving starch, it is employed in the preparation of arrack from rice. It produces large numbers of gemmae. A specially interesting form is Rh. japonicus, which, like M. Rouxii, is applied industrially for the conversion of starch, especially of maize starch, into sugar. It was isolated by Boidin in 1900 from Japanese koji, and, like the species discovered by Calmette, was called Amylomyces (/3) ; 206 MICRO-ORGANISMS AND FERMENTATION. it was described more exactly by Vuillemin. It shows a great resemblance to both the former species, and, like Eh. Oryzce, it forms gemmae. Vuillemin also described Eh. tonkinensis Thamnidium elegant (after Brefeld). 1. Sporangiophore (low magnification). •2. Three sections of above (high magni- fication). a. Terminal sporangium, c. Sporangioles. 3. Drooping fruit carriers with spor- angioles. 4. Free sporangioles. Fig. 39. {Amylomyces y), which has the same structure as the^foregoing, but shows a different behaviour towards the sugars. MUCOR. 207 Amongst other moulds standing in close relationship to the Mucor species, Phycomyces nitens is frequently referred to in the literature of this subject. It usually occurs on oily substrata, but also on bread, excrement, etc. It resembles Mucor, and its olive green sporangiophores with their metallic lustre attain to an extraordinary size. The sporangium is t>lack, the columella pear-shaped, and the spores yellowish. Thamnidium elegans (Fig. 39) frequently occurring on the •dung of various animals, on bread, etc., is a fine mould with sporangia recalling those of Mucor ; but in addition to the terminal sporangium formed at the apex of the main sporangio- phore, the latter gives rise to a number of forked side branches, on which sessile sporangia (sporangioles) are formed without columella and with fewer spores. In conclusion, we may allude to Sporodinia grandis, a fungus often met with on toadstools growing in woods. It forms a •dense felt of branching hyphae with numerous sporangia, and also produces zygospores. Hansen's determination of the limits of temperature for the three species, M. racemosus, neglectus, and alpinus, when grown on wort-agar gelatine and in wort, proved that the formation of sporangia and zygospores can go on at a slightly lower maximum than is required for vegetative growth (the behaviour is thus analogous to that of the Saccharomycetes). The development of sporangia can, however, go on at the same minimum as that required for vegetative growth. The species vary with regard to the temperature limits for sporangia, on the one hand, and for zygospores, on the other ; thus M. -alpinus exhibits a higher maximum for the formation of sporangia than of zygospores, but the reverse is the case with M. neglectus. It follows that the temperature limits may serve to determine the species. Mucor racemosus, for example, when grown on the media alluded to, gives as limits for vegetative growth a maximum of 32°-33° C., and a minimum of 0-5° C. ; M . alpinus a maximum of 29°-31° C., and a minimum of 0-5° C. ; M . neglectus, maximum 33° C., and minimum 3° C. The Mucor species are of special interest to us, because they can act, in varying degrees, as true alcoholic ferments. 208 MICRO-ORGANISMS AND FERMENTATION. Their fermentative power is not connected with the formation of budding gemmae, for these have not been observed in either Mucor Mucedo or M . stolonifer. This form of fermenta- tion has been regarded as a special kind of breathing. Unlike normal respiration, such as is performed by every organised being — the absorption of oxygen and exhalation of carbon dioxide — it can take place in the absence of free oxygen. The oxygen in the cell contents makes fresh intra-molecular linkings, with the result that the carbohydrates, and more particularly the sugars, become disintegrated, so that not only carbonic acid but also alcohol is produced. Adopting the term suggested by Pfliiger, the process is known as intramolecular respiration. This conception implies that the fermentative change pro- duced by Mucor, which is only possible in the absence of free oxygen, differs essentially from that brought about by yeast, which can go on either in the presence or absence of free oxygen, Wehmer's experiments with two species of Mucor (M. race- mosus and M. japonicus) have shown, however, that the pro- duction of alcohol was not diminished by the constant bubbling of air through the liquid, nor yet when the fermentation is carried out in very thin films of liquid with a large exposed surface. In other directions the two kinds of fermentation possess characteristics in common, and the collective evidence makes it difficult to regard the processes as essentially different. On the other hand, Palladin and Kostytschew and others have proved that the two fermentations are not identical. A special alcohol enzyme, such as that isolated from yeast, ha& not been isolated from Mucor mycelium. Kostytschew ob- served, however, that the mycelium of M . racemosus, which had been killed by treatment with acetone, was able to produce an amount of carbon dioxide equal to that evolved by the living cells. M . racemosus, Hansen's M . negl&ctus and (accord- ing to Saito) Rhizopus japonicus, var. angulosporus, and RJi. Tamari are the only species capable of inverting and fermenting a cane-sugar solution. This was proved by Fitz for M . race- mosus, and confirmed by Hansen and others. The great majority of species are, however, able to ferment maltose, invert sugar, and dextrose. Considerable diversity may be observed amongst the MUCOR. 209 different species in regard to the production of alcohol. The same rules which govern yeast fermentations seem on the whole to apply to these processes. Thus, according to Wehmer, when the general conditions are favourable, in presence of oxygen and at a medium temperature, the fermentation is practically completed in the course of a few days. A re- markable feature of the fermentation produced by these fungi is that the liquid remains clear throughout the operation. Some of the results obtained during Hansen's investiga- tions may be quoted to show the difference in the productivity of the various species. M. erectus possesses the greatest fermentative activity. In beer- wort of ordinary concentration (14°- 15° Balling), it yields up to 8 per cent, by volume of alcohol. It also induces alcoholic fermentation in dextrin solutions, and con- verts starch into reducing sugar. Mucor spinosus yields up to 5-5 per cent, by volume of alcohol in beer-wort. In maltose solutions distinct fermentation phenomena were observed, and after the lapse of eight months the liquid contained 3-4 per cent, of alcohol. Mucor Mucedo has a comparatively feeble fermentative power both in wort (up to 3 per cent, of alcohol) and in maltose and dextrose solutions. Mucor racemosus produces as much as 7 per cent, of alcohol in wort, develops invertase, and ferments the inverted cane sugar ; thus, like the two species above mentioned, it occupies a particular position. According to Gayon, Mucor circinelloides exercises a very powerful action on invert sugar (yielding 5-5 per cent, by volume of alcohol). According to Wehmer, M. javanicus produces 4 to 5 per cent, of alcohol in a few days. Whilst the Mucor species are of no technical importance as alcohol producers, those possessing powerful diastatic enzymes, capable of converting starch into sugar, occupy an important place in industry. It has already been mentioned that they have been used by the Asiatic races for centuries. Their systematic use in Europe began in 1892, when Calmette isolated M . Rouxii from " Chinese yeast." The diastatic enzyme of this fungus reacts most powerfully at 35°-38° C. and produces chiefly dextrose. The process, carried on as it 14 210 MICRO-ORGANISMS AND FERMENTATION. now is on a large scale in special factories, consists in first boiling the starch (maize or rice starch) under pressure, then liquefying the mass by the addition of a small quantity of green malt, or hydrochloric acid, and sterilising the fluid at a high temperature. By adding a culture of the fungus grown from spores at 38° C. the change into sugar is soon effected. In 1895 Went and Prinsen Geerligs published their re- search on Rhizopus Oryzce (Chlamydomucor Oryzce), which likewise converts starch into dextrose. Shortly afterwards Collette and Boidin announced the discovery of two similar species, Rhizopus tonkinensis and Eh. japonicus, which react more vigorously than M . Rouxii ; other species have subsequently been discovered. A number of Mucor species produce small quantities of acid in sugar solutions. A detailed investigation of this subject has not yet been carried out. Wehmer, however, has observed the formation of citric acid by M . pyriformis, and several species have been shown to produce oxalic acid. Most species liquefy gelatine, but quite slowly as a rule. Albumen-splitting enzymes occur in the different species, and some of these appear to play a part in the ripening of cheese. 5. Monilia (Figs. 40 and 41). A number of different fungi of comparatively simple structure are described under this name in works on mycology. From a mycelium, the colour of which varies according to the species, branches are thrown up which give rise to series of oval or elliptical spores. The genus has an interest for us on account of one of its species, named by Hansen Monilia Candida from Bonorden's description, which possesses very remarkable physiological properties. It occurs in nature in the form of a white layer covering fresh cow-dung, and on sweet, succulent fruits. When introduced into wort, it de- velops a copious growth of yeast-like cells. At the same time it excites a vigorous alcoholic fermentation, and whilst this is progressing forms a mycoderma-like film on the liquid ; the cells in this film extend further and further, and finally MONILTA. 211 form a complete mycelium. During the early fermentation the fungus produced only 1-1 per cent, by volume of alcohol, whilst S. cerevisice gave 6 per cent. ; but the Monilia con- tinued the fermentation, and produced at the end of six months 5 per cent, by volume of alcohol, whilst the culture yeast gave no further quantity. Hansen states that Monilia does not secrete invertase, but, nevertheless, ferments cane sugar, from which he con- cludes that cane sugar is directly fermentable. He suggests, A Fig. 40.— Monilia Candida, (after Hansen). — A , growth in beer- wort or other saccharine nutritive liquids ; B, cells of a young film-formation. however, the possibility that cane sugar may be converted into invert sugar in the interior of the cells, and that the latter is immediately fermented. Hansen's observations were confirmed by the work of E. Fischer and P. Lindner, and subsequently by Buchner and Meisenheimer. They proved that an inverting enzyme cannot be extracted either from the fresh or from the dried vegetation. 212 MICRO-ORGANISMS AND FERMENTATION. Fig. 41. MONILIA. 213 On the other hand, they were able to obtain preparations which inverted cane sugar actively either by using the dried fungus, by grinding the cells with powdered glass, by killing them with acetone, or by pressing out the juice (see Chap. v.). Thus the fungus contains an inverting enzyme, but it is com- pletely retained by the protoplasm of the living cell. In contrast with yeast invertase, Monilia invertase is insoluble in water ; it does not diffuse, as yeast invertase does, through the cell-wall, or through the protoplasmic lining of the cells, neither does it diffuse through parchment. In this respect, and in the ease with which it is decomposed, it possesses characters in common with Buchner's zymase (see Chap. v.). According to Fischer, maltose is split up both by fresh and by dried Monilia, and also by an aqueous extract of a dried growth ; he, therefore, infers that Monilia contains the enzyme maltase recently discovered by him in S. cerevisice. Fig. 41. — Monilia Candida (after Hansen).— Mould growths like a are frequent ; they consist •of chains of elongated cells, more or less thread-like, and rather loosely united ; at each joint there is generally a verticil of oval cells, which readily fall off ; b represents another form, also of frequent occurrence, but distinguished from the former by having no verticillate cells ; instead of these there generally issues from every joint a branch of the same form as the mother cell, but shorter; the links of these chains are often closely united, the constrictions in many cases disappear, and a very typical mycelium, with distinct transverse septa (c) is produced ; the forms b and c occur in the nutritive medium, a commonly on the surface. Forms like d have much resemblance to Oidiuin lactig. t shows a train of pear-shaped cells with verticils of yeast-cells resembling S. exiautm ; the chain of lemon-shaped cells represented at/closely resembles Ehren- berg s figures of Oidiuin fructigenum. Between the principal forms described there are numerous yeast-cells of different forms, variously arranged in colonies. According to Bau, Monilia also ferments dextrin formed from diastase. As recently as 1883, Monilia Candida was the only fungus* known to be capable of fermenting cane sugar, although not secreting invertase. Since then Zopf, Beijerinck, Behrens, and other investigators have observed this phenomenon in the case of a few other micro-organisms ; they form, however, rare exceptions. It forms another example of the unexpected gradations that exist in nature. A certain amount of carbon dioxide and ethyl alcohol is developed in liquids undergoing a Monilia fermentation. Finally, this fungus is distinguished by its power of with- standing high temperatures. In beer-wort and cane-sugar solutions it develops vigorously at 40° C., and induces an active fermentation at this temperature. The limits of 214 MICRO-ORGANISMS AND FERMENTATION. temperature for the development of Monilia in wort arer according to Hansen, maximum 42°-43° C., and minimum 4°-6° C. Many other species have been described, amongst which may be mentioned M. sitophila, discovered by Went, which grows on the earth nut (Arachis hypogaea) in West Java. Its mycelium extends by degrees throughout the entire fruit, the hypha3 assuming a yellow colour on exposure to air. By means of the various enzymes which the fungus contains, a change is brought about in the fruit contents. In this fermented con- dition the earth nuts are eaten in large quantity by the natives. Sachsia suaveolens, discovered by P. Lindner, is also an inter- esting fungus belonging to this group. It produces a high percentage of alcohol in wort, and develops a wine bouquet. 6. Oidium lactis (Fig. 42). Oidium lactis is a mould which has played an important part in the literature of the physiology of fermentation, and in that of medicine. It is known as the milk mould. Some authors have sought to establish the theory that this fungus is a stage in the development of species which, under other circumstances, occur in entirely different forms,, and with quite different properties. It was thus brought into- genetic relation with Bacteria, Chalara, Saccharomyces, etc. Both Brefeld and Hansen have carried out numerous investi- gations with this fungus, and have undertaken culture ex- periments, which were continued for a long time without producing any other than the ordinary Oidium form. Recently, it is true, Brefeld has discovered a formation of conidia re- sembling chains of Oidium cells in several higher fungi, but it has not yet been determined whether this also includes that particular species which we designate Oidium lactis. The transparent, thin-walled hypha?, often forked and branched, form a thick white felt ; in the upper part of the filaments transverse septa are formed close together, after which the single cells, filled with very refractive protoplasm, are detached as conidia (Fig. 42: 3 to 7, 11 to 14, 17 to 19). As a rule, the conidia, in longitudinal section, are rectangular OIDIUM LACTIS. 215 Fig. 42.— Oidiwn lactig (after Hanson).— 1, Hyphse with forked partitions; 2, two ends of hyphic — one with forked partition, the other with the beginning of a formation of a spherical link ; 3-7, germinating couidia ; 6-6"', germination of a conidium, sown in hopped beer-wort in Ranvier's chamber, and represented at several stages ; at each end germ tubes have developed ; after nine hours (6"') these have formed transverse septa and the first indications of branchings ; 11-14, abnormal forms ; 15, 16, hyphrc with interstitial cells, filled with plasma : 17, chain of germinating conidia ; 18, conidia which have lain for some time in a sugar-solution ; the contents show globules of oil ; 19, old conidia. 216 MICRO-ORGANISMS AND FERMENTATION. with rounded corners (Fig. 42 : 3, 6, 17 to 19) ; in a growth of this mould spherical, oval, pear-shaped conidia, and others of quite irregular form are, however, almost always present (Fig. 42: 4, 5, 11 to 14). These organs of propagation, the only ones known, send out one or more germ tubes. When the fungus grows on solid substrata, the hyphse unite and form remarkable conical bodies. Fresenius correctly gave to this species the specific name of lactis ; for universal experience shows that it has its ordinary habitat in milk, where it can usually be found. It also occurs spontaneously in various other liquids, and among these in the saccharine liquors which are employed in the fermentation industries : in the latter it is able to induce a feeble alcoholic fermentation. Thus, according to Lang and Freudenreich, it produces in milk and grape-sugar solutions, in the course of about ten days, 0-55 per cent., and in five weeks, 1 per cent, by volume of alcohol ; smaller proportions of alcohol are produced in cane-sugar and maltose solutions. Its maximum tem- perature is, according to Hansen, 37-5° C., and its minimum below 0-5° C. Cultures made in lactose nutritive solution develop a powerful odour, resembling that of soft cheese, such as Limburg cheese. Oidium is thought by Weigmann, Conn, and others to play some part in the ripening of Camem- bert cheese. It is believed that Oidium is of importance in the ripening of this and other kinds of cheese, because it absorbs the acids produced by lactic acid bacteria, and thus paves the way for peptonising bacteria. Casein, in sterilised milk, is rapidly decomposed by the fungus. According to O. Jensen, an Oidium is always present in rancid butter. The fungus may occur in beer, especially when poor in alcohol. As the amount of alcohol increases, the conditions for its growth become less favourable ; still, neither wort nor beer is exposed to the danger of being attacked to any extent by Oidium, since it is not able to compete in the struggle for existence with the crowd of organisms which at once appear when fermentable liquids are exposed to the atmospheric germs. In numerous investigations with top-fermentation yeast, the author has found that it offers a very favourable nutritive FUSARIUM. — CHALARA. 217 material for this fungus, especially when the yeast is in a quiescent state at the end of the fermentation. Sometimes a microscopic examination has shown an enormous number of conidia. It is not known what influence such a growth ex- ercises on the quality of the yeast and the beer, but without doubt it is advisable to avoid the fungus as much as possible. It forms vigorous growths on pressed yeast also, which have a deleterious action on the quality of the yeast. A large number of species and varieties find shelter under the name O. lactis. Weigmann has identified several, and Grimm also isolated a number of forms from sour milk, cheese, etc., which differ clearly from each other both in regard to their character- istic growths on gelatine, and especially on potatoes, and also in regard to their peptonising action on the substratum. 7. Fusarium. The red colour occasionally occurring on malt grains is due to various fungi, among which is a Fusarium described by Matthews and Klein. The mould formation begins on the germinating part of the grains, and thence spreads over its surface. The filaments of the mycelium, which show globular swellings, are connected by numerous bridgings. The red colouring matter is present in the contents of the filaments. On a moist medium the membranes gradually swell, forming a slimy envelope, which is coloured violet by iodine. The oval conidia germinate either directly, or previously grow into sickle-shaped multiple cells. Germinating filaments issue from the points of the latter, and by slow degrees the cells swell up. Both the mycelium and the sickle-shaped conidia are able to produce thick-walled spores like gemmae. The fungus does not appear capable of hindering the growth of sound malt grains, even if its mycelium spreads freely over their surface. Generally speaking, it only attacks diseased grains. 8. Chalara. Chalara mycoderma (Fig. 43) is described in Pasteur's Etudes sur la biere as one of the organisms commonly occurring 218 MICRO-ORGANISMS AND FERMENTATION. on grapes. The mycelium forms a film on liquids, and consists of branched, greyish filaments, which at different points develop conidia of unequal form and size filled with glistening and highly refractive protoplasm. The mycelium frequently divides up into separate Oidium-Uke cells. Cienkowski first gave a detailed description of Chalara in his memoir on the Fig. 43.— Chalara mycoderma (after Hansen).— 1, A branched hypha. the terminal limb of which is throwing off conidia ; 2, a hypha, at the upper cell of which a sterigma, which has thrown off conidia ; 3-9, various forms of hyphw links, which are separating conidia. film-forming fungi. Hansen found that this mould develops both in ordinary wort and lager beer, as well as in the diluted liquors. 9. Dematium pullulans. A mould about which a great deal has been written in the literature of our subject is Dematium pullulans (Fig. 44), which was first described by de Bary, and more minutely by Loew. DEMATIUM PULLULANS. It frequently occurs on fruits, especially grapes, and has a branched mycelium from which buds are thrown out ; these have a striking resemblance to ordinary yeast cells (4), and are able either to propagate through many generations, by yeast-like budding, or to produce germinating threads giving; Fig. 44.— Dcmatium pullulans (after Loew).— 1, 2, Full-grown mycelial threads with yeast-like cells ; 3, cells of the latter developing to mycelial threads ; 4, cells with yeast-like buds ; fv appearance of yenst-like cells on the germ tubes of the cells, with brown covering. rise to a mycelium (3). Skerst states that the mycelium develops more particularly at low temperatures, whereas the separate cells form at temperatures of 19°-32° C. In a. strongly concentrated grape-sugar solution the fungus chiefly 220 MICRO-ORGANISMS AND FERMENTATION. develops mycelium. When this has attained a certain age, it forms numerous, closely contiguous, transverse septa, and gradually turns brown or olive green (5) ; this forms the resting stage of the plant. In Hansen's air analyses, Dematium was frequently found from spring until late autumn in wort to which air had access. He observed that when the mould was sown in a saccharine liquid it at first developed only mycelial threads ; after some time, however, yeast-like cells separated, without inducing alcoholic fermentation. P. Lindner states that one Dematium, species produces a ropiness when cultivated in wort, owing to the formation of slime from the cell membrane. Dematium species are also found in milk and dairy products. A great development of Dematium occurs in the sap which oozes from the cut stem of the vine, and, according to Wortmann, this is the main reason Fig. .45. — Dematium specie* (Jorgensen). — Spore-formation in mycelial threads. why the sap is gradually converted into a slimy, gelatinous mass. Wine must may also turn slimy, for the same reason, if it is allowed to ferment too slowly. In isolated cases Wortmann observed that Dematium exercises a destruc- tive influence on grapes. The author observed in 1895 endo- genous spore-formation in Dematium-like moulds occurring on dried grapes, but the organism showed no development of the resting cells described above (Fig. 45). The spores de- veloped nothing but a yeast growth in saccharine liquids. The yeast thus developed was capable of spore-formation, and is, therefore, a true Saccharomyces. CLADOSPORIUM HERBARUM. 221 10. Cladosporium herbarum (Fig. 46). This mould occurs along with others in fermentable liquids r in the fermenting rooms, and also on hops, malt, etc. It sometimes occurs in very large quantities in the fermenting rooms. The author found, in one case, that the ceiling and a portion of the walls in a bottom-fermentation room were thickly covered with small black patches ; these consisted of Cladosporium, the conidia of which were consequently always- present in the yeast. The plant consists of a yellowish-brown- mycelium with short, straight filaments, stiff and brittle ; those growing erect can produce at their upper extremities conidia of very varying form — spherical, oval, or cylindrical, straight or curved. In contrast to Peni- cillium, where the new conidia are formed basi-petally (i.e., below those which have already been cut off), they rise, in the case of Cladosporium, either apical or lateral, from a kind of budding of the mother conidium, the development being thus basi- fugal. The name Cladosporium herbarum doubtless includes several closely related species. According to Janszewski's re- searches, the same species can appear in different forms and with a varying size of cell. He showed that the commonly occurring species represents a stage in the development of an Ascomycete (Mycosphcerella}, the perithecia of which bear some resemblance to those of Aspergillus. Wortmann includes Cladosporium amongst those fungi, the mycelium of which, growing through the corks of wine bottles, give rise to the corked flavour of wine. These and other species of fungi occur during the ripening of cheese which r Fig. 46. — Cladosporium herbarum. — Conidia-forming hyphte (Loew) and Conidia (Holm). •222 MICRO-ORGANISMS AND FERMENTATION. through their development, acquires a dark brown or black •colour. Fungi belonging to this group play a part in bringing about the decomposition of eggs. Zopf has identified a species which recent investigations have shown to be capable of sending germinal hyphse through the egg shell and membrane, .and of gradually decomposing the albumen. O. Jensen has observed that a species of Cladosporium promotes the rancidity of butter on account of its power of splitting up fatty sub- stances. Eriksson states that rye is sometimes attacked by Cladosporium, and that the mould when consumed in rye bread or in beer may prove pathogenic. Concerning these, or certain closely related forms, Zopf detailed exact morphological investigations, accompanied by numerous illustrations, in his memoir on Fumago, and also in his work on the fungi. This black, soot-like fungus occurs very frequently on plants. Frank correctly says : — " We are still quite in the dark with regard to specific differences, due •especially to the frequent polymorphism of these organisms, and to the fact that the different evolution forms are scarcely ever found together." Among the various fungi occurring on the vine, the two following parasites have obtained an unenviable notoriety, on .account of the great damage they cause : — 11. Oidium (Erysiphe) Tuckeri. This fungus, which is also called " the true mildew," forms whitish spots, changing to brown, on the leaves and shoots of the vine. These consist of mycelial filaments, from which •elliptical or oblong, colourless conidia separate, 8 /u long and 5 fji. thick. The mycelium spreads over the fruit, which is gradually covered with a delicate growth of a grey colour, while it thrusts through the fruit skin roundish suckers, killing the epidermal cells. When grapes are attacked at an earlier .stage, the epidermis is unable to keep up with the growth of the contents ; it then gradually splits open like skin affected by .scurf , the contents exude, and the grapes either dry up or putrefy. 'They may impart to wine a very unpleasant smell and taste. PERONOSPORA VITICOLA. 223 On the full-grown grapes the fungus does not do so much harm, but may still prevent the further maturing of the fruit. The best remedy for this dangerous parasite is sprinkling with finely powdered sulphur, but this only takes effect in sunny weather. Kig. 47. —Peronospora viticola (after Cornu). 12. Peronospora viticola. The second vine fungus is " the false mildew," Peronospora viticola, which penetrates to the interior of the leaves and fruit, where it spreads and kills the cells. The conidiophores (Fig. 47) burst out from the stomata of the leaves in tufts. The upper part is branched, and both the branches and the 224 MICRO-ORGANISMS AND FERMENTATION. principal axis end in short conical apices. The conidia are oval, 12 to 30 /m. long, and have a smooth, colourless membrane. In the conidia, as a rule, five or six swarming spores are formed, which burst out when the conidia are immersed in water, and penetrate through the epidermis of the leaves and fruit. The growth forms thick, prominent whitish spots on both leaves and fruit. In the interior of the plant, big, globular oospores are formed (30 //. diameter), which have a brownish membrane, smooth or slightly fluted, and are surrounded by the thin, colourless, or yellowish, oogonium wall. This fungus causes great injury, because the grapes either wither away or putrefy according to the stage at wrhich they are attacked ; moreover, it destroys the foliage. The species is indigenous to North America, and was introduced into Europe in the year 1878 along with American vines ; it has now spread to all vine- growing countries. Vine growers are endeavouring to suppress this pernicious parasite by the application of copper sulphate and calcium hydrate (Bordeaux mixture), and by similar remedies. 225 CHAPTER V. YEASTS. ACCORDING to modern usage the word " yeast " is used to describe those alcohol-forming fungi which are formed, as a rule, by a process of budding, and which under special con- ditions form spores in the interior of the budding cells. The old name Saccharomycetes has been retained to describe these fungi, which are of such great technical importance. It has already been stated that both the bacteria and mould-fungi possess alcohol-forming species, whilst among the moulds certain bud-forming species also occur. Mycelium formation has been shown to exist in not a few Saccharomycetes, and since an endogenous spore-formation also occurs in certain of the moulds, it would appear doubtful whether it is correct to class the yeasts as an independent group of fungi. The direct observation of genetic connection between typical Dematium-like mould-fungi on the one hand, and Saccharomycetes on the other, makes it difficult to accept the earlier view. This observation at ,all events proves that species exist which cannot be classed in an independent group. Doubtless future investigations will bring to light further instances of species which represent stages in the development of higher fungi. The genera Mycoderma and Torula, which include no members exhibiting endogenous spore-formation, but include a number of species known only in the budding stage, will be dealt with in an appendix to this section. The Nutrition of Yeasts. Some account of the nutrition of fungi in general has already been given in the chapter on moulds. In the following 15 «~226 MICRO-ORGANISMS AND FERMENTATION. paragraphs a review is given of the special features which yeasts present in this respect. The inorganic substances — phosphorus, potassium, mag- nesium, and sulphur — have been enumerated by A. Mayer as indispensable for the nutrition of yeasts. His statement is based on the results of his analyses of yeast (not pure cultures) and of his nutritive experiments. As is well known, yeast contains considerable quantities of phosphoric acid. In Munich brewery yeast, the proportion of phosphoric acid represents about 3| per cent, of the dry residue. If malt- wort or other nutrient medium contains too small a quantity of phosphoric acid, this defect may be remedied by adding . potassium phosphate. Potassium is also an essential food element for yeast. It is readily absorbed, both in the form of phosphate and sulphate. Magnesium is an element of equal importance. Sulphur can always be detected in yeast, and must, therefore, be regarded as essential to its metabolism. Calcium, on the contrary, does not appear to be necessary for the propagation of yeast. This element plays an important part, however, in the fermentation process, for it has been shown by Seyffert that brewery yeast quickly degenerates in a wort poor in lime. According to recent work carried out by Delbriick, Lange, Henneberg, Hayduck, Seyftert, and others, calcium carbonate is of importance by rendering certain poisonous substances innocuous which are present in the raw materials, and are believed to be of an albuminoid character (see Chap. i.). In the preparation of artificial nutrient solutions for yeast, these substances should be added in the form of salts, the total quantity not exceeding about 1 gramme per litre.* * Ad. Mayer made use of — Acid potassium phosphate, KH.,P04, Crystallised magnesium sulphate, MgS04, 7HoO, Tribasic calcium phosphate, Ca3P.,Os, beginning with greatly reduced quantities. Laurent made use of a solution containing per litre — 0*75 gramme potassium phosphate. 5'0 grammes ammonium phosphate or sulphate. O'l gramme magnesium sulphate. I'O tartaric acid. NUTRITION OF YEASTS. 227 Molisch found that small quantities of iron exert a favour- able influence on the propagation of pressed yeast. Kos- sowicz showed, moreover, that in this respect ferrous sulphate has a much more favourable effect than ferric chloride. With regard to the importance of carbon compounds for the nutrition of yeast, Laurent, in particular, proved that yeast can assimilate, in addition to sugar, large quantities of such compounds as lactic acid, glycerine, dextrin, tartaric acid, etc., but not oxalic acid and its potassium and ammonium salts. In practice, sugars play the chief part in nutrition, but the species of yeast differ amongst themselves in this respect, as well as in their power of fermenting these carbohydrates. Thus, according to Beijerinck, Schizosaccharomyces octosporus can readily assimilate maltose, glucose, and Isevulose, but not saccharose and lactose. Glycogen constitutes an essential part of the cell contents at a certain stage of its life. This is specially the case when the liquid is rich in carbohydrates, which are stored up as reserve material in this form. Glycogen was discovered by Errera in yeast, and has been more closely studied by other investigators. Laurent proved that yeast can store up very considerable quantities. It appears in the cell as minute semi-fluid drops, with no definite form, and gives a reddish- violet coloration with iodine, which disappears on warming. When the food supply shows signs of becoming exhausted, the yeast cell falls back upon its glycogen. With regard to possible sources of nitrogen for yeast, it may iirst be noted that amongst inorganic sources the ammonium salts are readily absorbed. It was proved by Pasteur that yeast can grow in a nutritive fluid containing no organic nitrogen, but only nitrogen in the form of ammonium tartrate (100 c.c. water, 10 grammes sugar, 0-1 gramme ammonium tartrate, and the ash from 1 gramme of yeast). Subsequently Willdiers Hanson's artificial culture fluid contained — 88'5 grammes distilled water. 0*2 gramme magnesium sulphate. 0*3 .. monopotassium phosphate. 1-0 „ peptone (Witte). 10*0 grammes saccharose. 228 MICRO-ORGANISMS AND FERMENTATION. proved by exhaustive experiments that it is not sufficient to supply nitrogen in inorganic compounds. Kossowicz reached the same conclusion, and showed that by sowing a single cell in a saccharine fluid containing mineral matter no development took place, whilst with a greater infection of cells development may proceed. It has been definitely proved in the case of the Mycoderma species that their demand for nitrogen can be fully satisfied by ammonia in the form of inorganic compounds. Organic nitrogenous compounds occur in considerable quantities in most of the liquids fermented in practice. Thus the cereals which are utilised in breweries and distilleries contain a series of proteins which possess a nutrient value for the yeast cell. Rye is specially rich in these substances, and for this reason an admixture of this cereal is always used in the manufacture of yeast. Peptones and amides are formed during the mashing process, both of which appear to be ab- sorbed by yeast with special facility. Asparagin, which is present in considerable quantities in sprouted corn, malt, and potatoes, plays an important part as a source of nitrogen ; the yeast converting it into protein. Cider musts are notably poor in nitrogenous food for yeasts. Miiller-Thurgau, there- fore, recommends the addition of ammonium chloride (about 20 grammes per hectolitre) or of ammonium tartrate. A large number of analyses have been made to determine the albuminoid contents of yeast. Wijsmann found that the proportion varies greatly, even at different stages of fermenta- tion. At first the quantity of nitrogen rises rapidly, but afterwards it gradually diminishes. Thus the nitrogen may rise from about 7 to 10 per cent., calculated on the dry sub- stance, during the first hour. Amongst the albuminoids, special reference must be made to the nucleins, substances which owe their name to the fact that they are the principal constituents of the nucleus of the yeast cell. They were shown to be present in considerable quantities in Kossel's extensive researches on pressed yeast, and others have since demon- strated their presence in yeast. They are very complex substances, and appear to play an essential part in the develop- ment of the yeast cell (division of the nucleus). Their occur- THEORIES OF FERMENTATION. 229 rence in the cell may be demonstrated by micro-chemical methods — e.g., by the action of pepsin (dissolved in 0-2 per cent, hydrochloric acid) — which, according to Zacharias, attacks the other albuminoids, but not the nucleins ; they are, however, dissolved by weak alkalies. The great importance of yeasts, both from the scientific and practical standpoint, is due to their power of forming alcohol from the sugars. From time to time numerous attempts have been made to explain the actual processes which go on during the course of fermentation. It is only quite recently that a starting point has been found for the investigation, which has made it possible to subject this physiological activity of the yeast cell to experimental treatment. The labours of previous workers in this field have, however, produced results of the highest scientific and practical value, and not a few of the investigations in this and adjacent fields of research have laid the basis upon which modern views are built. Our statement must, therefore, be based on a resume of the entire development which has led, during the course of years, to the various theories of fermentation. Theories of Fermentation. It was long ago observed that when a sugar solution or fruit juice is exposed to the air, fermentation phenomena occur after a certain lapse of time. The liquid becomes turbid, an evolution of gas takes place, a precipitate is formed, and the surface is covered with a layer of yeast. The liquid gradually loses its sweet taste, clarifies at the same time, and then proves to contain a new substance with a stimulating action. What exactly the process might be was the object of many speculations in olden times, which were not based upon any true investigation of the processes. We will put all these speculations on one side, and start with the end of the eighteenth •century, the time of the renowned Lavoisier, the founder of modern chemistry, who gave the first explanation of the phenomena based on facts, the first link of a theory of fer- mentation. He proved that simultaneously with the dis- 230 MICRO-ORGANISMS AND FERMENTATION. appearance of sugar, spirit of wine, carbon dioxide, and acetic acid were formed. He explained the process as the splitting up of an oxide into substances both poorer and richer in oxygen. As the yeast played no part in determining the quantitative ratio of these he did not concern himself further with it. At the beginning of the nineteenth century Gay-Lussac published the well-known equation of fermentation, which still holds good, according to which a molecule of grape sugar *" was decomposed into two molecules of carbon dioxide and two- molecules of alcohol. The basis was thus given for a definition of fermentation — viz., a breaking down of complex bodies into bodies of simpler construction. The question now arose in what way this transformation was brought about ; what was the true cause of the decom- position of the liquid ? In the literature of the seventeenth, and still more in that of the eighteenth century, allusions are made to a " ferment " (Willis, Stahl) which was declared to be " a body existing in a state of internal motion which transfers its motion to other bodies present in the liquid, whereby the coupling of the compounds present is torn apart. The fragmentary particles are, however, through constant friction, attenuated and trans- formed into a new and more stable compound." These indica- tions, however, remained unheeded. In 1810, Gay-Lussac,. encouraged by the brilliant chemical discoveries of Lavoisier, undertook experiments to elucidate the process of fermentation, starting from Appert's method of preparation, which consisted essentially of preventing organic matter from undergoing fermentation by boiling it, and immediately afterwards sealing it tightly in vessels so that no air could penetrate to it. This process was, however, no new one, for as early as 1782 the Swedish scientist, Scheele, proved that acetic acid can be preserved unaltered after subjection to heat. Gay-Lussac examined the air contained in such hermeti- cally sealed vessels, and found that it contained no oxygen. In his Zeitdlter des Sauerstoffs, this observation led to the view that oxygen itself was the true cause of the process of * Not cane sugar, as Gay-Lussac believed. THEORIES OF FERMENTATION. 231 fermentation, a view confirmed by the practical experience that sulphurous acid could be used for fuming out casks to arrest the fermentation of must, because the conversion of sulphurous acid into sulphuric acid brought about the removal of oxygen from the air of the casks. The importance of yeast for the fermentation process was quite overlooked. It was regarded as a precipitation from the liquid of no further importance for the comprehension of the process. The first indication of the true relationship had, however, been discovered at a much earlier period. About the year 1600 two Dutchmen, Hans and Zacharias Janssen, invented the microscope, and in the latter half of the seventeenth century another Dutchman, Leeuwenhoek, issued his renowned letters on the investigation of different substances undertaken with the help of this instrument for the Royal Society. In 1680, in the course of one of his letters, a description and drawing of beer yeast appeared for the first time, and later in the same year one of wine yeast. Shortly after the first clear sketches of bacteria appeared. He held the view that the globular yeasts were derived from the flour of cereals used in brewing, and he compared them with starch granules. He had, however, no glimmering of the importance of yeast for fermentation. The observations of the learned Dutchman regarding the microscopical " animals/' for so he named the bacteria, origin- ated the great discussion which has extended into our time, involving researches and explanations of the important question as to whether these organisms can be derived from inorganic and dead matter, or whether they are derived from external fermenting and living matter. The whole of the following development is based upon the investigation of this question, ' which naturally had its influence in the domain of fermentation. After the fairy tales of earlier times had been disproved, Needham came forward in 1745 with definite experiments designed to show that the lowest microscopical forms of life, the " infusoria," were created in the following way : — He exposed decoctions of meat and other organic substances to such a high temperature that, according to his views, all forms of living matter must be killed, and the vessels were 232 MICRO-ORGANISMS AND FERMENTATION. then hermetically sealed. When he opened them later he found living " infusoria '* in the material, and he naturally argued that they were spontaneously generated, and that the substances liberated during decay had combined again and formed these microscopic forms of life. Needham 's experiments were sharply criticised by Spallan- zani in 1765, who proved that if decoctions were maintained for three-quarters of an hour at the boiling point, no living forms were developed until air was admitted. These experi- ments, incidentally, gave a rational basis to the processes of Scheele and Appert. Needham replied that this result could be explained in a perfectly natural way by assuming that the air present in the vessels was so altered in its character by continued heating that it was no longer able to maintain life. Spallanzani was unable to combat this view experimentally, and so the matter remained undecided, and each view had its supporters. The Needham school was supported by the observation of Gay-Lussac that air in hermetically-sealed vessels contained no oxygen. No progress was made until the year 1836. From this time on begins a period of rational investigation. Franz Schulze proved for the first time that oxygen does not play the part that had previously been assumed, and his experi- ments also led to the introduction of the first indications of a biological theory alongside the dominant chemical theory. Schulze vigorously boiled a mixture of water and organic matter in a glass flask, and then allowed air to pass through the flask after bubbling through sulphuric acid. This was carried on daily for a long time. The result showed that the contents of the flask could be preserved unaltered for months together, whereas living forms of matter appeared in the de- coction as soon as the vessels were opened and exposed to the access of air. At the same time Schwann carried out a similar experiment, with the exception that air was passed through a red-hot tube into the decoction. He obtained identical results. But, although these experiments proved that the air present in the flasks contained oxygen, and that living matter was not produced, they proved unconvincing to the supporters THEOKIES OF FERMENTATION. 233 of spontaneous generation. Tfyey fell back on the theory that the powerful treatment the air had undergone had so altered its composition that it was no longer able to produce life. Schroeder and Dusch took up the subject in 1853-1861, Avith the object of proving that air containing all its gaseous constituents, unaltered, may be allowed to react on boiled fermentable material without effect. It is unnecessary to expose the air to any vigorous chemical treatment with strong reagents, if it is first separated as far as possible from solid particles. For this purpose they made use of a filter of cotton wool, through which the air was led before it came into contact with the boiled organic mixtures. Boiled meat and meat- broth, as well as malt-wort, were unaltered when filtered air was introduced into the flasks. On the other hand, the experiments did not succeed with milk or the yoke of egg stirred up in water and boiled. A completely decisive proof could not be furnished by Schroeder until, in 1861, he succeeded in sterilising this substance. About this time Pasteur had begun a number of his epoch-making researches, in which the principle of sterilisation was clearly established. It appeared clear, therefore, to Schroeder that in certain cases before filtration the air must contain something that could bring about fermentation and decay. Whether these are " floating, microscopic, organised germs in the air, or a chemical substance, as yet unknown, which is separated by contact action and fixed on the cotton wool, must remain to be determined." It also appeared to be probable, after correction of the unsuccessful experiment, " that lower in- fusorial ferments exist, produced and separated either from living plant cells or from living animal tissue, which are capable of exercising certain organic functions and trans- formations/' Mention must also be made of the experiments begun by Hoffmann in 1860 regarding decay and fermentation. He boiled the organic matter in a flask with a long drawn-out neck bent several times at an acute angle. The subsequent inflow of air during cooling deposited dust by gravity so that none could fall into the liquid.* The result was exactly that obtained by filtration ; the liquid remained unaltered. Not- * A similar arrangement was made use of by Chevreul. 234 MICRO-ORGANISMS AND FERMENTATION. withstanding all these observations, the school of spontaneous generation maintained their belief, and still numbered many adherents. In 1857 Pasteur, the distinguished French scientist, entered the field. He submitted the problem to such conclusive experimental treatment, from every side, that his conclusions were generally adopted, and have been held ever since. He proved that the many unsuccessful experiments designed to- overthrow the doctrine of spontaneous generation were occasioned by the fact that the organic liquids concerned had not been exposed to a sufficiently high temperature, or heated for a sufficiently long period. Moreover, he showed that in such cases the liquid under treatment was not so greatly altered that it was no longer fit for the development of the germs, as the supporters of spontaneous generation maintained. Thus, if the liquid is boiled in a flask, the neck of which is drawn out into a tube and twice bent (the same idea as that of Hoffmann and Chevreul) so that the liquid remains sterile, and if a small portion of the liquid is then allowed to run into the tube, it soon begins to ferment, owing to the germs deposited in the tube coming in contact with the liquid. The same thing occurred when the air is passed through cotton- wool and a small quantity of the wool is introduced into the sterilised liquid. Pasteur also employed gun-cotton in place of ordinary cotton-wool. Air passed through gun-cotton was sterilised, and the fluid, after boiling sufficiently, remained sterile for an unlimited time. The gun-cotton was afterwards dissolved in alcohol and ether, and it was proved that it contained the same microscopical organisms that develop in liquids undergoing fermentation and putrefaction. This great work of Pasteur's resulted in the overthrow of all proofs previously adduced on behalf of the school that maintained the spontaneous generation of microscopical life in organic liquids. He established the extremely important result for industry which embodied all the essential principles of the technique of sterilisation. A high stage of development of this technique has since been reached, both in its purely scientific and practical aspects. Thus was laid the foundation of the belief that fermenta- THEORIES OF FERMENTATION. 235 tion is brought about by living matter, the vitalistic theory of fermentation, in contrast to the chemical theory which found its best-known advocate in the distinguished Liebig, who- built largely on the theories propounded by Willis and Stahl, after Gay-Lussac's idea that oxygen was the direct cause had been given up. Although Liebig's theory has been abandoned, it is neces- sary, even in a brief historical description, to touch upon it, because it held the field for a long time, on account of its- author's great renown. Experimental chemistry had won great triumphs in the last twenty years of the seventeenth century. Chemists had succeeded in ascribing extremely complex organic processes, previously ascribed to the mysterious vital energy, to a simple action of chemical affinities. As an obvious consequence, the attempt was made to explain fermentation phenomena in the same way, without the help of living beings. Liebig, however, regarded the yeast which appeared in the fermenting liquid as a substance constantly undergoing decomposition, by which the chemical motion incidental to these processes was trans- mitted to the sugar, and brought about the decomposition of the latter. It will be seen at once that this theory could not be held when the presence of living and vigorous- yeast cells was recognised. Liebig, however, did not regard yeast as a plant ; it represented to the chemist a substance without life, and microscopical investigation, according to- Liebig, could contribute nothing of importance to the under- standing of the process. We shall now proceed to discuss how the knowledge of yeast developed and led to the vitalistic theory which prevailed for such a long time. The Austrian Plenciz declared, as early as 1762, that decay only takes place in a body when " germs of a wormy character develop and begin to multiply." Probably we have here the first definite announcement regarding the cause of such decom- positions. A long time elapsed before Leeuwenhoek's observations on yeast cells, in 1680, carried us a step further. As far as we can judge from the known literature, it is believed that the 236 MICRO-ORGANISMS AND FERMENTATION. Austrian Erxleben in 1818 was the first who definitely expressed the thought that fermentation " appears in no way to be a simple chemical operation, but rather is in part a process of growth, and should be regarded as the link in the long chain •of nature which combines those actions that we describe as •chemical processes with those of vegetative growth." But this must be regarded only as a hypothesis without further foundation. Twenty years later, and almost simultaneously, three scientists expressed clear and definite views based on direct experiments regarding the dependence of alcoholic fermenta- tion upon yeast cells. It may be of interest to see how they arrived at the same result in three different ways. Cagniard-Latour was the first to publish his work on yeast, in 1835-37. In his studies of beer and wine fermentation, both in practice and on the small scale, he observed that the yeast globules rise to the surface of the beer-wort on account of the entangled gas wrhich they produce. They possess the power, by budding or by elongating their own tissues, of multiplying, and in this way producing manifold globules, which separate from each other when fully grown. He thus confirmed his view that yeast cells are organic, and belong to the vegetable kingdom. During propagation they are nour- ished by the beer-wort, and when the fermentation has come to an end the liquid contains many times the quantity of yeast that was added to it, whereas the earlier view was that the substantial precipitate consisted mainly of secretions. He also found that yeast will not propagate in pure sugar solutions. His researches enabled him to conclude that in all pro- bability it is the yeast cells that destroy the stability of the components of sugar, and bring about its decomposition into alcohol and carbon dioxide ; that fermentation, in fact, is .a result of vegetable activity. The same observation regarding the vegetable character of yeast was made simultaneously, or a short time after, and •quite independently by Theodor Schwann. It has already been mentioned that he made important contributions to the discussion of the generation of living matter, and it was these THEORIES OF FERMENTATION. 23T investigations that brought about his exact study of yeast under the microscope in 1837-39. Schwann arrived at th& result that it is not atmospheric oxygen, but a substance conveyed in air, and destroyed by heat, which brings about fermentation. To determine whether this substance is of animal or vegetable character, he enquired whether the sub- stance is destroyed by those poisons that are capable of killing infusoria, or by those that kill moulds. The latter proved to be the case, for a solution of potassium arsenite arrested the fermentation of wine ; therefore, he argued, the substance must be of a vegetable character. Under the microscope the yeast resolved itself into the " recognised granules which constitute the ferment." Then he observed how they form continuous rows, with other rows placed diagonally. He also observed that small, granules appeared on the sides of the cells, which form the starting point for new rows, and usually on the last granule of a row appeared a tiny and sometimes elongated body. It will b& seen that this constitutes an exact description of a budding colony of yeast resulting from direct observation under the microscope. Schwann observed that the similarity between this picture and that of many other kinds of fungi was con- siderable, and this strengthened his belief that yeast is a plant. At his instigation Meyen examined " this substance," and gave the plant the name it has since retained of Saccharomyces (sugar-fungus). Schwann also demonstrated that the feeble evolution of gas in grape juice may be regarded as a sign of fermentation ; immediately afterwards the first individuals of the sugar-fungus made their appearance ; these plants grew and multiplied throughout the period of fermentation. As it had also been shown that fermentation ceased through every treatment which brought about the destruction of the fungus (boiling, addition of potassium arsenite, etc.), the connection between fermentation and the sugar-fungus cannot be denied, and " it is extremely probable that the latter brings about the pheno- mena of fermentation through its growth." He declared that fermentation was carried out in such a way that " the sugar- fungus absorbs sugar and a nitrogenous body necessary for its- 238 MICRO-ORGANISMS AND FERMENTATION. nutrition and its growth, whereby those elements which are not taken up by the vegetable body, are principally com- bined to form alcohol (probably along with many other substances)/' F. T. Kiitzing was the third who dealt with this important problem at the same time (1834-37). Within the scope of his •elaborate investigations concerning the lowest microscopical plants he included the yeasts and other micro-organisms that usually occur in brewery wort and distillery mash, and pub- lished good drawings of these growths. It is of particular interest that Kiitzing was the first to investigate the mother of vinegar, the slimy skin which forms on the surface of a liquid that is undergoing acetic fermentation. He examined this film from its earliest stage, and found that it consisted of very small plants, which gradually increase in length. He realised the extraordinary importance which the study, of the lowest forms of life would have for organic chemistry, and for the whole field of natural science. Chemistry must rule out yeast from amongst its chemical compounds, as it proves to be an organism, and he regarded it as certain that " the whole process of the spirituous fermentation is dependent on the formation of yeast, and that of acid fermentation on the formation of mother of vinegar " ; " fermentation is synony- mous with the vital process/' Thus he supplied a clear and •definite form for the vitalistic theory of fermentation in opposition to Gay-Lussac's oxygen hypothesis, and to Liebig's theory of the breaking down of yeast cells as the cause of fermentation. Mitscherlich's work is also of a fundamental character. In 1841 he described the yeast as consisting of round and oval globules, and he solved the question of their importance for fermentation through the following beautiful experiment : — A little yeast is placed in a glass tube, closed at the lower end with a sheet of paper, and this is placed in a sugar solution. In the course of several days it will be seen that fermentation has actually taken place in the tube, owing to the sugar solu- tion having diffused through the paper. Alcohol gradually •diffuses out throughout the liquid, which becomes saturated •with carbon dioxide, but the greater quantity of carbon THEORIES OF FERMENTATION. 239 dioxide is evolved from the tube. It is only after some time, when the paper softens and allows the yeast globules to pass through, that the fermentation process begins to take place on the surface of the paper. He concludes that " fermentation only takes place at the surface of the globules." He also published beautiful drawings of yeast, showing their methods of growth and propagation, and described the contents o£ the •cell after staining with iodine. All these observations did not suffice to establish the new theory. The great authority of the chemist, which still prevailed, required an equal authority in the region of biology to take up every point of the discussion, and by convincing experiments along the whole line, to compel attention ; lacking such an authority, the earlier disputants were unable to win the victory. This great work was carried out by Pasteur with the same conclusive results as in the case of generation. The investi- gations begun by Pasteur did not consist, like those of the earlier experimenters, of short, isolated pieces of work, but ranged over a series of years from 1857 onwards, and were published in a number of memoirs. In this short review it is impossible to do more than indicate a few isolated and especially important experiments taken from the series, which ranges over the whole field of fermentation. At an early stage he made the important observation that the amount of sugar dissolved during fermentation is greater than that corresponding to the carbon dioxide and alcohol produced. The remainder of the sugar that disappears is utilised by the yeast during fermentation, partly for its pro- pagation— a circumstance which cannot be reconciled with Liebig's view, who demanded as a condition of fermentation that yeast should be in a state of decomposition. Shortly after, Pasteur proved that during fermentation yeast not only produces alcohol and carbon dioxide, but simultaneously succinic acid and glycerine, the latter derived from a further part of the disappearing sugar. He also showed that by the addition of ammonium tartrate to the fermenting liquid yeast can be brought to more rapid development, and the liquid can be more highly fermented than usual, proving that this salt 240 MICRO-ORGANISMS AND FERMENTATION. must be a food-stuff for yeast. In general, he proved that no decay of yeast takes place during fermentation, and that the presence of assimilable albuminoids in the liquor is unnecessary, for by sowing a minute portion of yeast in a liquid which only contains sugar, ammonium tartrate and a few salts, fermenta- tion can be brought about with development of young cells capable of propagation. At the same time, he showed that the reason why many of the early experiments, which should have refuted the older theories, did not succeed was due to the fact that it was im- possible at the time to secure absolute sterilisation of the liquids. He then produced further proofs that the acetic acid fermentation, already recognised by Kiitzing as due to physio- logical activity, must certainly be regarded as having this character. One further observation must be mentioned on account of its wide-reaching importance. He proved that calcium lactate can undergo fermentation resulting in the formation of butyric acid, and that the active organism can exist without access of air. He gradually extended his observations in this entirely new field (anaerobiosis = life without air), and de- finitely distinguished between aerobic and anaerobic life. It was this remarkable discovery, which at a later stage included the alcoholic yeasts, that led the distinguished scientist to a, solution of the problem under what conditions yeast cells can decompose sugar. In 1876, in his Ktudea sur la Here, he formulated his cele- brated theory of fermentation, based upon a series of actual .experiments, details of which cannot be given here, a theory which has served both as the basis and the starting point by which progress has been made throughout a long series of years, and one which will always retain its importance. It starts essentially from the thought that living yeast cells under certain conditions are obliged to live apart from air, and that they then react as exciters of fermentation. Fermentation is, therefore, bound up with the life of yeast cells ; it is life without air. As yeast under these conditions is obliged to obtain its necessary demands of oxygen from sugar in order THEORIES OF FERMENTATION. . 241 that it may continue to develop as a living organism, it splits up the sugar, and the residue of the oxygen, as well as the carbon, constitute new compounds — viz., the fermentation products, alcohol, carbon dioxide, etc. At the same time, Pasteur emphasised the idea that for each kind of fermentation — alcohol, acetic acid, butyric acid, etc. — a specific kind of organism occurs. It will be seen that Pasteur's theory consists both of a biological and a chemical portion. The yeast cells fulfil their normal existence with generous access of atmospheric oxygen, and under these conditions develop, according to his view, most vigorously, and prepare themselves in the best possible way to continue their existence without air, and this is the necessary condition for their existence as alcohol formers —i.e., decomposers of sugar. The first statement, which clearly explained an important biological problem, still holds good ; the second, which endeavoured to supply an answer to the requirements of the chemical process, can no longer be accepted. That Pasteur did not apply his definition in the narrowest sense of the word is shown by the fact that he himself em- phasised the fact that yeast can exercise fermentative power in presence of a limited supply of air as well as in its absence. This was established under certain conditions for low ferment- ation beer-yeasts by Pedersen in 1878, and Hansen in 1879. They arrived at the result that the quantity of dry substance in beer-wort which a given quantity of yeast can convert into alcohol, carbon dioxide, etc., is smaller when the liquid is aerated during fermentation than when it is not. A similar result was obtained by Eduard Buchner in 1885 in his experi- ments on bacteria. Hansen so arranged his experiments that the cells during aeration were in constant motion, carried hither and thither by the vigorous blast of air. As they, nevertheless, continued to give a distinct alcoholic fermentation, there can be no doubt that this is not determined by life without air. Nageli, in 1879, in his Theory of Fermentation, proved that access of oxygen is always favourable to alcoholic fermentation in a sugar solution, if no nutritive material is present, and 16 242 MICRO-ORGANISMS AND FERMENTATION. consequently the quantity of yeast is only slightly increased. Nageli says (p. 26), " The theory of Pasteur, that fermenta- tion results from a lack of oxygen, forcing the yeast cells to secure their requirements of oxygen from the fermenting material, is opposed to all the facts brought to bear upon this subject." This view is shared by A. J. Brown. He arranged a set of experiments in which fermentation proceeded in presence of full access of oxygen, and a parallel set in which oxygen was excluded. In both series the same number of yeast ceUs were used, and they were kept under such conditions that it was impossible for them to multiply. Otherwise every condition was the same. It proved, contrary to Pasteur's theory, that the cells in the first case developed a higher fermentative activity than when oxygen was excluded. ERieppe and his pupils have also rejected Pasteur's theory of fermentation, and have brought forward examples of ferment organisms " which are able to bring about the specific fermentation much better, on the whole, in presence of atmo- spheric oxygen." Similar experiments were undertaken by H. Buchner and Rapp, with the object of ascertaining by exact quantitative methods to what extent free access of air brings about the replacement and suppression of the fermentative power of yeast cells by their oxidising function. With this object in view, they prepared pure surface cultures of yeast with the greatest possible access of air, and carried out parallel experi- ments with limited access. The first lot of cultures were grown in large cylindrical vessels, the inner wall of which was covered with a thin lining of wort-gelatine containing 10 per cent, of grape sugar. This was infected with a coating of pure yeast, and in each experiment a current of air was passed through the vessel for five days. The carbon dioxide was absorbed in caustic potash, and after each experiment the amount of alcohol and the ratio between the yeast and the fermented sugar were determined. Parallel experiments were carried out, in which the same quantities of beer-wort and grape sugar were allowed to ferment in Erlenmeyer flasks. As a consequence of the rapid and abundant growth of THEORIES OF FERMENTATION. 243 yeast on the surface of the gelatine, the fermentation on gelatine ceased much more quickly than in wort, where the yeast collected on the bottom of the flask. It was further proved that considerably more carbon dioxide was formed in the surface cultures than in the parallel experiments with wort. This carbon dioxide must be due to the respiration of the yeast. Nevertheless, only about one-seventh of the sugar was decomposed by oxidation, whilst more than six-sevenths were fermented. Although the yeast had been submitted, according to Pasteur's view, to the most favourable condi- tions for life without bringing about fermentation, this did not prove to be the case. As is now universally known, a free supply of oxygen exercises a favourable influence on the propagation of cells, but these experiments served to establish the fact that oxygen has scarcely any influence on the process •of fermentation, and that the absence of oxygen must not be regarded as being of immense moment for the fermentative activity, for even in presence of a full quantity of oxygen the fermentative power of yeast still exceeds the respiratory power. From Nageli's many-sided work on the lower organisms, we can only refer, in connection with the preceding, to his " molecular-physical " theory of fermentation, which may be regarded as a modification of Liebig's theory. Whilst Pasteur regarded fermentation as the result of an activity taking place in the cell, Nageli defined fermentation as a transference of states of motion of the molecules, groups of atoms or atoms of different compounds, constituting living protoplasm (which otherwise undergo no change) to the fermenting material, whereby the stability in these molecules is destroyed and •disruption is brought about. During fermentation the vibra- tions of the molecules of protoplasm are transferred to the fermenting material. The cause of fermentation is to be nought in the living protoplasm in the interior of the cells, but its activity extends for some distance outside the cells. The decomposition of sugar takes place to a slight extent inside the yeast cells, but principally outside them. This theory is opposed to that of Pasteur, and is related to that of Stahl and Liebig. 244 MICRO-ORGANISMS AND FERMENTATION. We now return to the epoch-making researches of Pasteur, He proved in the clearest and most unmistakable manner in his Etudes sur la biere what power is possessed by microscopic- life, and he strongly emphasised the fact that bacteria may have a far-reaching influence on the course of alcoholic fer- mentation and on the character of the beer. The budding organisms were dealt with in a similar way. He indicated that certain fungi of this group, which are not described in detail, may react in different ways on the products of fermentation, as Bail had previously experienced. Pasteur's communications r however, only traversed the nebulous views of his predecessors, and his assumptions led to two opposing lines of thought. This is seen, for instance, in his observations on the so-called cheesy and at'robiotic yeast. It is possible that we have to- do in this case with independent and peculiar types of yeast,, but it is also possible that we are dealing with forms which are brought about by a particular treatment of the usual brewery yeast. It should not, however, be overlooked that he himself indicated the direction in which the solution of the question must be sought — viz., that it was at the time impos- sible to determine whether one or more species was present ; an exact method for the pure culture of yeast species had not been discovered. Thus a true orientation ' in the world of micro-organisms cannot be found in his work. It was im- possible at any point in Pasteur's thesis to find characters described for the budding fungi that would enable an analysis, to be based upon them. He believed that all budding fungi may to some extent possess the power of bringing about alcoholic fermentation like the Saccharomycetes. It is never possible to tell whether he is referring to true Saccharomycetes or to other budding fungi. Pasteur did not differentiate- between the several kinds of budding fungi (Saccharomycetes, Torula, Dematium, etc.). Pasteur took the standpoint that every individual fer- mentation, lactic, butyric, acetic acid, etc., is produced by a particular exciter of fermentation. It was only when the technique of pure cultivation had been further developed that an explanation of the true connection was possible — viz., that each one of these processes must be carried out by different THEORIES OF FERMENTATION. 245 kinds of organisms. This was proved by E. C. Hansen in 1878 for acetic acid bacteria, by Miquel for uric acid bacteria, and by Hansen in 1883 for the alcohol yeasts. The chief reason why the reform in brewing technique •could not be carried out was that the existing scientific stand- point made it impossible to clearly define the relations existing between the different fungi concerned in alcoholic fermenta- tion. Pasteur was, therefore, unable to escape from the indistinct assumptions and the contradictory views of his predecessors. In a review given in his book (pp. 4-7) regarding the micro-organisms that bring about diseases in beer, he speaks only of bacteria, and this belief is reiterated by Duclaux in 1883, and by other French, German, and English workers. As a result of his studies, Pasteur recommended brewers to undertake the purification of their yeasts, to rid them of bacteria by cultivating them in a sugar solution with tartaric acid or in wort with a little carbolic acid. In contrast to all this, in 1883 Hansen published his doctrine that some of the most dangerous and most commonly occurring •diseases in low-fermentation beer are not produced by bacteria, but by certain species of Saccharomycetes, and that the names £. cerevisice, S. Pastorianus, and S. ellipsoideus, suggested by Beess, do not indicate one, but several different species and races. Hansen proved that species which had been incorrectly grouped under the systematic name S. cerevisice yield different products in the brewery. From this standpoint he elaborated Ms system, utilising a stock yeast derived from a single species. After some opposition, this system was adopted in all brewing countries, and introduced into the industry. Hansen's experi- mental demonstration showed that Pasteur's process for purifying yeast by means of tartaric acid furthers the develop- ment of disease yeast to such an extent that they are capable of completely suppressing the true culture yeasts. Pasteur greeted Hansen's system as an advance, and wrote, " Hansen was the first to realise that beer yeast should be pure, and that not only in regard to microbes and disease ferments in the narrower sense, but also that it should be free from cells of wild yeast." The main problem regarding the actual cause of the decom- 246 MICRO-ORGANISMS AND FERMENTATION. position of the sugar molecule and the special conditions under which it took place still awaited solution. Meanwhile, in the last decade of the nineteenth century,, new views regarding the fermentative forces were gaining ground, when it proved possible to separate the exciter of fermentation in certain cases (diastase from malt, pepsin from gastric juice). The characteristic effect of these ferments was that minute quantities were able to split up large amounts of the given material, and that they completely lost this power when subjected to heat. The name enzyme was applied to> the substances isolated from the living cells of the barley corn, the mucous membrane of the stomach, etc., and gradually a large number of these ferments were distinguished, amongst them some of great technical importance. The thought naturally suggests itself that it must be possible to find such an enzyme amongst the many elements of which the living yeast cell is constituted which would be capable of splitting up sugar. As early as 1858 we find & suggestion of this kind put forward by Traube that " the chemical processes going on in living organisms originate mainly in the circumstance that protein substances are liable to undergo decomposition in the presence of water, and that under the peculiar conditions actually obtaining they are also- apt to give rise to peculiar ferments." A direct outcome of this view was Miquel's discovery in 1890 that the bacterium causing the ammoniacal fermentation of urine contained an enzyme which can bring about this fermentation on its own account. In 1894 Emil Fischer, by purely chemical research, resulting in his celebrated work on the synthesis of the sugars, on the use of phenyl-hydrazin, and the osazone-reaction, diverted the current views on fermentation phenomena into new channels. His researches led him to explain the behaviour of the yeast cell towards the particular sugar of the nutritive liquid in the same way as that of the enzymes (invertase, emulsin), so that the chemical activity of the living cell does not differ from the action of chemical ferments. According to Fischer, fer- mentation of polysaccharides is always preceded by hydrolysis of the sugar. But there exists an exact relation between the THEORIES OF FERMENTATION. 247 molecular structure of a given sugar and the sugar-inverting enzyme of a yeast cell ; if a sugar comes into contact with the albuminoids of a yeast cell, which play the most important part among the agents utilised by the living cell, the sugar is decomposed only if its configuration, the geometrical structure of its molecules, does not deviate too much from the con- figuration of the molecules of the albuminoid. Thus, accord- ing to Fischer's theory, the function of the living cell depends much more upon its molecular geometry than on the com- position of the nutritive material. Another way in which Fischer, as well as Thierfelder, obtained confirmation of his fermentation theory was by examining the behaviour of Hanson's and other yeast species towards the artificial sugar species, synthetically obtained by Fischer. They found, indeed, that the yeasts are quite fastidious regarding the geometrical configuration of the sugar molecule, whilst they often remain unaffected by other altera- tions in its composition. Among the various synthetically-prepared sugars examined by Fischer with regard to their behaviour towards yeasts, melibiose is especially mentioned. It is fermented by brewers' common bottom-fermentation yeasts, but not by many brewers' top-fermentation yeasts. In harmony with this, Fischer found that bottom-fermentation yeast contains an enzyme capable of extraction from the dried yeast in aqueous solution, which decomposes melibiose, converting it into glucose and galactose ; but in a corresponding treatment of the brewers' top-fermentation yeasts no decomposition of this sugar could be observed. As brewers' top-fermentation yeast contains invertase, it follows that the ferment which splits up melibiose cannot be identical with invertase. C. J. Lintner and Fischer showed, by methods devised by the latter, that natural maltose is split up into two molecules of glycose, if acted upon by an aqueous extract of dried yeast, or by cells, the membrane of which has been torn by grinding with powdered glass, and that there is a marked difference between this enzyme and invertase which hydrolises cane- sugar. The former enzyme is termed yeast-glycase or yeast- maltase. Its optimum temperature is about 40° C., whilst 24S MICRO-ORGANISMS AND FERMENTATION. that of invertase, according to Kjeldahl, is 52°-53° C. In a similar way, a lactose-cleaving enzyme (lactase) and an enzyme resembling invertase were isolated from Monilia. At the same time, Hans and Eduard Buchner were endeavouring to prepare a juice by a treatment of the yeast cells similar to that adopted by Emil Fischer — i.e., by grinding the wall of the cells, hoping to apply it to therapeutic experi- ments. To preserve the juice, it was mixed with sugar, and E. Buchner thus observed that a vigorous development of gas took place in the mixture. A further examination showed that the gas was carbon dioxide, and that alcohol was simul- taneously produced in the juice. This was the basis of the extended researches which led to the discovery of the alcohol enzyme, which was successfully separated from the living cell (communicated first in 1897). Buchner's process is as follows : — Fresh washed and strongly pressed yeast is ground with quartz and kieselguhr in a mortar. The cells are torn and broken open by the sharp sand, and the liquid absorbed by the kieselguhr. In a few moments the whole mass cakes together to form a dough. This is wrapped in a strong press cloth, and subjected to very high pressure in a hydraulic press, up to 90 kilogrammes per square centimetre. For every kilogramme of yeast about 500 c.c. of clear yellow or yellowish-brown juice is obtained. When the juice is mixed with a solution of saccharose, grape sugar or maltose, a strong frothing takes place within a few minutes, due to the development of carbon dioxide, and at the same time almost the same quantity of ethyl-alcohol is produced. By the addition of minute quantities of alkalies (potassium carbonate, disodium phosphate, etc.), the process of fermentation is quickened. It can be shown that the fermentation is not caused by living cells remaining in the juice, for it is possible to add strong antiseptics like chloroform, thymol, or toluol.* which would arrest every living function of the cells, or again the juice may be filtered free from germs through a porcelain filter, without destroying its activity. It might be supposed * On the other hand, n>ercuric chloride destroys the fermentative power of the juice. THEORIES OF FERMENTATION. 249 that the fragments of protoplasm torn from the cells could be regarded as carriers of this power, and that the enzyme itself had not been separated. This cannot, however, be the case, for if the juice is treated with precipitants like alcohol-ether or acetone, the active substance is thrown down, and this, along with other precipitated substances, on drying, forms an amorphous and very stable powder which, on treatment with water, can once more be employed as an exciter of fermentation. It has since been shown that high pressure is not essential. R. Albert has recently shown that by treatment of yeast with alcohol-ether, or, better still, with acetone in such a way that all the cells are destroyed, a very active powder can be prepared (zymin). The yeast is partially dried and soaked for a quarter of an hour in acetone (ten times its volume). It is then spread on filter paper to dry, washed with ether, and dried at 45° C. The preparation takes the form of a white powder. The powder, which consists of dead cells, that are still whole, produces almost immediate fermentation in a sugar solution. If it is washed with water, the water does not acquire any fermentative power. If, however, the cells are first disintegrated, it is possible by simple suction with a water pump to obtain a juice from which a precipitate is thrown down by means of ethyl alcohol, which can be dis- solved in water and immediately produces a vigorous fer- mentation in a sugar solution. Whilst yeast that has been killed in the usual way does not retain any alcohol enzyme, it is possible by this method to fix the enzyme so that it remains intact in the dead cells. These remarkable observations only permit of one explana- tion, viz., that living yeast cells are not essential for the production of fermentation, and that it is possible to separate an active enzyme from the disintegrated yeast cells which is soluble in water, and is much more resistant to antiseptics and other strong influences than the living cells, a property that it possesses in common with other enzymes. This substance, which is contained in pressed-yeast juice, Buchner named zymase (alcoholase). Buchner declares in very interesting fashion that this discovery proves that both Pasteur and Liebig were correct 250 MICRO-ORGANISMS AND FERMENTATION. in certain respects ; Pasteur in so far as zymase can only be produced by living cells, Liebig in so far as the fermentation is excited, not by living cells, but by a separate enzyme. Thus, for the first time, we have a solid basis for a true theory of fermentation established by the study of that par- ticular enzyme which brings about fermentation. A short resume is given below of the properties of the enzyme, so far as they have been clearly defined in the short space of time succeeding its discovery. It must, however, be understood that very little is known concerning its chemical character. Zymase cannot be regarded as consisting of living matter. It can be distinguished from invertase of yeast cells, which converts saccharose into fermentable sugar, by the fact that it does not diffuse through the cell wall. If the juice is heated to 40°-50° C., a flocculent pre- cipitate of albumen forms, and the clear liquid loses its fer- mentative power. Invertase has been identified in yeast- juice, and it must also contain an enzyme hydrolysing maltose and one hydrolysing glycogen, as it is capable of bringing about fermentation with these carbohydrates, neither of them being directly fermentable ; but, according to Hahn, it also contains a substance of importance, a proteolytic enzyme hydrolysing albumen. If a test tube containing fresh yeast- juice, and another containing juice that has stood for a week at room temperature in presence of toluol (to prevent the growth of micro-organisms), are placed in a water-bath at 40°-45° C., it will be found that in the former a strong coagulum separates out in a few minutes, whilst in the latter only a few flocculent particles are visible. The coagulable albumen, when kept for some time, disappears by a species of auto-digestion. Hahn has named the enzyme yeast- endotryptase. This enzyme reacts best in presence of acid, whilst the activity of zymase is improved by the addition of weak alkali. The presence of oxygen is advantageous to proteo- lysis. The enzyme can be isolated in a comparatively pure state, and is found in yeast cells. According to Hahn, it cannot be separated from quite normal cells, and such cells can only deal with those albuminoids which are forced through the cell walls. Endotryptase has a powerful action on zymase, and even THEORIES OF FERMENTATION. 251 when the juice is kept at a low temperature a marked loss in. its fermentative power is observed in the course of a few days, owing to the influence of endotryptase. It is quite possible that it is this enzyme, more strongly developed, which attacks the enzyme of the yeast cells when they are exposed to un- favourable conditions. Buchner certainly believes that this- accounts for the fact that yeast- juice prepared from one and the same species of yeast may contain very variable quantities of zymase. Zymase is extraordinarily sensitive both to variations in temperature and to the presence of strong alkalies. To protect the juice from the action of endotryptase large additions of cane sugar have been employed. Thus when mixed with 75 per cent, sugar solution the activity of the enzyme has been prolonged for several weeks. As a result of a number of fermentations, Buchner notes that the fermentative power of 20 c.c. of yeast-juice with the addition of 8 grammes of sugar and 0-2 c.c. of toluol results in a yield of 1-87 grammes of carbon dioxide. Compared with the fermentative power of fresh yeast, the action of the juice appears trifling. Thus, 1 gramme of good pressed yeast produces in an 8 per cent, cane-sugar solution 1-5 grammes of carbon dioxide in six hours at 30° C., whilst 20 c.c. of yeast-juice is produced from about 40 grammes of yeast, but it should not be forgotten that during the fermenta- tion with living cells new zymase is constantly being produced, and that by no means all the existing zymase is extracted from the cells in the preparation of the juice. To avoid the rapid decomposition of the juice, it may be dried in a vacuum at 25°-35° C. It forms a yellowish powder, which remains unchanged for a long time, and when dissolved in water displays an almost undiminished fermentative power. In contrast to the action of weak alkalies, the addition of acid is prejudicial to the juice. With regard to the best conditions of temperature, it has- been shown that the highest fermentative activity is reached at 12°- 14° C. The most favourable temperature for zymase undoubtedly lies higher, but it must be remembered that at the higher temperature endotryptase immediately comes into* action and attacks zymase. 252 MICROORGANISMS AND FERMENTATION. As to the concentration of the liquid, the largest amount of carbon dioxide is obtained by fermentation of liquids •containing 30 to 40 per cent, of sugar, doubtless because in such concentrations the action of endotryptase is restricted. The fermentation lasts longer under these circumstances, and to secure a rapid fermentation, 10 to 15 per cent, of sugar should be employed, but under these conditions the action soon comes to an end. Amongst the many attempts that have been made to isolate zymase from the juice, we may mention that by pre- cipitation with alcohol-ether the whole of the zymase can be •converted into a dry form without loss of activity. By the treatment of the dry substance with water and glycerine, the liquid, even when filtered, possesses the full fermentative power. This constitutes, therefore, a true solution of the active substance ; a further treatment with alcohol-ether does not yield any increase of zymase in the precipitate. The living yeast cells contain varying quantities of zymase. Thus the content of zymase often increases perceptibly in quiescent pressed yeast when kept at low temperatures. It is a remarkable fact that yeast cultivated in a strong sugar solution with inorganic salts exhibits a comparatively small amount of zymase at the moment of greatest fermentative activity with the greatest production of froth. If, however, the yeast is removed at this stage, washed, pressed, and stored for a few hours at a low temperature, it will be found that the zymase content has considerably increased. In the same way, yeast taken fresh from the brewery shows an increase of zymase in some cases after storing. In other cases no such increase is observed. These facts can be explained on the assumption that the endotryptase is influenced by the low temperature, even when the other conditions are favourable. A short review of the chemical changes that take place during the fermentation of yeast- juice follows. The first problem is to discover whether the phenomena caused by the addition of yeast-juice to sugar solutions are identical with those of alcoholic fermentation of sugar. The attempts to solve this have led to remarkable results. The chemical action of the enzymes already discussed — invertase, THEORIES OF FERMENTATION. 253 maltase, lactase, diastase — consists in the hydrolysis of the polysaccharides into simpler compounds, the monosaccharides, an action which can also be produced by purely chemical treatment. Zymase is distinguished from these enzymes by bringing about the complete breaking down of the sugar molecule and the formation of new compounds, exactly like the alcohol enzyme of the living yeast cell. As is well known, this splits up sugar into almost equal parts of alcohol and carbon dioxide. This is also the case with zymase. A part of the sugar, however, is not converted into these products. During the pressed-juice fermentation, glycerine is produced to the extent of from 3 to 8 per cent, of the fermented sugar ; it is derived from the sugar. On the other hand, no succinie acid is produced. Acetic acid is formed in minute quantities, but somewhat more than in the fermentation with the living cell. This is probably due to the action of a special enzyme. It is of great interest to know that lactic acid is often produced in the zymase fermentation, whereas in other cases the lactic acid originally present or that added to the liquor disappears. This observation suggests a possible solution of the way in which the sugar molecule is decomposed into alcohol and carbon dioxide. It is reasonable to suppose that lactic acid is an intermediate product, and that the zymase consists probably of two enzymes, one of which (zymase in the narrower sense) converts sugar into lactic acid ; the other (" lactacidase ") converts the lactic acid so formed into- alcohol and carbon dioxide. The results could then be ex- plained by supposing that an excess of one or other enzyme in the juice causes either the production or the decomposition of lactic acid. It has been stated that part of the sugar is not decomposed into alcohol and carbon dioxide. This cannot be detected by the use of Fehling's solution, and it is certainly not present as reducing sugar. The experiments of Harden and Young have proved that a polysaccharide is formed by a synthesising enzyme present in the juice. By suitable hydrolysis this- substance may be converted into reducing sugar. The later work of Harden and Young has carried the investigation further. They found that by adding boiled 254 MICRO-ORGANISMS AND FERMENTATION. .and, therefore, inactive yeast- juice to fresh juice, the activity of the latter is considerably increased. It follows that the juice must contain an enzyme which is stable at the boiling point, and another which cannot withstand this temperature, a,nd that it is only in conjunction that they can exercise fer- mentative activity. Buchner and Meisenheimer have obtained a pressed juice and also a stable preparation from yeasts fermenting lactose, which are capable of carrying on the fermentation. It has not yet proved possible to isolate zymase, and nothing is yet known regarding its composition. Its properties may be summarised as follows : — It is soluble in water and -dilute glycerine, and is not very sensitive to chemical reagents. In solution it is decomposed at 60° C. When yeast juice is kept at low temperatures (down to 0° C.), the zymase gradually disappears, whilst in a frozen condition it remains unaltered for some time. In a dry condition it may be stored for months with unaltered activity, and withstands a temperature of 110° C. It is precipitated along with albuminoids by treat- ment with alcohol, acetone, and ammonium sulphate. It can be dialysed with difficulty or not at all, and occurs in very variable quantity in the living cells according to their stage of development. H. Fischer describes zymase as the fermenting enzyme to distinguish it from the other enzymes. In addition to the enzymes already mentioned, the alcoholic, the hydrolytic (maltose-, cane sugar-, and glycogen-splitting), .and the proteolytic enzymes, yeast-juice contains an oxidising, .a reducing, a fat-splitting, a hydrogen peroxide-splitting, and a clotting enzyme. The vitalistic view of alcoholic fermentation and of the other changes brought about by the yeast cell must be given up in the light of Buchner 's and Fischer's discoveries, for it has been established that they can take place quite independ- ently of the living cell. At the moment this has no direct or important bearing upon the fermentation industry. The particular action of selected species of yeast on the individual substrata, wort, must, etc., is undoubtedly a product of the .action of the complicated forces in the cell, of which only a ENZYMES OF YEAST. 255 small number are known. To secure the results required in practice, it is still necessary, therefore, to make use of living growths. The Enzymes of Yeast. In addition to the general sketch included in the foregoing chapter on the enzymes occurring in fungi, a few particulars must be given regarding their special relations to yeast. Invertase is commonly found in all species. It is prepared by treatment of the yeast with alcohol or ether, or by drying and heating to 100° C. The enzyme is then extracted with water or glycerine, and precipitated with alcohol ; the pre- cipitate is afterwards dried. It hydrolyses cane sugar, which is split into one molecule of glucose and one of Isevulose, and it is only after this decomposition that yeast can ferment sugar. The optimum temperature of invertase is about 55° C., and it is destroyed at 75° C., but in a dry state it can stand much higher temperatures. It is resistant to small doses of antiseptics, and to vigorous action of the proteolytic enzyme of yeast. Its action is increased by very dilute acids, but considerably diminished by treatment with alkali. Maltase also occurs in many yeast species. It decomposes maltose into two molecules of glucose. It has its optimum at 40° C., and is destroyed at 55° C. According to Bokorny and others, it is much more sensitive to chemical reagents than invertase. Melibiase. — By careful treatment of raffinose with dilute acid, it is hydrolysed into laevulose and melibiose. The latter is decomposed by melibiase, which, according to Bau, is usually present in bottom-fermentation yeast, but is often absent in top-fermentation yeast. Lactase decomposes lactose into one molecule of glucose and one of galactose. It is only after this enzyme has reacted that lactose can be fermented. It has been found in a number of yeast species. According to van Laer, P. Lindner and others, several yeast species are capable of fermenting dextrin. It is assumed that the fermentation is preceded by a similar hydrolysis to 256 MICRO-ORGANISMS AND FERMENTATION. those previously mentioned. Such an enzyme (amylase) has been detected in a few moulds. Amongst the enzymes found by Buchner and Hahn in yeast-juice, the proteolytic enzyme or endotryptase plays an important part in the life of the cells, especially during the so-called auto-fermentation. Its presence was mentioned in the early literature of the subject. Its optimum is at 40°- 45° C., and it is completely destroyed by one hour's heating at 60° C. In a dry condition it is more resistant. It can withstand the action of weak antiseptics, and weak acids. Neutral salts react favourably. Saccharose, even in a 5 per cent, concentration, restricts its activity, and in a 35 per cent, concentration it is completely arrested. Will examined a series of yeasts, and proved that the rate at which they liquefy gelatine is variable, and that those' that act rapidly are also- those that require a free supply of oxygen. Reducing enzymes are also found in yeast cells, and amongst these must be classed the enzyme which converts sulphur into sulphuretted hydrogen.* According to Nastukoff, Osterwalder, Schander, Will, and others, it occurs in very different degrees of activity in the various yeast species. A clotting enzyme in yeast has been detected by Rapp. The Action of the Saccharomycetes and similar Fungi on Carbohydrates and other Constituents of Nutritive Liquids. Diseases in Beer. The first decisive proof that species of Saccharomyces may produce very different reactions on the nutritive liquid was given by means of pure cultures of yeasts prepared by E. C. Hansen in 1883, and afterwards by the author. * Both in beer and wine a formation of sulphuretted hydrogen occurs (in the latter case called " Bockser "), more particularly at the end of the fermentation.. As both the grapes and the wine and beer casks are treated with sulphur, it can find its way into the liquid, but even in the absence of free sulphur or sulphur compounds, the albuminoids of the nutritive liquid and the contents of the cell protoplasm ir>ay provide material for the formation of sulphuretted hydrogen. Certain experiments appear to indicate that a diseased condition of culture yeasts- may be connected with this fact. DISEASES IN BEER. 257 Hansen's epoch-making researches on disease yeasts proved that amongst the wild yeasts there are groups which bring •about detrimental changes in beer, whilst others proved to be harmless. Amongst the former there are some which impart a bitter taste and disagreeable odour to beer (Sacch. Pastorianus I.), usually without producing turbidity, whilst others (Sacch. Pastorianus III. and Sacch. ellipsoideus II.) •only fully develop their activity at a late stage of the secondary fermentation, and then make the beer turbid. This effect is •due to the abundant yeast deposit formed a comparatively short time after the finished beer has been drawn off, which rises at the slightest movement of the liquid. These disease yeasts cannot produce turbidity if they only come in contact with beer at the end of the principal fermentation. It is possible, however, if the beer comes in contact with the two species after storing, that an infection with young cells of S. •ellips. II. may produce turbidity. The disease yeasts which influence the odour and flavour of beer are only of importance when they occur at the beginning of the principal fermentation. The chief danger lies in the pitching yeast. Weakly fermented beer is much more liable to attack than other beer. Hansen's observations on the disease yeasts have been confirmed and extended by Gronlund, Will, and others. Becker made the interesting observation that certain wild yeasts, which impart .a bitter taste to beer, are capable of influencing the attenuation when mixed with culture yeasts. The fermentation is increased and in certain cases extends over a longer period than with the pure culture yeast. Wild yeasts can also bring about disturbing effects in top-fermentation breweries. For instance, according to de Bavay, the " summer-cloud " of Australian beer is caused by a Saccharomycetes, which causes turbidity, and imparts a bitter and slightly acid taste. In English high- fermentation beers the author found yeasts of the Saccharo- myces anomalus type which produced turbidity ; in weakly- fermented, Danish high-fermentation beers, Torula species having similar properties occur. Similarly van Hest found species of Torula producing turbidity in top-fermentation Dutch beers. Chapman found that Sacch. Past. I. occurs in English beer, and gives the well-known bitter taste known as 17 258 MICRO-ORGANISMS AND FERMENTATION. " yeast bite." Frew observed that the " stench " in English beers, which have undergone secondary fermentation, due to sulphuretted hydrogen or a similar substance, is derived from a special wild yeast, Sacch. foetidus I. It is well known in practice that 8. ellips. II. and other species can produce- diseases even when the beer is first infected in the storage casks, transport casks, or bottles. Reference must be made to the fact that mixtures of culture yeasts, each capable of producing a good product, may, according to Hansen, produce diseases in beer. By the use of mixtures for the pitching yeast it was found that the species present in smaller quantity rendered the beer more liable to turbidity, in comparison with fermentation carried on with the leading species alone. Even, when the two species were separately applied, and the beers mixed for the first time in the storage casks, similar phenomena were observed. Pichi has found species producing disease in wine. In the storage casks of lager beer, Lafar found a budding fungus of the Mycoderma type, which produced acetic acid. Just as the moulds react differently upon various carbo- hydrates, so it has been shown by the exact researches of Hansen and others that the yeasts exhibit pronounced char- acteristics. In addition to the true Saccharomycetes, Myco- derma cerevisice, Sacch. apiculatus, the Torulas, and Monilia,. are reviewed in the following paragraphs : — Hansen's six Saccharomycetes (Sacch. cerevisice I., Sacch.. Pastorianus I., II., and ///., Sacch. ellipsoideus I. and II.)- behave as follows : — They all develop invertase ; they convert saccharose into invert sugar and ferment the latter ; they ferment maltose and dextrose, but not lactose. All the bottom yeasts used in practice react similarly with these four sugars. Sacch. Marxianus, Sacch. iMdwigii, and Sacch. exiguus do not ferment maltose and lactose ; they invert saccharose and ferment nutritive solutions of invert sugar and dextrose. Sacch. membrancefaciens and Mycoderma cerevisice possess no inverting enzyme, and do not ferment any of the four sugars. Sacch. apiculatus does not invert saccharose, and of the ACTION OF FUNGI ON CARBOHYDRATES, ETC. 259 four sugars it only ferments dextrose. It only induces, there- fore, a feeble alcoholic fermentation in beer-wort. Amongst the Torulas there are many which do not secrete invertase, are incapable of fermenting maltose, and only yield about 1 per cent, by volume of alcohol in beer-wort. Other species invert saccharose. In nutritive dextrose solutions the different species induce a more or less vigorous fermenta- tion. Monilia Candida, although possessing no inverting enzyme soluble in water, ferments saccharose, maltose, and dextrose. It ferments beer-wort, but at ordinary room temperature it only yields the higher percentages of alcohol at a much slower rate than the Saccharomycetes. In milk, various budding fungi have been found. Of these, Grotenfelt and the author have described certain Saccharo- mycetes ; Duclaux, Adametz, Kayser, and Beijerinck several non-Saccharomycetes. They all decompose lactose. Fermi found that certain red and white yeasts exercise a diastatic action. Morris arrived at similar results in experiments with pressed yeast. If we now review all these different properties of the Saccharomycetes, we shall see that they fall into two groups :— I. Those which possess an inverting enzyme and induce alcoholic fermentation. This group is further subdivided into :— (a) Those which not only ferment saccharose and dextrose, but also vigorously ferment maltose (the six species first described by Hansen, and the yeasts employed in the brewing industry) ; (b) Those which ferment saccharose and dextrose, but not maltose (Sacch. Marxianus, Ludwigii, and exiguus). II. Those which do not possess any inverting enzyme, and do not induce alcoholic fermentation (Sacch. membrancejaciens). The budding fungi which do not form endospores (non- Saccharomycetes) show the most varied characters with refer- ence to the properties of inversion and fermentation. I. The great majority do not ferment maltose. Many of these induce a more or less vigorous fermentation in solutions of dextrose and invert sugar. Some Torulas invert saccharose. 260 MICRO-ORGANISMS AND FERMENTATION. and many possess no inverting ferment (Mycoderma cerevisice, Torulas, and Sacch. apiculatus). II. The Torula novae carlsbergice, and a few of the species isolated by Will, ferment maltose. One species (Monilia Candida) resembling Torula ferments maltose, as well as both saccharose and dextrose. It contains no inverting enzyme soluble in water. The lactose-fermenting Saccharomyces and Torulas demand a special classification. When we consider the behaviour of these fungi in the fermentation industries, it is at once seen that it is only in the genus Saccharomyces that species occur which rapidly and vigorously ferment maltose. The yeasts for breweries and distilleries must, therefore, be selected from the true Saccharo- mycetes. The non-Saccharomycetes, the great majority of which cannot ferment maltose, are scarcely capable of playing any important part in these industries ; on the other hand, they may be employed in the manufacture of wines from grapes, currants, and other fruit, since several are able to induce just as vigorous a fermentation in solutions of dextrose and invert sugar as the Saccharomycetes. It is, therefore, of the utmost importance that a suitable species should be selected. Amongst the carbohydrates synthetically prepared by E. Fischer, isomaltose may be mentioned. For some time it has played a great part in the literature of the subject. As is well known, he discovered this sugar in the products of the reaction at a low temperature of hydrochloric acid on grape sugar, and it received the name of isomaltose because it appeared to have a constitution similar to that of maltose. The sugar is known only in the form of an osazone. Even the existence of Fischer's isomaltose has been questioned, because it was regarded as impure maltose. By a fresh investigation, however, Fischer succeeded in proving biologically that this sugar is sharply distinguished from maltose by the fact that isomaltose is neither fermented by fresh yeast nor split up by the enzymes of yeast, and he asserts that it is only possible to differentiate with certainty between the two sugar species in this way. ACTION OF FUNGI ON CARBOHYDRATES, ETC. 261 The different action of the Saccharomycetes on the same nutritive liquid (wort or must) under identical conditions, has been further studied by Borgmann, Amthor, and Marx. According to Borgmann, the chemical reactions brought about in wort by the two Carlsberg bottom yeasts, No. 1 and No. 2, show a striking difference. These two species — which had been in use for some time in the fermenting room, and were still practically pure — were employed for pitching two fermenting vessels containing wort from the same brew ; the fermentation took place under conditions which enabled a true comparison to be made, and the resulting beer was stored as usual. The differences in the chemical reaction were especially noticeable in the proportion of free acid. Thus : — No. 1. No. 2. Free acid (calculated as lactic acid), . 0'086 0'144 per 100 c.c. Glycerine, 0'109 0'137 „ As a result of these experiments, Borgmann pointed out that the ratio between the alcohol and glycerine in these two beers differs from that previously found in beer, the ratio obtained from previous analyses being : — Alcohol. Glycerine. Maximum, 100 5*497 Minimum, 100 4-140 Whilst the Carlsberg beers gave the following ratios :— Alcohol. Glycerine. No. 1, 100 2-63 No. 2 100 3-24 It must be admitted, as Borgmann observed, that good beer may be produced by a method not open to criticism, in which the ratio of alcohol to glycerine may sink below the previously-admitted minimum. A series of the eight different species of Saccharomyces, and amongst them six culture yeasts, all in absolutely pure cultures, were examined by Amthor with reference to their chemical action on beer-wort. The fermentations were conducted in Pasteur flasks of one litre capacity under identical conditions, and formed two series, one corresponding to the primary fermentation in the brewery, and the other to the secondary 262 MICRO-ORGANISMS AND FERMENTATION. fermentation. The amount of alcohol, extract, the specific gravity, attenuation, glycerine, nitrogen, reducing substance, and degree of colour, were determined in the fermented worts. The tables show palpable differences in the chemical reactions brought about by the different species. The percentage of alcohol varied within the limits of 4-34 and 6-02 by volume (3-55 to 5-94 at the end of the primary fermentation), the extract from 8-27 to 11-23 (8-49 to 11-61 at the end of the primary fermentation), the attenuation from 36-7 to 53-3 (28-8 to 52-1 at the end of the primary fermentation) ; the percentage of glycerine showed very striking differences, and fluctuated between 0-08 and 0-15; likewise the amounts of nitrogen, of reducing substance, and to some extent even the colour intensity, showed considerable variations. Hiepe drew some interesting parallels between the behaviour of a number of culture yeasts and wild yeasts with regard to the sugars. For this purpose he instituted fermentations in sugar solutions containing yeast decoction. He took out the first sample five minutes after the fermentation has been induced, and then fresh samples every day, till the fermenta- tion had subsided. In each sample the amount of (1) inverted sugar, (2) fermented extract, (3) fermented dextrose, and (4) fermented laevulose was determined. In these four respects well-marked, specific differences developed in the course of a day. Thus, in five minutes an English high-fermentation yeast had inverted 1-95 per cent, sugar, whilst a low-fermenta- tion yeast from the author's collection had inverted 58-85 per cent. A complete inversion of the sugar with two low- fermentation brewery yeasts took place in the course of about twenty-four hours, whilst in the case of Sacch. exiguus this reaction required eleven days ; for the other species the time required lay between these two limits. The detailed tables, given by Hiepe, show that the successive fermentation of the total quantity of extract, as well as that of the two sugars, takes place according to a scale peculiar to each individual species. A glance at the numerous details of the experiments further shows that the fermentation of dextrose, as a rule, begins much more vigorously than that of laevulose ; but whilst the fermentation of the former reaches its maximum ACTION OF FUNGI ON CARBOHYDRATES, ETC. 2fi3 • on the second day, the fermentation of laevulose does not reach its highest activity until later, in some species even as late as the fifth day ; by slow degrees the proportionate amounts of sugar fermented approach each other, and finally both sugars disappear simultaneously. The yeast species also behave differently with regard to the amount of acid produced in the nutrient liquid. From this point of view Prior examined the fermentation products of a number of brewery yeasts and wild yeasts in hopped wort, and found that the amounts of acid formed varied from 4-7 to 10 c.c. of decinormal caustic soda solution per 100 c.c. of fermented wort ; the fixed organic acids varied from 2-1 to 5-4 c.c., the volatile organic acids from 2-1 to 5-8 c.c. The evidence shows that, in culture yeasts, the amounts of fixed organic acids usually exceed those of volatile acids, whereas in Hansen's wild yeast species (Sacch. Pastorianus I., II., and ///., and S. ellipsoideus I. and //.) the reverse is the case, the volatile acids exceeding the amount of the fixed acids ; this is specially the case with Sacch. Pastorianus I. A large number of Saccharomycetes occurring in must — absolutely pure cultures of which were prepared by Hansen's method — were examined by Marx in 1888 both botanically and with reference to their chemical action on the nutritive liquid. They showed distinct differences in fermentative power, and in their capacity for producing volatile substances which impart a special bouquet to wine, and finally in their power of resistance to different acids and to high temperatures. Amthor subsequently investigated a number of absolutely pure cultures of wine yeasts, and detected typical differences with regard to the time taken by the fermentation, as well as in the chemical composition of the wines. Similar results have also been obtained by Jacquemin, Rommier, Martinand, and Rietsch in France ; Miiller-Thurgau in Switzerland ; Wortmann and Nathan in Germany ; Mach and Portele in Austria ; Forti and Pichi in Italy ; some of the comparative •experiments conducted by these authors having been carried out on a large scale. The most thorough and extensive investigations into the different behaviour of wine yeasts with regard to must are due 264 MICRO-ORGANISMS AND FERMENTATION. to J. Wortmann. He states, as the general upshot of his investigations, that the differences in the divers types of genuine wine yeast are sometimes so great that they can be detected merely through the chemical analysis of the products- of fermentation or metabolism ; in other cases, however, they are of such a kind that we can only convince ourselves directly of their presence by their odour and flavour. Every type of yeast shows some individual peculiarity more or less charac- teristic in its action on any must, regardless of its nature or origin. The number of yeast cells formed in a given must, apart from the nutritive contents of the must, depends on the specific power of propagation of the chosen type ; on the other hand,, it is in itself independent of the origin of the particular must. In any given must, whether it be an excellent or an indifferent nutrient medium for the wine yeast, one yeast type will multiply more freely than another. An extensive comparison of the amount of extract contained in a number of wines fermented with three different yeast species showed that in the same must the " Wiirzburger " yeast consumed the smallest quantity of extract ; next came the " Johannisberger," whilst the " Ahrweiler " yeast used up the largest amount of extract, and, accordingly, left the smallest residue in the wine. The specific activity of wine yeasts is clearly brought out in the formation of glycerine, which has a predominant in- fluence in determining the flavour of wine. The three species mentioned above were compared in a large number of musts of different origin, and, on the average, the Wiirzburger yeast formed more glycerine than the other two ; of these, the Johannisberger yeast was superior to the Ahrweiler, which,, as already stated, ferments the extract most vigorously. The difference observed between the chemical activities of these species was emphasised by the fact that the Wiirz- burger yeast had multiplied most feebly. This example, amongst others, shows that the alcoholic fermentation of must is independent of the formation of glycerine. It isr therefore, impossible to establish a definite relationship between the contents of glycerine and alcohol in wine. ACTION OF FUNGI ON CARBOHYDRATES, ETC. 2(55 Both the percentage of nitrogen and of ash proved to be different in wines fermented with the three kinds of yeast. The acid content was highest in the wines fermented with Wiirzburger yeast, and practically equal for the other two kinds. In accurate comparative experiments, a large number of species differed widely with respect to the amount of alcohol produced in the liquid ; those yeasts having the shortest fermentation period yielded the smallest percentage of alcohol, and conversely. With regard to the bouquet in wine, Wortmann and Miiller- Thurgau distinguished between that which originates in the grape — " grape bouquet " — and that which is produced as a result of the activity of yeast — " fermentation bouquet." In some wines the grape bouquet is so strongly developed that the fresh bouquet formed by the action of yeast on some of the substances present in the grape plays only a subordinate part in determining the character of the wine. In other wines where the grape bouquet is not so strongly developed the fermentation bouquet may have a great influence on its character. The result of applying a pure culture of a wine yeast will, therefore, differ with different wines. In the first group of wines the influence must be regarded as an indirect one in that the pure culture suppresses foreign organisms, which might mask the true grape bouquet, whereas the fer- mentation bouquet will have but little influence on the wine. In the fermentation of such wines the best results will be obtained by using yeasts from the locality. It is quite other- wise with the second group of wines. With such musts possessing no outstanding characteristics, the application of specially selected yeasts will exercise a directly favourable influence on the flavour and on the whole character of the wine. The long experience of the author has fully established the fact that both the quantity and the variety of the fermentation bouquet may be increased by such means. The fermentation bouquet differs with each yeast species, but neither its quality nor quantity stands in direct relation- 266 MICRO-ORGANISMS AND FERMENTATION. ship to its fermentative power, but a certain uniformity appears to exist among the yeast species in their action on any given wine. A complete alteration of the fundamental character of a wine by the use of pure cultures of wine yeasts is impossible, but by a suitable selection of species and the right method of application important advantages can be secured in every •case. The same applies to the different kinds of fruit wines, which may be fermented both with grape wine yeasts and with pure cultures of the species growing on the respective fruits. Kayser compared the chemical properties of several types of wine yeast, and found that the formation of volatile acids at higher temperatures differed for each species. Thus, with a rise in temperature, the quantity of these acids increased in one species and decreased in another. Forti, basing his conclusions on comparative experiments with wine yeast, has drawn attention to the existence of typical differences in the fermentative power of the species, power of resistance to high temperatures, and both quantity and quality of the nitrogenous constituents required in the nutritive liquid. According to his view, there is a well- marked distinction in the character of the fermentation produced by yeasts in the primary or vigorous fermentation, on the one hand, and those of the secondary or quiet ferment- ation on the other. The numerous investigations carried out continuously since 1884 in the author's laboratory with pure cultures of yeasts, as applied in the various branches of the fermentation industry, have furnished ample opportunit}'- for collating experience relating to the chemical activity of species, and to their respective powers of retaining their peculiarities intact during storage, a matter of importance to every branch of the industry. Numerous instances have been met with in which even feebly-pronounced characters, manifested through taste or smell, remain inherent after several years' preservation of the growth ; they may be restored by suitable development of the culture under favourable circumstances. PRODUCTS OF ALCOHOLIC FERMENTATION. 2G7 The Products of Alcoholic Fermentation. It has already been stated that saccharose can only be fermented after the intervention of invertase has caused absorption of water and decomposition into glucose and Isevulose. The same holds good with regard to maltose, which is split up into two molecules of glucose. In a similar way lactose is split up by certain species of yeast before alcoholic fermentation takes place. Other sugars (the hexoses) are directly fermentable. Of these, the commonest is glucose or dextrose (grape sugar), which is fermented by every known species of alcoholic yeast. This also applies to Isevulose or fructose, which is so widely distributed in the vegetable kingdom, and usually occurs in conjunction with dextrose. Invert sugar is a mixture of the two. The principal product of fermentation is alcohol, more particularly ethyl alcohol. In 1815 Gay-Lussac first estab- lished the true character of the reaction when he showed that cane sugar (more correctly grape sugar) gave 51-11 per cent, of alcohol and 48-89 per cent, of carbon dioxide on fermentation. Pasteur showed that by-products always occur, and that part of the sugar is utilised for the nutrition of the yeast, so that it is never possible to convert the whole amount of sugar into alcohol and carbon dioxide. Pasteur's results were 48-3 per cent, of alcohol and 46-4 per cent, of carbon dioxide, which agrees well with recent determinations, showing that practically equal quantities of alcohol and carbon dioxide are formed. It has already been stated that in all probability lactic acid is formed as an intermediate product in the fermentation, as shown by Buchner and Meisenheimer's work on the action of yeast juice. Rayman and Kruis proved that beer which had been subjected to fermentation with absolutely pure cultures, and kept for some years at the usual temperature, contained only ethyl alcohol, but when air was introduced and the yeast formed a film, the alcohol was decomposed into carbon dioxide and water. Glycerine occurs in varying quantities, and, according to Wortmann and Laborde, this does not depend entirely on the 268 MICRO-ORGANISMS AND FERMENTATION. decomposition of the nutritive liquid, but more particularly upon the yeast species. Its production is favoured by a high temperature of fermentation, and by a greater sugar concen- tration, or in general by the use of a rich nutritive fluid. It is almost impossible to give limits for the proportion of alcohol and glycerine. In wine fermentations the formation of glycerine varies usually from 2-5 to 14 per cent, of the amount of alcohol produced, whereas in beer it represents only 1-65 to 4-3 per cent, of the alcohol. By fermenting saccharose with zymase Buchner and Rapp obtained even smaller quantities of glycerine. Succinic acid is another by-product which varies in quantity. According to Rau, the quantity increases with increasing temperature, and apparently the composition of the nutritive fluid has no influence on the result. Lactic acid is always found as a by-product in fermentations carried on in the absence of living cells. Prior's detailed researches prove that the different races of yeasts produce very varying quantities of volatile and non- volatile acids. He found that acetic acid was a constant product of fermentation. Formic acid is produced, according to Rayman and Kruis, by the oxidising action of yeast on the albuminoids of the- nutritive liquid. Aldehyde (acetaldehyde) also occurs regularly and must be regarded as an intermediate product between the fatty acids and the alcohols. Rayman and Kruis proved that, especially in the case of distillery yeasts, considerable quantities are formed when free access of air is permitted, and the surface of the fermented liquid is then covered with a film of yeast. They assume that acetaldehyde is produced by oxidation of ethyl alcohol. Methyl alcohol, often found in bacterial fermentations, may also be developed during a yeast fermentation, especially by the fermentation of glucosides present in fruit juices. It also appears possible that propyl and butyl alcohol may be produced in a normal alcoholic fermentation, the former from lactic acid. Especial interest is attached to the presence of amyl alcohol (isoamyl alcohol), which forms the main constituent AUTO-FERMENTATION. 269 of fusel oil ; according to Rayman and Kruis, it is produced in larger quantities at high temperatures and in the absence of air. Amyl alcohol is freely produced in liquids containing grains which have been treated with sulphuric acid. According to Ehrlich, fusel oil is formed in the ordinary growth of yeast from leucin and isoleucin, two cleavage products of albumin. It appears to be produced also in the auto-digestion of yeasts. We must also record the production of acetic ether and other volatile and non-volatile ethers which help to impart the particular character to the fermented liquids. Auto-fermentation. Pasteur's researches indicated that yeast is capable of forming alcohol and carbon dioxide under certain conditions, even in the absence of sugar from the surrounding liquid. By boiling yeast with dilute sulphuric acid, he prepared a ferment- able sugar which he believed to be derived from the cell- wall. Salkowski has proved that in reality glycogen plays a part in auto-fermentation. As glycogen can be fermented by Buchner's yeast- juice, it is concluded that the yeast cells contain an enzyme which can hydrolyse glycogen before it is fermented. Salkowski states that by treatment with chloro- form-water glycogen is split up, but auto-fermentation does not take place. C. J. Lintner found that sodium chloride has a similar action, and in the presence of chlorides of sodium, calcium, magnesium, or ammonium no such fermentation takes place. On the other hand, sodium sulphate and mag- nesium sulphate react favourably. It is necessary, therefore, in fermentation experiments to include in the calculation the amount of alcohol and carbon dioxide produced from the yeast cells. It is not only the carbohydrates, but also the nitrogen compounds that are gradually resolved in the yeast cell. Hahn proved the presence of a proteolytic enzyme in Buchner's yeast-juice, and it is known that yeast has the power of liquefying gelatine. In auto-digestion Kutscher and Lohmann detected a number of cleavage products of the proteins, more especially guanin and adenin, and also leucin, 270 MICRO-ORGANISMS AND FERMENTATION. ammonia, etc. Their experiments were carried out in presence of toluol to prevent an infection with bacteria. The process of auto-digestion begins if yeast is kept for a long time at a high temperature without nitrogenous food. In practice, auto-digestion may take place in the manu- facture of pressed yeast where decomposition of the yeast is- frequently encountered without the occurrence of any bacterial infection. In this case, owing to lack of nourishment, the cells gradually resolve their albuminoids, and doubtless a proteolytic enzyme is simultaneously secreted. The yeast mass is then more readily exposed to infection by bacteria. Cells rich in glycogen appear to be less liable to such a decom- position. Fermenting Power ; Fermentative Energy ; Raising Power. The work carried out by yeast can be distinguished under three heads : — The activity of the enzymes ; metabolism ; synthesis of material. The activity of the enzymes is of a sugar splitting, hydro- lysing, and proteolytic character. As proposed by Neumann Wender, the fermenting power of yeast may be expressed in terms of the quantity of sugar which is split up at a given temperature in unit time by unit quantity of yeast. The fermentative energy may be defined by determining the time required within which a given quantity of sugar is decom- posed by unit mass of yeast under special conditions. The '' raising power " is a function of the carbon dioxide formed by pressed yeast, whereby the dough is raised. Carbon dioxide is developed during both true and auto-fermentation. A true and practicable determination of the " raising power " of baker's yeast can only be carried out in so far as it is possible to prepare a kind of normal dough. The Biological Relationships of Yeast. The problem of the occurrence of yeast in nature was raised as soon as its vegetable character had been established. The first researches on this question were undertaken by Brefeld in 1875, who arrived at the result that the yeasts are very BIOLOGICAL RELATIONSHIPS OF YEAST. 271 widely distributed in nature, and that their germs are present in atmospheric air, in dust, and in vegetable matter, and that their breeding places are specially to be sought in the excre- ment of herbivorous animals. Here they can exercise their fermentative power. It will be seen from what follows that this view can no longer be accepted. It is true, as the author has proved by his own investigations, that the excrement of herbivorous birds contains numerous budding fungi, and amongst them Saccharomycetes, but their breeding places must be sought in quite a different direction. In 1876 and 1879 Pasteur published complete memoirs regarding the occurrence of yeasts on grapes, and stated that they were to be found only on ripe grapes. At the same time he did not succeed in answering the important question as to where the yeast fungi found a habitat during the remaining part of the year. He expressed the view that Dematium pullulans, which is found everywhere on grapes ,. lives through the winter in the form of thick-walled and coloured resting-cells, and produces new yeast cells in the following summer, but it is now recognised that these budding cells are not wine-yeast cells. On the other hand, it was shown by the author in 1895 that other mould forms occurring on grapes, which resemble Dematium, but do not possess thick- walled resting-cells (Fig. 45), produce internal spores which develop budding Saccharomyces cells. What part these moulds play in the preservation of the yeast vegetation has not yet been determined. Great uncertainty still existed regarding the most important question as to where the yeast remained during the different seasons of the year. It was established for a single species by E. C. Hansen in 1880-81, and his further researches, of a very detailed and fundamental character, have cleared up the question for so many other species that this important phase of the biology of yeasts is now fully understood. The researches of Hansen were first carried out on the small lemon-shaped yeast-fungus S. apiculatus, which always appears in the earliest stage of wine fermentation. By a microscopical examination and culture experiments it was shown that during the summer months the organism appeared in vast quantities with the 272 MICRO-ORGANISMS AND FERMENTATION. ripening of the sweet juicy fruits (cherries, gooseberries, straw- berries, grapes, plums, etc.). On the other hand, it was only quite exceptionally that they were found on the unripe fruit. As the organisms were found vigorously budding on the ripe fruit, but never, or only very rarely on other fruit, and on the leaves, branches, etc., the fact may be accepted that these ripe fruits act as a true host to S. apiculatus. This was further established by the observation that they are to be found without exception in the soil under cherry and plum trees, vines, and other fruit-bearing trees upon which the organism grows, but that they are extremely seldom found in samples of soil taken in other localities of a most varied character. The fruit falls to the ground, and the rain carries the fungus into the soil ; the problem, then, is whether it is able to winter there. The answer was obtained in two ways. First, numerous samples of soil were taken during the course of the winter and spring at these places, and in the vast majority of cases these gave a vigorous growth of the organism in wort. Secondly, cultures of S. apiculatus were placed with every precaution in the earth, and allowed to remain throughout the winter. They were removed in the spring and early summer, and culture experiments proved that the organism was alive in every sample. In this way it was established that the organism is able to winter in the earth, just as it had been previously shown that it only occurred in the soil at these particular localities. In later experiments of Hansen's, vigorous growths of the organism were placed on the surface of the soil in well-sealed Chamberland filter tubes. Three years later the contents of these tubes were introduced into sterilised wort, and a vigorous growth of the organism developed. The cycle of operations may, therefore, be spread over more than one year. It still remained to be proved whether the earth is the true habitat in winter time. This was carried out as follows : — : Hansen examined dust in a great variety of places from January to June, and also the dried fallen fruit of many trees, and lastly, many kinds of excrement. These analyses gave a negative result, and thus furnished the desired proof. The soil under the particular fruit trees must, therefore, be regarded BIOLOGICAL RELATIONSHIPS OF YEAST. 273 as the true winter habitat of the fungus. It preserves its usual appearance throughout the long winter time, and is then carried up into the air by the combined agency of insects and of wind, and by these means of transport it is distributed from fruit to fruit. It is obvious that during the period when a large number occur on ripe fruit the currents of air may carry the fungus to other places, and also on to unripe fruit. Hansen stated in his first memoir that the rare occurrence on unripe fruit must be due to the fact that the organism quickly dies off, partly through want of nourishment, and partly through the drying up of the cells. He subsequently proved by experiment the correctness of this view. He distributed both old and young cells in water, and placed them either in a thin layer on an object glass or on a tuft of thinly spread cotton- wool ; thus allowing evaporation to go on while the cells were pro- tected from the sun. In less than twenty-four hours the whole of the cells were killed. It is quite obvious that the individual cells spread over the surface of unripe fruit are ex- posed to more unfavourable conditions than in his experiments. If, however, thicker layers of the cells are covered by cotton- wool or filter paper, they remain living just as they do in the soil for a long time. Thus they live for more than eight months in filter paper. It was then possible for Hansen to demonstrate that the greater number of yeast species must pass through a similar cycle in nature. Their most important breeding places are the sweet juicy fruits. Their winter habitat is the soil, and they are carried by wind, rain, insects, and other creatures on to the fruit. They then multiply once more on sweet fruit, and obviously more particularly where the juice oozes out from the fruit. Hansen further found that these yeast species often occur in the ground at places far removed from orchards, where 8. apiculatus can no longer be found. Miiller-Thurgau arrived at the same results as Hansen with regard to 8. apiculatus during an examination of the wine species. He found that grapes are their chief breeding places, and that their presence may be distinguished in the soil throughout the year. On the other hand, they seldom occur 18 274 MICRO-ORGANISMS AND FERMENTATION. in the air. He further proved that the wine-yeast cells may occur in soil at a depth of from 20 to 30 cm. In 1897, Wortmann's researches, recorded in his work on the preparation of wine, were directed to determining the behaviour of wine yeasts in soil at different seasons of the year. The experiments were continued for two years, and consisted in taking samples of soil every fourteen days from one and the same part of a vineyard. By sowing the soil in sterile must, he obtained an idea of the vegetation. His main observation was that directly after the vintage (in November and also in December) the samples of soil developed a growth of yeast in must so rapidly that no other fungi were able to develop. In January, February, and March also a develop- ment of yeast was always obtained from the samples, but it occurred more slowly. In the spring and summer the con- ditions were always less favourable, and a longer period elapsed before fermentation began. Some samples, indeed, gave no yeast development, but only other organisms. The least favourable conditions were observed in the late summer (August and September), but from the time the grapes began to ripen, a vigorous growth was again observed in the flask. Wortmann concluded that while the wine yeast remains in the soil its nutritive state is of the greatest importance. The vegetation is most vigorous during the early stages, when it has been enriched with cells fresh from the grapes — i.e., in the autumn, winter, and the beginning of spring — whereas during the summer, the most favourable period for vegetation gener- ally, its power is constantly diminishing, the cells having drawn upon their reserve material. According to this view, the yeast is dependent upon its own body-material during its habitat in the soil. The lower temperature ensuing after the vintage allows metabolism to go on so slowly that it enables the cells to maintain life throughout the winter and spring. At the beginning of summer, with increasing temperature, the cells rapidly assimilate the remainder of the reserve substance, and consequently die off slowly. The cells that are still alive are weakened, and the samples of soil, therefore, give a very feeble growth in the flasks. The cells are con- tinuously carried by insects and other means from the soil to BIOLOGICAL RELATIONSHIPS OF YEAST. 275 the vegetation, and those which light upon the grapes when they are ripe find full nourishment, and produce a new vigorous growth. Wortmann was able to confirm Miiller-Thurgau's observation that no wine yeasts are to be found in a vineyard which has not been worked for a long time ; they are gradually killed out by exhaustion. In those wine districts where the culture of grapes has been continued for centuries, the yeast oells which are brought from the soil when the grapes are ripe adapt themselves more and more to the excellent nutrient material, and in this way specially good races of wine yeasts .are developed. In 1903 and 1905 Hansen obtained results which differed from those of Wortmann in one important point relating to the condition of yeast cells during their abode in the soil which the latter regarded at a state of starvation. This new and very detailed research led to the result that elliptical and Pastorianus forms of Saccharomyces (but not S. apiculatus) are to be found throughout the year in all kinds of soil in the neighbourhood of {Copenhagen. Their number diminishes, however, at a distance from the orchards. A similar condition of things was found by examining soil in the Harz Mountains and in the Alps. The soil in vineyards is specially rich in yeast species, and the greater the elevation the smaller is the number of organisms found. Above a certain height no organisms are found. The reason for this wide distribution lies, as Hansen showed, in the fact that, in addition to the normal breeding places for yeast, there are others which he called secondary breeding places — e.g., aqueous extracts from fruit and other vegetable matter and from excrement. In the former, the cells multiply very rapidly, in the latter, feebly or not at all. If yeast cells from sweet juicy fruit and from the upper layers of soil, where they form spores, are carried by insects or by wind to distant places, they may, unlike S. apiculatus, maintain life even when dried, on account of their greater power of resistance. In the same way they can multiply more readily in soil in the aqueous extracts already referred to, and may even preserve life for a longer period in presence of nothing but moisture. Thus the fact is fully explained that the larger species occur much more widely distributed throughout the soil than the 276 MICRO-ORGANISMS AND FERMENTATION. small lemon-shaped wine yeasts. S. anomalus and 8, mem~ brancefaciens are especially resistant to the effect of drying. They are, therefore, found at great distances from the primary habitats. In this way the fact may also be explained that fewer yeast species are sometimes found in the soil of vine- yards than in the neighbouring meadows. The cells in the vineyards are dried up and killed, whereas in the meadows where the cells are protected from drying, life is maintained, and the cells multiply. In such places cells also occur during the hot season of the year, and here their propagation goes- on most vigorously. Where the ground is subject to drought the variation brought about in the course of years may be altogether extraordinary. The soil must, therefore, be considered the chief habitat of yeast at every time of the year. They are carried from the earth by means of wind and rain, as well as by the action of insects and other creatures, to the sweet juicy fruits, where they multiply vigorously ; a few fall to the earth again, whilst others are carried to secondary places of incubation. When the fruit is ripe the wild yeasts thus strongly developed find their way into the fermentation industry. It is only if they are allowed to remain, to multiply, and to obtain a secure footing, that they are capable of bringing about any disturbance in the industry. Otherwise they are immediately suppressed by the large quantity of the culture yeast added to the nutritive liquid. During their development on grapes and other juicy fruit the yeast cells compete for nutrition with many other organ- isms, including bacteria and moulds. These observations led Wortmann to adopt the view that the true importance of alcoholic fermentation is biological. Most of the competitors of yeast can multiply much more rapidly, and would soon suppress it if no means existed for restricting their growth. This means is supplied by the alcohol produced by the yeast cells, whereby they are able to poison their enemies. Wort- mann showed how the poisonous action of alcohol is apt to- support yeast in competition with other organisms. During the early stages of the development of yeast in must a surface growth of various organisms can be observed. Amongst these the small apiculate yeast is especially prominent, and this soon BIOLOGICAL RELATIONSHIPS OF YEAST. 277 brings about a fermentation. The alcohol so formed sup- presses most of the moulds. The true wine yeasts now gradually begin to develop, and simultaneously the develop- ment of wild yeasts, of bacteria, and of the Dematium species, ceases. As soon as the alcohol content rises above 4 per cent., as a result of the activity of the true yeasts, S. apiculatus is suppressed, and the wine yeasts immediately take command of the field to such an extent that, in an ordinary microscopical examination, nothing but their cells can be observed. The most powerful alcohol - formers amongst the yeasts again gradually supersede the weaker species. Temperature plays a great part in the life of yeast cells, and Hansen has made use of this relationship as one of the most important means for characterising the species. In 1883 he proved that both the spores and vegetative cells of different species possess different powers of resistance to heating in water. In this respect the spores are more resistant than the vegetative cells. In such determinations the condition of the cells has a marked influence, and the result depends largely upon their age. Thus the two-day-old cells of S. ellipsoideus II. grown in wort at 27° C. were killed on warming to 56° C. for five minutes in sterilised distilled water, whilst cells similarly prepared, but two and a half months old, were heated to 60° C. for five minutes without being destroyed. Ripe spores of this species, developed at 17° -18° C., and partially dried for eight days at the same tempera- ture, withstood heating for five minutes at 62° C., but not at 66° C. The vegetative cells of S. cerevisice I. were killed by five minutes' heating at 54° C., and the spores at 62° C. An interesting classification of Hansen's six species in relation to any given temperature, is obtained by cultivating them in wort under conditions favouring the formation of films. Thus, if the development is carried out at 36°-38° C., the three Pastorianus species are killed in eleven days, whilst S. cerevisice I. and the two ellipsoid species remain alive. From this and similar experiments, it may be argued that the rule formerly accepted that top-fermentation yeasts can 278 MICRO-ORGANISMS AND FERMENTATION. develop at a higher temperature than bottom-fermentation yeasts has no general application. Kayser's more recent work along the same lines has con- firmed these results. He also proved that the species with- stand considerably greater heat in a dry than in a moist con- dition. Thus a yeast species isolated from pale ale was killed in a moist condition by heating for five minutes at 60°-65° C., whilst in a dry condition it withstood a temperature of 95°-105° C., and in the case of a wine yeast (St. Emilion), the corresponding temperatures were 55°-60° C. and 105°-110° C. The spores withstand temperatures 10° and even 20° higher than the vegetative cells. Vegetative cells which are derived from the heated spores- show a somewhat greater power of resistance than normal vegetative cells. This increased power of resistance is not transmissible ; by cultivation in beer - wort it disappeared entirely in the second generation. The temperature limits within which budding of cells can take place in wort were investigated by Hansen. The upper limit for S. Past. I. is 34° C., for S. membrancefaciens 35°- 36° C., for S. anomalus and 8. Ludwigii 37°-38° C., for 8. Past. II., III., and S. ell. I., II., and for S. cerev. I. about 40° C., and for S. marxianus 46°-47° C. The lower limit for each of these species is 0-5° C., with the exception of 8. cerev. I. and 8. Lvdwigii with a limit of l°-3° C. Miiller- Thurgau found that the wine yeasts that he examined are incapable of propagation at temperatures above 40° C. It is, of course, impossible to establish any one temperature that shall serve as the optimum for the growth of yeast cells, because the composition of the nutritive liquid has a greater effect than it has on other determinations. The formation of new cells in the same liquid goes on at a diminishing rate when the development proceeds at a constant temperature, because the increasing quantity of the products of metabolism and the simultaneous impoverishment of the nutritive fluid acts restrictively upon the growth, especially at higher temperatures. An approximate temperature of 28°- 30° C. is found to be favourable for the development of many species. Without doubt the species behave differently in this respect as well as BIOLOGICAL RELATIONSHIPS OF YEAST. 279 in regard to the maximum production of yeast which can be developed from a given inoculation. Many fermentations take place in the industry at lower temperatures ; indeed, in the case of bottom-fermentation breweries, very considerably lower than the optimum for the multiplication of the cells. In order that fermentation may be completed at so low a temperature within a reasonable time, and before other organisms have an opportunity of infecting the liquid, relatively large amounts of yeast are introduced, and propagation is assisted by aeration. At times the pitching yeast is first placed in a smaller quantity of the liquor at a higher temperature (about 20° C.), allowed to grow for a few hours, and the newly- formed and vigorous cells are then introduced into the cold liquor. There appears to be a tendency to forego the extremely cold fermentations once customary in many places. In dis- tilleries, where fermentation proceeds at a higher temperature, it is often necessary to take special precautions to avoid a considerable rise in temperature during the first stages of the fermentation ; otherwise the propagation of cells ceases too soon. Consequently the growth would be so enfeebled that it would be impossible to carry the fermentation to completion. It has already been stated that the composition of the nutritive fluid plays an important part in the propagation of the yeast cells. Liquids containing a large percentage of sugar have a weakening effect on the cells. According to Laurent, growth ceases in a decoction of malt germs containing 60 grammes of sugar per 100 c.c. A few species of yeast, nevertheless, appear to retain their activity even in the presence of greater quantities of sugar. The aeration of yeast, as carried out in practice, is of real importance for propaga- tion. Exact conclusions regarding this were published by Hansen in 1879. He used the cell-counting chamber, already alluded to, and found that a beer yeast grown in wort at 12°- 14° C. showed the formation of eleven cells from a single cell in sixty hours without aeration, whereas with aera- tion thirty-six cells were formed from each individual in the same time. The importance of aeration depends not only upon the fact that oxygen reacts more intensely on the indi- 280 MICRO-ORGANISMS AND FERMENTATION. vidual cells, but also upon the removal of the products of metabolism. The stirring up of the cells brought about by air bubbles brings them constantly into contact with fresh portions of the nutritive fluid. In the air-yeast factories this fact is utilised, and a considerably higher yield of yeast is obtained than in the older process (foam yeast). According to Delbriick, it is found in practice that 100 parts of malt yield 21 to 23 parts of pressed yeast in a non-aerated wort, and 30 parts in an aerated wort. He further proved that the higher yield was reached after four and a half hours' aeration. For brewery yeasts the aeration of the wort is of special importance, as the clarification is dependent upon it. In a badly-aerated wort the yeast does not readily settle out. This circumstance must not be neglected even during the growth of the pure culture in the flasks. Amongst the products of metabolism removed by aeration, carbon dioxide deserves special mention, for it exercises a restrictive effect on the multiplication of the yeast. This has been proved by parallel experiments in open and closed vessels under conditions otherwise identical. If, however, a com- parison is made between the amount of alcohol formed in the two vessels and the quantity of yeast produced, it will be found that the individual cells in the closed vessel have produced a larger quantity of alcohol than those in the open vessel. The carbon dioxide appears, therefore, to exercise a favourable influence on the fermentative power of yeast. The action of light on yeast cells is described in the general review of the physiological properties of fungi. Variations in the Saccharomycetes. Hansen's numerous investigations proved that the Saccharo- mycetes are affected in varying degree by external agents, and that it is possible by suitable treatment to bring about variations along different lines. Even the individual peculi- arities of cells in a pure culture may be of importance in this respect. Some of these changes are only evanescent. By suitable cultivation they disappear, and the species returns to its original condition. Others are more deeply seated, and VARIATIONS IN THE SACCHAROMYCETES. 281 it is only by a special treatment that the culture can be deprived of its newly-acquired properties. In certain cases, it is found impossible, even after years of methodical treatment, to cause a growth to revert to its original state. 1. The times given for the appearance of the first indication of spores are based upon the understanding that the growth has been cultivated at 25° C. for twenty-four hours in wort. In 1883 when Hansen published temperature curves for his six species, he found that growths which had been developed for two days instead of one, at the same temperature, developed spores more slowly and less freely than usual. If, however, they are subsequently treated in wort in the way described, the normal conditions are re-established. This forms an example of a very feebly-rooted variation. 2. In a gelatine culture, Carlsberg bottom yeast No. 1 is often found in both oval and elongated sausage-shaped cells. If a colony derived from each of the cell forms is transferred to flasks containing wort, a growth is again obtained consisting partly of oval and partly of elongated cells. Hansen's experi- ments proved that the latter when cultivated in new flasks retained to some extent the sausage-like form, and when transferred to the pure culture apparatus the growth con- tinued to show a mixture of such cells, but when the yeast was conveyed to an ordinary fermenting tun they disappeared. The variation in this case is, therefore, a more deeply seated tme. It only ceases when the yeast has been transferred through a series of fermentations. Another example is shown by a bottom yeast which, after a long period of stunted growth, had been propagated in wort at about 27° C., and formed cells with a normal appearance, whilst the growth cultivated at 7|° C. gave entangled colonies with mycelial branchings. This forms a striking example of the effect that temperature has upon the form of cells. 3. Hansen's observations of S. Ludwigii supply an illus- tration of a far-reaching change in the character of the cells. If single individuals are grown as pure cultures, growths are obtained which show a marked difference in their power of .spore - formation. By systematic selection of single cells, Hansen succeeded in producing growths which gave no spores 282 MICRO-ORGANISMS AND FERMENTATION. under the usual conditions, and conversely, it was possible to select a yeast colony derived from a cell containing spores y and by further cultivating the colony to obtain a growth which possessed the power of freely generating spores. By such systematic choice the species was divided into three forms- one distinguished by its vigorous spore-formation, another by the fact that this power had almost disappeared, and a third, which could not form spores. By frequent infections in wort the third form reverted to the power of forming spores. This took place slowly, but when Hansen transferred it to a 10 per cent, dextrose solution with yeast decoction this property was instantly restored. In other species, varieties which have lost their power of spore-formation completely, or in part, may make their appear- ance, without any known cause, both in liquid and on solid nutrient media. In some cases (e.g., S. Ludivigii) that power is restored if dextrose is added to the nutrient liquid. Similar observations regarding asporogenesis have been recently made by Beijerinck on 8. octosporus. If a pure culture of brewery yeast is developed in a wort which has not been aerated after sterilisation, it generally loses its normal " breaking " and clarifying properties, under brewery conditions, and this to a degree dependent on the species. These new variations must often be cultivated through a great many generations in ordinary brewery wort before regaining the original qualities of the species. As- aeration brings about changes in the chemical composition of the wort, it is evident that the effect on the protoplasm is- due to such circumstances. The author of this book showed in 1890 that when a. brewery top-fermentation yeast which has given a good clari- fication in practice is kept for some time in wort-gelatine at room temperatures, it tends to lose its clarifying properties for a considerable time. At the same time, it brings about a con- siderably stronger attenuation than in its original condition. As an additional instance of the effect of the chemical composition of wort in producing new varieties, we may" mention the observation, due to Hansen, that S. Pastor i- anus /., which imparts an unpleasant taste and smell to beer- VARIATIONS IN THE SACCHAROMYCETES. 283 wort, is apt to lose this power for a time if preserved in an aqueous solution of cane sugar. A similar proof of a variation in brewers' low-fermenta- tion yeast, due to the composition of the nutrient liquid, was furnished by Seyffert, who found in the case of a selected type which, after long use in breweries, had lost its good properties with regard to clarification, that it was possible to restore it to its original condition by treatment with lime. Gypsum was added either to the wort, the brewery water, or the steeping vat, and from wort prepared in this way wort- gelatine was concocted, in which the degenerated yeast growth was sown for fresh pure - cultivation. On development of the colonies in small flasks, these new growths showed true " breaking " and the power of adhering to the bottom of the flask ; the qualities thus regained were retained during the use of this yeast in practice. Another example of physiological transformation is the following : — The three species described by Hansen under the name Saccharomyces Pastorianus form a dough-like sediment under certain conditions similar to those of the other Saccharo- mycetes ; under other conditions, however, a film-like, wrinkled, or caseous sediment consisting of small lumps (Pasteur's levure caseeuse) — a sediment of very different appearance. In the latter case, the fermenting wort also assumes a charac- teristic appearance, and, contrary to what ordinarily occurs, remains bright throughout the fermentation, so that yeast flakes may be observed rising to the surface and sinking again to the bottom. If this curious sedimentary yeast is repeatedly cultivated by new fermentations in wort, it can be again transformed into the dough-like condition. Both Hansen and the author established the fact that by long storage under ice, and subsequent growth in wort, a brewery bottom-fermentation yeast exhibited top-fermenta- tion phenomena, which, however, by continued pitching gradually but entirely disappeared. Similar observations have been made by Will. We also find a transitory physiological transformation in film-formations of the Saccharomycetes. 4. In 1889, Hansen published the results of a series of 284 MICRO-ORGANISMS AND FERMENTATION. •experiments which were undertaken with the hope of dis- covering the conditions causing variation, and of experi- mentally bringing about the formation of new races, and if possible new species, He has since published additional work on the subject. (a) He found in the case of typical Saccharomyces that when their cells were cultivated in aerated wort * at a tem- perature above the maximum for their spore-formation, and near the maximum for their vegetative growth, they were affected in such a manner that they lost their power of forming spores and films (Asporogenesis). This was also true of the innumerable generations successively formed in new cultures under the most varied conditions. The starting point was always a growth which showed not the slightest trace of asporogenous cells. For example, it may be noted that 8. Past. I. loses its power of forming spores by treatment at 32° C. In the case of the wine yeast, Johannisberg II., this occurs at 36° C. In the seventh culture of S. Past. I. all the cells were asporogenous. Hansen succeeded also in bringing about a transformation by cultivation on solid media. Such asporogenous growths were formed in the case of S. Past. I. on wort-gelatine at 32° C., when inoculations were made at shorter intervals, as is commonly the case when liquids are used. In some of the species treated in this way, it was also observed that they yielded a more abundant crop of yeast in wort-cultures, but a slower fermentation. This was, for instance, the case with Carlsberg low-fermentation yeast No. 2. The newty-formed variety attenuated more slowly and weakly than the original species ; but at the same time the clarification was better. Rayman and Kruis have shown that the cells present in films possess the power of oxidising alcohol produced during fermentation, into carbon dioxide and water. Hansen's varieties, while completely losing the power of forming films, are rendered incapable of performing this oxidising action. Thus, while a flask, containing the original species, which had developed a luxuriant film after six months' standing, showed only 1-5 per cent, by volume of alcohol, a parallel flask, which * By repeated shaking of the successive cultivations. VARIATIONS IN THE SACCHAROMYCETES. 285- showed no film-formation, contained 5-5 per cent, of alcohol — a quantity equal to that found at the end of the first month. In another series of experiments Hansen showed that the action of higher temperatures upon the cells without aeration was capable of producing radical and lasting alterations of a different kind in the nature of the protoplasm. When Carls- berg yeast No. 1 was cultivated in wort at 32° C. through eight cultures, each successive culture being inoculated from the preceding one, which had been left undisturbed until the end of the fermentation, a variety was evolved in the ninth culture which produced 1 to 2 per cent, by volume less alcohol than the original form, in wort of 14° Balling, containing 10 per cent, of saccharose. The new variety clarified better under brewery conditions, and gave a weaker attenuation at the end of the primary fermentation ; a similar behaviour was noted in the case of other species. (6) Hansen also succeeded, by cultivation in nutrient gelatine, in producing new stable varieties. Thus, two varieties of Carlsberg low-fermentation yeast No. 1, each generation of which was transferred to the surface of wort-gelatine, attained a fermentative power superior to that of the original forms. The difference is still more marked when cultures are developed from spores of the top-fermenta- tion yeast S. cerevisice I. on yeast-water gelatine. The new varieties produced 3 per cent, more alcohol than the parent form. The observations already detailed regarding asporogenesis lead to the interesting conclusion that a species can lose one of its characteristic properties as a result of external influence, and that virtually a new species is produced. In the course of Hansen's experiments on spore transfor- mations brought about by the action of temperature and aeration, it was observed that if cells of successive generations were removed many were affected even in the first growths under the new conditions ; this modification, however, is tem- porary in character ; it is only after successive generations have been allowed to develop through continued inoculation under the new conditions that the acquired characters become 286 MICRO-ORGANISMS AND FERMENTATION. •constant. It appears from this that the transformation does not depend on temperature or aeration alone, but also on the nutrition and propagation of the cells. A comparison of these different factors has, however, shown that they contribute unequally to the result. Both the nutrient liquid and the aeration are only of importance in bringing about vigorous new formations, and may, therefore, vary consider- ably in strength without materially affecting the result. This, however, is not true of temperature ; a fluctuation of a few •degrees is sufficient to prevent the variations described from •coming into existence. Hence, it follows, that temperature plays the principal part in these transformations. As previously stated, these remarkable changes are only brought about by a long-continued and violent interference with the vital processes of the cells ; they do not occur so long as development takes place in the normal manner. An example of the way in which the Saccharomyces cells retain their power of forming spores under ordinary conditions is supplied in breweries and distilleries. Here culture yeasts have existed continuously for centuries, and untold generations have been produced under conditions which would not allow, .as a rule, of this function being brought into play, and yet the power remains intact. Lepeschkin observed a well-developed mycelium formation in Schizo-saccharomyces Pombe and mellacei, which he regarded as a stable variation brought about by the alteration (mutation) of certain cells. Hansen observed a remarkable variation when young growths of S. ellips. II. and the wine yeast Johannisberg II. were preserved for a few months in Freudenreich flasks in shallow layers of wort at 0-5° C. A few cells of these bottom- fermentation yeasts gave top-fermentation phenomena. The further investigation showed that selection had taken place. The top-fermentation yeast cells remained continuously as top yeasts, the bottom-fermentation yeast cells as bottom yeasts. A similar state of things was observed during the examination of a large number of cells from old cultures of brewery bottom-fermentation yeasts. On the other hand, .similar cultures both of wild top-fermentation and of brewery VARIATIONS IN THE SACCHAROMYCETES. 287 top-fermentation yeasts yielded only a small number of cells which displayed bottom-fermentation phenomena. We are not dealing, therefore, as in the previous cases (asporogenesis) with the action of definite factors producing a transformation, but with unknown causes, and probably with sudden variations of the same kind as the mutations studied by H. de Vries. According to these researches the two physiological forms, top- and bottom-fermentation yeasts, are not independent. On the contrary, they may both occur in a growth derived from an individual cell. They can exist together in the same liquid, one or other securing the upper hand in their compe- tition and thus determining the character of the growth. Since 1887 the author, who has long enjoyed the co-opera- tion of his laboratory superintendent, H. Rafn, has treated as one of his principal problems the study of the variations of yeasts during their application in the different branches of the fermentation industry. The number of his investigations has now increased to many thousands. The difficulty in work of this character, where large masses of yeast are under in- vestigation, is to make sure that the growths observed by the separation of a certain number of cells with abnormal characteristics are real varieties of the parent cells, and have not been derived from infection by foreign species. The botanical and biological investigation can never form more than part of the examination, and must, moreover, be carried out with the utmost care. We must take refuge to a great extent in the different characteristics that are developed, partly during large scale fermentations and partly during parallel fermentations carried out in the laboratory with small quantities. For such experiments it is obvious that only yeast masses can be used which have been derived from a single cell. As a result of observations carried out during a series of years, it has been definitely established that variations do very frequently take place. They occur without any obvious cause, and on occasion they may develop in such quantities that the whole mass of yeast changes its character or " de- generates." This expression, which is used in practice, only indicates that the yeast mass in the special brewery or distillery concerned no longer suffices for the particular requirements. 288 MICRO-ORGANISMS AND FERMENTATION. It does not indicate what the true value of the yeast may be> for this branch of the industry. Thus occasionally such a yeast mass that has altered its character has produced excellent results when applied in other places where the requirements are different. A selection of a cell from the yeast mass that has not degenerated has often proved the basis for regeneration, in that the new culture possesses the properties of the original stock. A very cautious treatment of a sample of purely culti- vated yeast will throw some light upon this question. If a number of cells are separated from a yeast mass derived from a single cell, which has been in use in the industry for some time, the pure cultures from these cells will show differ- ences in a set of parallel fermentations, and sometimes im- portant differences in respect to taste, smell, and other char- acteristics of the fermented liquid ; also as regards the attenu- ation, the character of the yeast layer, etc. Varieties may, for instance, occur which produce a penetrating and unpleasant bitter flavour, but in every other respect give a result in agreement with the culture yeast. Thus it is interesting to record a case where a selected variety gave considerably more rapid clearing than the original race, whilst in every other respect, practical and biological, it was identical. In other cases the power of attenuation varied greatly. By studying a number of selected growths a series of inter- mediate forms could be detected, and by a proper selection cultures were prepared which gave the normal attenuation, in wort of the same character. A problem of great practical and theoretical importance is to decide if such variations occurring in the yeast mass in practice are constant or of a purely transitory nature. Hansen adopted the view that, as a rule, " the races prepared from industrial yeast cannot be maintained, but disappear," and that " so long as the beer yeasts are kept under brewery conditions, they only display slight alterations, which are of a transitory character." This view, however, is in contradiction to the results repeatedly obtained in the author's laboratory. Strongly marked abnormalities may occur in practice in the work of single cells, and certain of these variations prove to VARIATIONS IN THE SACCHAROMYCETES. 289 be of a stable character both when applied on the large scale, and also when stored for years in a 10 per cent, cane-sugar solution. There are variations still kept in the laboratory which after preservation in such a solution for more than ten years still retain their properties. These races, therefore, do not disappear. It follows that in the preparation of pure cultures in yeast to be applied in a brewery, a distillery, a wine fermentation, etc., we cannot reckon on dealing simply with a type ready to hand in a pure condition, but rather with a mixture of elements, often of a highly different character, even if the mass of yeast has been originally derived from a single cell. By the process of pure cultivation based upon a detailed knowledge of the special practical requirements, a form can be prepared of the required type. Such work can never be attempted at random, but must consist of systematic research carried out with rigid rules. How long such a type may be preserved in practice before it develops such pronounced varieties and in such quantities that the character of the yeast mass experiences a change, depends to a great extent upon circum- stances which are still unknown. It will be seen from all this that the principle applied in the author's laboratory in carrying out the pure culture of brewery, distillery, and wine yeasts, etc., is based on a reliable starting point, and the experience gained during the long time that has elapsed since the laboratory was instituted has only served to confirm the correctness of the author's view. The improvement of yeast, about which the author has published his views, consists in selecting cells taken from a mass of yeast which has given satisfactory results, and pre- paring growths which display the desired characters in greatest perfection. This treatment is carried on through several generations, and in each case after the mass of yeast has been applied for some time in practice. These observations have no connection with any variation in the composition of a nutritive fluid. They are simply concerned with comparative experiments with selected and absolutely pure cultures. 19 290 MICRO-ORGANISMS AND FERMENTATION. Morphology and Anatomy of Yeast Cells. Yeast Deposits. — Hansen's investigations in 1881-1883, which took the form of a direct study of the growth of a single cell under the microscope, and of growths derived from a single cell, made it possible for the first time to give exact descriptions of the different species of yeast. He proved that the shape, relative size, and appearance of the cell are not sufficient in themselves to characterise a given species, for the same species may exist in different forms under differing external influences. At the same time he established the fact that the shape may provide valuable indications, as the various species may react in a different way and with a different shape when the same influence is brought to bear. As an example of the results which may be obtained by a comparison of young deposits of yeast, the six varieties isolated by Hansen may be quoted (8. cerevisice I., S. Pastori- anus /., //., ///., 8. ellipsoideus /., //.). The growths are developed in the following manner : — The cells, after short cultivation in wort, are introduced into fresh wort, and brought to vigorous development at 25° to 27° C. in twenty-four hours. If then 8. cerevisice /. is compared with the three 8. Pastorianus species, the general appearance -is strikingly different. 8. cerevisice I. consists predominantly of large round or oval cells, and 8. Pastorianus chiefly of elongated sausage-shaped cells, but it is a very different matter if the cells of the first are mixed with cells of one of the second species. It then proves to be impossible, by simply noting the form, to distinguish between the larger and smaller oval and roundish cells of Pastorianus and many of the cerevisice cells. The two species, 8. ellipsoideus I. and //., are pre- dominantly oval and round. Sausage-shaped cells occasionally occur, and here again it is impossible, simply by studying the form, to determine the species when 8. cerevisice or 8. Pastori- anus are mixed with them. By direct measurement of the sedimentary forms it is also impossible to discriminate them. On examining pictures of these six pure cultures, it will be seen that we are dealing with three different divisions of MORPHOLOGY AND ANATOMY OF YEAST CELLS. 291 budding fungi, one of which is represented by S. cerevisice /., the second by the three Pastor 'ianus species, and the third by both the ellipsoideus species. So much and no more •can be established by a purely microscopical observation, and this only under the particular culture conditions described. The development of the yeast cell takes place through budding, a slight swelling appearing in the mother cell, which increases in size. According to Kny, budding follows with equal rapidity both in light and darkness. As soon as the new cell has attained a certain size it can form a new bud, and this process of budding continues until a group of budding cells is formed. The cells may break away from each other at an earlier or later stage, so that the group may consist of a varying number of individuals. The development of the yeast cell was observed by Mitscherlich in 1843. The daughter cell may assume a totally different form from the mother cell. This may also take place in the industrial species, including those which give fairly uniform oval cells in the large fermenting vats. For example, ordinary brewery, low-fermentation yeast may, for reasons unknown, produce cells with the appearance of Pastorianus and ellipsoideus, so that it is impossible, under the microscope, to distinguish whether such a culture yeast is infected with a foreign yeast or not. As an example of the change of form brought about by an unknown cause in the case of industrial yeast, it may be men- tioned that, by excessive treatment with air, .the air-yeast of the pressed-yeast factory alters from an oval or elliptical to a much elongated Pastorianus shape. In general, it may be stated that low-fermentation yeasts form groups containing fewer cells than is the case with top- fermentation yeasts. There are, however, many exceptions to this rule. It is impossible to indicate any universal type of microscopical picture for the two groups of yeasts, and the same holds good for the general picture of a single race of culture yeast. It is only by exactly comparable growths carried out in parallel experiments in the laboratory that it is possible to establish differences between the general appear- ance of the races. When applied in practice, so many different factors come into play that the appearance of the growth may 292 MICRO-ORGANISMS AND FERMENTATION. entirely alter its character. On these lines no starting point can be found for an analytical examination of yeast to determine its purity. A peculiar group of yeasts, the Schizo-saccharomycetes, are distinguished from others by the formation of daughter cells through division of the mother cell, a cross-section being formed in the latter. Film Formation. — It is well known that fermenting and! fermented liquids are covered with film growths. It was. first shown with certainty that Saccharomyces (in the strict sense) are able to form films by Hansen's observations on cultures derived from single cells. The universally occurring Mycoderma species form films easily and rapidly. Some also give fermentation phenomena ; others do not. Such a film is greyish on beer and wort, with a dry appearance, and in its later stages wrinkled and lighter in colour. Amongst the cells there is a considerable admixture with air. Similar films are formed by a few of the Torula- cells. The film of Chalara Mycoderma is gelatinous, and has a bright appearance. In the case of Monilia, which may occur with budding cells, the film formation is peculiar. During- the vigorous fermentation, a film forms on the froth, which, gradually spreads over the whole surface, and is occasionally wrinkled. The cells in the flask form a deposit, produce- a vigorous fermentation, and rise with bubbles of carbon dioxide to the surface again, where they begin a new stage of development. If sterilised lager beer is inoculated with this fungus, no fermentation takes place, and a thin dusty film is- formed, but under other circumstances the fungus forms white, floury, and woolly layers like Oidium. The films of true Saccharomyces differ somewhat from these. As a rule, they are produced in the following way : — If cultures are allowed to stand undisturbed for a longer or shorter period in wort at room temperature, it will be found that small specks of yeast appear on the surface of the liquid at th& completion of the primary fermentation. These collect to- gether at a later stage to form islands of varying size and shape, with a flat upper and arched lower surface. Finally these fuse together to form a light greyish-yellow and slimy film,. MORPHOLOGY AND ANATOMY OF YEAST CELLS. 293 which often spreads up the wall of the vessel forming a com- plete ring. Such a complete film-formation only takes place when the primary fermentation is completed. If the flask is shaken, shreds of the skin are loosened and sink, and in this way a complete layer may be collected on the bottom, whilst the skin reforms and assumes a mottled appearance, the younger portions being thin and dark, whilst the older are thick and pale in colour. The necessary condition to enable the film to form is the presence of a free and undisturbed surface with access of air. A vigorous film-formation assumes a free access of air. The function of film-formation is subject to the same conditions as the formation of endospores. Along with film-formation a bleaching of the wort takes place, which now assumes a light yellow colour. This occurs more rapidly at a high temperature, and is most readily observed in those species which bring about the most vigorous film-formation. Erlenmeyer flasks half-filled with wort and oovered with filter paper are admirably adapted for such •cultures. A few drops of a young and vigorous growth of yeast should be introduced. Hansen undertook the following determinations : — (1) The temperature limit for the formation of films. (2) The approximate time required for the first appearance of the film at different temperatures. (3) The microscopical appearance of the growth at different temperatures. The main object of comparative observations of this kind lies in determining the microscopical appearance of films at similar temperatures. The examination of the film was undertaken when it had just developed sufficiently to be visible to the naked eye. A glance at the illustrations representing these film-growths (see description of species) will show that their general character differs from that of the sedimentary forms. For instance, the sedimentary form of S. cerevisice I. is oval or spherical, whilst in the film, elongated and mycelial cells quickly appear, and the growth gradually assumes an appearance quite distinct from that of sedimentary yeast. 294 MICRO-ORGANISMS AND FERMENTATION. If we compare the film-formation of the six species, we find that the films developed at the higher temperatures offer very little scope for discrimination, S. cerevisice I. and S. ellipsoideus II, alone being distinguishable from the remainder. It is quite otherwise, however, when young films developed at 13°-15° C. are examined. The two species, 8. Pastorianus II. and S. Pastorianus III. — both top-fermentation yeasts, the cells of which in ordinary cultures cannot be distinguished from each other with certainty — exhibit in this case entirely different forms of growth. An equally striking difference is found between the otherwise similar species, 8. ellipsoideus^ I. and //. Observations of the limits of temperature for the formation of films show that for 8. cerevisice I. and 8. ellipsoideus /. these lie approximately within 38° and 5°-6° C. ; the limits for the three Pastorianus species are 34° and 3° C. ; 8. ellip- soideus II. has the same lower limit as the last species, but its maximum temperature is 38°-40° C. The time limits, compared with those given for ascospore- formation, show that in both cases development takes place more slowly at low than at high temperatures. At temperatures above 13° C. the film of 8. ellipsoideus II. develops so rapidly and vigorously that flasks containing this yeast can be recognised by this alone. Thus, at 22°- 23° C. the film had completely covered the surface in six to twelve days, whilst the other five species required three times- as long to form a film, and this was generally more feebly developed. This species and 8. Pastorianus III. also develop a vigorous film with comparative rapidity at the ordinary room temperature, the other species being left far behind. A further important biological relationship is the following : — Hansen's investigations have proved that the temperature maximum for budding in wort is higher than the maximum for film-formation, and that this again is higher than the maximum for spore-formation ; in other words, with a rising temperature, a point is reached at which spore-formation ceases, then a higher point at which film-formation ceases, and lastly, a still higher at which budding is no longer possible. On the other hand, the experiments indicate that the tern- MORPHOLOGY AND ANATOMY OF YEAST CELLS. 295 perature minimum for film-formation is lower than that of spore-formation. In brewers' low-fermentation yeasts, and in some wild yeasts, Will observed round and oval cells, having a thick membrane and containing a number of small oil-drops (Fig. 48). These occurred in the rings of yeast and in the small surface patches preceding true film-formation. If treated with con- centrated hydrochloric acid, the membrane splits into two a. Fig. 48.— Resting cells (after Will).— The outer layer is partly or completely detached, a, b, in wort ; c-f, in mineral nutrient solution. layers. In cultures, especially in artificial nutrient liquids, the outer layer of this membrane gradually detaches itself ; sometimes in such a way that the outer layer is not torn, so that it appears as though the one cell were contained within the other. The cell contents are coloured green or brown by concentrated sulphuric acid. The glycogen reaction '"with iodine has been occasionally observed in the cells. They appear to play a certain part in the life economy of the growth, 296 MICRO-ORGANISMS AND FERMENTATION. as resting cells, for these cells are sometimes found alive in old growths when most of the other individuals have perished. In artificial nutrient solutions containing mineral salts, sugar, Fig. 49.— Besting cells (after Will).— A, usual mode of germination ; B, resting cells, with club or sausage-shaped daughter cells with transverse walls. and asparagin, with addition of citric or tartaric acid, such resting cells occur also in the sediment. Globular or oblong MORPHOLOGY AND ANATOMY OF YEAST CELLS. 297 yeast-cells germinate from the resting cells, either singly or in large number (Fig. 49, A). Club-shaped cells with trans- verse-wall formation frequently arise, especially in older •cultures of resting cells produced in mineral nutrient solution. This phenomenon may recur in derived growths (Fig. 49, B). During germination on a solid nutrient medium, Will also observed a splitting up of these transverse walls (Fig. 49, B). According to Rayman and Kruis the cells of the film have a, marked respiratory power, oxidising the alcohol formed into •carbon dioxide and water, and at the same time splitting up the albuminoids of the liquid into amides and ammonium salts of organic acids. Cultures on Solid Substrata. — After Schroeter and Koch had shown that by cultivation on solid media, species of bacteria display distinct characteristics, Hansen succeeded in proving that a similar relationship holds good for yeasts. For this purpose he utilised beer- wort, to which about 5- 5 per cent, of gelatine had been added, contained in flasks closed by means of cotton- wool plugs. When these flasks are inoculated with the six species (S. cerevisice /., S. Pastorianus /., //., ///., 8. ellipsoideus I., II.), and allowed to stand at a temperature of 25° C., the growths which develop (streak-cultures) show such macroscopic differences in the course of eleven to four- teen days that four groups may be more or less sharply dis- tinguished. S. ellipsoideus I. stands alone, for its growth exhibits a characteristic net-like structure on the surface, which enables it to be distinguished from the other five by the unaided eye. When gelatine with yeast- water is employed for such cultures and the experiments conducted at 15° C., jS. Pastorianus II. yields growths after the lapse of sixteen days, the edges of which are comparatively smooth, whilst the growths obtained from S. Pastorianus III. are distinctly hairy. A microscopical examination shows that the two species are also distinguishable morphologically. This is by no means always the case with cultures on solid media ; in fact, the differences are often less marked under such condi- tions than when nutritive liquids are employed. For the Mycoderma species and S. membranes faciens, Hansen discovered a characteristic behaviour in wort-gelatine in which 298 MICRO-ORGANISMS AND FERMENTATION. they form shield-like colonies readily distinguishable from those of the Saccharomycetes. In this connection we may mention Hansen's observation that some species — e.g., S. Marxianus and S. Ludwigii — can develop a mycelium when grown on a solid medium, while others are unable to do so. The characters which can be obtained in this way fluctuate greatly, for both the behaviour of the living material and of the substratum may be strongly modified by other external influences. This fact was brought out by Will in a special study of four species of bottom - fermentation beer-yeasts. He discovered that when the inoculating material contained cells from the film they exercised considerable influence on the appearance of the colony. The giant colonies investigated by P. Lindner were prepared by transferring a drop-culture containing a vast number of cells to a spot on the nutritive gelatine. It gradually develops a large rounded colony which can be photographed. Even under these conditions the picture fluctuates for one and the same species, according to the differing circumstances. In certain cases, by the use of such growths, strongly marked differences can be produced, as is the case with the usual plate and streak cultures. Frequently, however, the differences between these giant colonies are so minute that it is impossible adequately to describe them. Aderhold, during an examination of gelatine-growths of German eUipsoid wine yeasts, found that in puncture-cultures and giant-cultures two types were distinguishable, one of which showed colonies with funnel-shaped depressions and with marked concentric lines, whilst the other showed conical growths with indistinct concentric structure, but very pro- minent radial streaks. A great number of yeast species liquefy nutrient gelatine. This was proved by the author in 1890 with respect to brewers' high - fermentation yeasts. Subsequently Will, Wehmer, and others made the same observations with other yeasts. Structure and Character of Yeast Cells. — During the growth of the cell the membrane gradually becomes more distinct. MORPHOLOGY AND ANATOMY OF YEAST CELLS. 299 When the cell is fully grown the strength of the membrane depends on the concentration of the nutritive fluid. It has a tendency to thicken in liquids with a high precentage of extract ; especially marked thickening is met with in the resting cells occurring in films. By treatment with concen- trated hydrochloric acid a division of the cell wall can often be observed. The gelatinous network first observed by Hansen may be regarded as a special development of the membrane, remini- scent of the zoogloea formation of bacteria. Under certain conditions, which have not yet been defined, the colonies brought about by the budding of yeast cells may com- bine to form irregular clots which sink more rapidly than individual cells (" break " and clarification in the brewery). This doubtless stands in relationship to a feature of the de- velopment of the yeast cell discovered by Hansen in 1884, He found that both Saccharomycetes and other budding fungi may secrete a gelatinous network which may take the form of strands or plates in which the cells are em- bedded (Fig. 50, A, B). If, for example, some thick brewery yeast is placed in a glass and allowed to remain under cover in such a way that it slowly dries, and then a trace of this yeast is mixed in a drop of water, the network can be clearly seen (Fig. 50, A). The formation also occurs in the gypsum block and gelatine cultures. The author has frequently observed this formation in the yeast samples despatched to his laboratory in filter paper enclosed in envelopes.* Hansen also found it in the film-formations of nearly all species. An ordinary microscopic examination of the pitching yeast in a brewery does not show this fermentation ; with the help of staining, however, its presence can be readily detected (Fig. 50, B). When the yeast is repeatedly washed, it is no longer possible to detect the network by staining ; but if the water is removed, and the yeast set aside for a time and then suitably treated, the gelatinous masses can be readily seen. By varying * This method of preserving a sample of yeast is very convenient. A small piece of filter paper is rapidly passed through a flame several times, and a few drops of yeast are poured on to it ; it is then folded up, and afterwards wrapped in several layers of paper which have been similarly treated. 300 MICRO-ORGANISMS AND FERMENTATION. the conditions of nourishment of the cells, the development •can be promoted or retarded, and the chemical composition modified. The whole behaviour suggests the zoogloea for- mation of bacteria. The chemical nature of the wall of the yeast cell is unknown. It is soluble in concentrated sulphuric acid and in concentrated chromic acid. It swells up and becomes transparent in potash and soda solutions. «SU»hT)OXA U /.' Fig. 50.— Yeast cell with gelatinous network (after Hanseu).— A, Network obtained by partial drying ; 1, portion formed of threads from which the cells have become detached ; 5 and 3 show that the network can also form complete walls, such a formation is seen between a and 6 — a is a negative cell, b is a cell with two spores ; U shows three cells, a, embedded in the network. B, network with yeast cells, the latter stained by methyl violet, network is not £tained. Some of the yeast cells are still in the meshes, but most have detached themselves. The most important part of the contents of the yeast cell is the cell-nucleus, which is not visible in a direct microscopical examination. It can, however, be detected by a suitable micro-chemical treatment of the cell. (The process for its detection is given in Chap, i.) As early as 1879 F. Schmitz de- tected a body in the cell by staining, which was undoubtedly the cell -nucleus. His observations were confirmed and MORPHOLOGY AND ANATOMY OF YEAST CELLS. 301 developed by Hansen and Strassburger, and later by Dangeard, Janssens, Wager, Buscalioni, Hoffmeister, Rayman, Kruis, and Guilliermond. We owe much to the fundamental studies and the beautiful and careful drawings of Guilliermond. The nucleus has a rounded form, and appears to enclose a still smaller nucleus. When the yeast cell begins to bud or to form spores, the cell nucleus propagates by division (Fig. 51), and this division takes place before the budding or formation of spores becomes visible. In connection with the fusion of spores observed by Hansen in S. Ludwigii, Guilliermond proved that a fusion of the nuclei takes place. He also observed this phenomenon in the case of S. octosporus. The vacuoles constitute another essential part of the cells. They separate gradually from the protoplasm of the young cells, and appear in increasing numbers as pale, feebly-re- fractive specks. In the older cells they are sharply defined, and may assume highly irregular forms. In the Mycodermctr Fig. 51.— S. Ludwigii.— Nucleus division during •pore-formation (after Guilliermond). species one or two very large vacuoles are usually found, and this applies also to the old Saccharomyces cells. The vacuolea are filled with an aqueous liquid, and often enclose fine granules. The yeast cells also contain larger and smaller particles of different refractivity, both in the protoplasm and in th& vacuoles, which are classed together under the name of granules. They are produced even in quite young cells. At a later stage, when the cells are filled with glycogen, they are not so obvious, but they are sharply defined when the glycogen has disappeared, and they may assume large proportions in the dying cells. Amongst those who have specially investigated the granules Eisenschitz, Raum, Zimmermann, Will, and Guilliermond may be mentioned. In many of these bodies, which doubtless vary in composition, oily or fatty substance have been detected,. 302 MICRO-ORGANISMS AND FERMENTATION. as well as albuminoids. Guilliermond showed that by fixing with alcohol and staining with haematoxylin or with methylene blue, some of the granules assume a red colour, whilst the protoplasm is coloured blue. As to their importance to the life processes of the cells, Guilliermond and others at present .assume that they are chiefly of service as reserve material. Glycogen also constitutes an important constituent of the cell contents. It has already been stated that it is assimilated by the cell when it has a rich supply of available carbohydrates. Its presence can be distinguished by a reddish-brown colora- tion with iodine in potassium iodide, whereas the albuminoid substances of the cell assume a yellowish colour. On heating the cell, the brown colour disappears, but reappears on cooling. In the fifth section of this chapter it has been shown that glycogen plays an important part in the auto-fermentation of yeast. Ascospore Formation. — In 1839 Schwann discovered that yeast cells can form new cells in their interior, and that these .are liberated by the bursting of the wall of the mother-cell. De Seynes gave a clear description of spores in 1868, and in 1870 Reess proved that they are produced by yeast cells of •different shape, and that the germination of spores takes place by budding. In 1872 Engel indicated moist gypsum blocks .as a specially favourable substratum for the development of spores. Reess, who did not work with pure cultures, regarded these spore-forming yeasts as a special group, which he indi- cated by the name Saccharomyces, a name proposed by Meyen, but he included along with these a large number of species in which no endogenous spore-formation had been observed. .Similar conclusions were published by de Bary in his celebrated work Vergleichende Morphologic und Biologie der Pilze (1884), which also contains admirable observations regarding yeast fungi. In 1882-3, Hansen undertook the first experimental in- vestigations concerning spore-formation, and his work made it possible to establish a sharp limit to the group of Saccharo- myces. The results of his investigations concerning the neces- sary conditions for spore-formation may be shortly stated .as follows : — SPORE-FORMATION. 303 1. The cells must be placed on a moist surface and have a plentiful supply of air. 2. Young and vigorous cells can exercise this function most easily and rapidly. Old cells which lack nutritive material can only develop spores with free access of oxygen. 3. The optimum temperature for most of the species yet examined is about 25° C. This temperature favours spore- formation in all known species. 4. A few Saccharomycetes likewise form spores when they are present in fermenting nutrient fluids. A lack of food cannot, as Klebs assumes, be regarded as a direct condition for spore - formation, since young and well-nourished cells can also be induced to form spores immediately — without previous budding — when they are placed under conditions which favour spore-formation, but are unfavourable to budding — e.g., in water saturated with gypsum, but with access of air and at a favourable tem- perature. A growth of yeast is developed in the way described on p. 290. Older cultures, developed in saccharine solution or in wort, must be cultivated several times in aerated wort before showing a normal formation of spores. A small quantity is transferred to a previously sterilised gypsum block ; this block takes the shape of a truncated cone ; it is enclosed in a flat glass dish covered by a larger inverted dish, and is kept moist by half-filling the dish with water.* If it is desired merely to bring about the formation of spores, the apparatus may be allowed to remain at the ordinary room temperature. The transferred cells develop through a few generations by means of budding, and then spore-formation begins in the mother-cells. Hansen was the first to give an accurate description of the structure of spores and a detailed account of their evolution founded upon observations of individuals. He distinguished three typically different groups of Saccharomycetes which are * Ascospores can also be obtained when yeast is spread upon sterilised solidified gelatine, prepared with or without nutritive solution (or on filter paper), and kept in a damp place ; likewise in yeast-water and in sterilised water. Spore- forming cells may also occur in the films of the Saccharomyces. 304 MICRO-ORGANISMS AND FERMENTATION. characterised either by their mode of germination or by the form of their spores (S. anomalies, etc.). After a lapse of time, dependent on the species, roundish particles of protoplasm appear in the cells ; these are the first indications of spores (Fig. 52). In their further development they are surrounded by a wall, which is more or less clearly defined in the different species. In most species the spores are spherical. S. anomalus forms an exception with its hemispherical spores, S. Marxianus and S. fragilis with kidney-shaped spores. Two distinct types of germination may be distinguished. In the first type, to which S. cerevisice I. belongs, the spores may expand during the first stages of germination to such an extent that the pressure which they exert on each other, while Fig. 52. — The first stages of development of the spores of Saccharomyces cerevisice 1. (after Hansen).— a, b, c, d, e, rudiments of spores, where the walls are not yet distinct ; /, •/, h, i, j, completely-developed spores with distinct walls. Fig. 53.— Spores of Saccharomyceg cere- visice 1. in the first stages of germination (after Hansen).— At a, d, e, and g, formation of partition walls; e, f, and g, the walls of the mother-cells have become ruptured ; g, a compound spore divided into several chambers, the coherent wall is ruptured in three places. they are still enclosed in the mother-cell, brings about the formation of partition walls (Fig. 53). This is caused by the wedging or squeezing together of the protoplasm between the spores, or else the walls of the spores may be brought into close contact. During further development, a complete union of the walls may take place, so that a true partition wall results ; the cell then becomes a compound spore divided into several chambers. During germination (Fig. 54) the spores swell and the wall of the mother-cell, which was originally fairly thick and elastic, stretches out and consequently grows thinner. It is finally ruptured, and then remains as a loose or shrivelled SPORE-FORMATION. 305 skin, partially covering the spores ; or it may gradually be absorbed during germination. Budding can occur at any point on the surface of the swollen spores ; it usually takes place after the wall of the mother- cell has been ruptured or absorbed, but it also occasionally takes place within the mother-cell. After the buds have formed, the spores may remain connected, or they may soon break away from each other. Fig. 54. — Budding of the spores in Saccharomycfs cerevisice 1. (after Hansen).— a, Three snores without the wall of mother-cell ; 6, cell with four spores ; b', the wall of mother-cell is ruptured ; e, cell with four spores, three of which are visible ; c' and c" shows the ruptured wall of mother-cell ; d, cell with three spores ; d"', the ruptured wall of mother-cell ; e-e'"", development of a very strong colony ; f-h, other forms of development ; A", the wall between the two spores has disappeared. Certain spores display a very remarkable behaviour (see Fig. 54, e-e'"" and h-h") ; the absorption of the wall separating two neighbouring spores causing them to fuse together. It is possible that the biological significance of this phenomenon lies in the fact that the spores may thus have a greater chance of forming buds under unfavourable conditions. One spore plays the part of a parasite to the other. The amalgamation of the spore is, perhaps, the beginning of the process. A similar fusion of spores was observed by Hansen in the 20 30G MICRO-ORGANISMS AND FERMENTATION. case of a wine yeast (Johannisberg II.). He placed spore- forming cells in a shallow layer of wort. In the course of a few hours they swelled up and burst the mother-cells. They were then transferred to a shallow layer in a saturated aqueous solution of calcium sulphate at 25° C. Under these conditions no budding took place, but several spores fused together and formed new endospores. Similarly, Guilliermond detected a fusion of spores in S. Ludwigii. In this connection we may mention the fusion (copulation) of vegetative cells observed by Schionning, Guilliermond, and Barker, in the case of S. octosporus, Pombe, mellacei, and Zygosaccharomyces. Further details are given in the systematic description of these species. The germination of the spores of the known species of the groups S. Pastorianus and S. ellipsoideus takes place in essen- tially the same way as that just described. S. Ludwigii forms a second and very different type (Figs. 55, 56), where germination does not take place through budding, but through a germinal tube, called a promycelium. Two such germinal tubes frequently fuse together, and the propa- gation of yeast cells takes place through division and not through budding, after the formation of a clearly defined septum. Similarly these yeast cells produce new cells. In this case, unlike the first type, it is not the spores, but the new formations springing from them that fuse together. Guilliermond observed such a fusion of germinal tubes in spores which were derived from different mother cells. In older spores this curious fusion is more uncommon (Fig. 56). A few germ-filaments develop into a branched mycelium (group b). The spores of S. anomalus have a remarkable shape similar to those of Endomyces decipiens* They are almost hemi- spherical with a rim round the base. During germination the spores swell and the projecting rims may either remain or disappear. Buds then crop out at different points on the surface of the spore. * A fungus which is parasitic on the lamellae of certain mushrooms. A similar species was described by P. Lindner as E. fibuliger. SPORE-FORMATION. 307 a a' Fig. 55. — Germination of the spores of Saccharomyceg Ludwiqii (after Hansen).— a-c, Gypsum- block cultures twelve days old ; d-h, a similar culture six weeks old. Fig. 56.—Saccl>aromycts Ludwigii (after Hausen).— Germinating spores from old gypsum- block cultures ; a and b, each spore has developed a germ-nlaraent ; c, shows different forms produced by fusion. 308 MICRO-ORGANISMS AND FERMENTATION. One object of Hansen's work was to determine to what extent the formation of spores was influenced by different temperatures, with a view of ascertaining whether the various species behave alike, or whether it might not be possible in this way to discover characteristics. It was necessary to determine : — First, the limits of temperature — i.e., the highest and lowest temperatures at which spores can be formed ; secondly, the optimum temperature — i.e., the temperature at wMch spores appear most rapidly ; and, thirdly, the relation between the intermediate temperatures. To determine the time required, the moment was registered at which the cells showed distinct indications of spores (com- pare Figs. 52 and 58). It is not possible to make use of ripe spores in these determinations, since no criterion exists for complete ripeness. Fig. 57.— Germination of spores of Saccharvmyces anomalug (after Hansen). The results obtained by Hansen are as follows : — The formation of spores takes place slowly at low tem- peratures, more rapidly as the temperature rises, until a point is passed at which their development is retarded and finally ceases. The lowest limit of temperature for the six species first investigated was found to be 0-5°-3° C., and the highest limit 37-5° C. Hansen also determined the intermediate tempera- ture and time relations for the six species, and found that when these two values are graphically represented, with the degrees of temperature as abscissae, and the time intervals as ordinates, the curves are almost identical for the six species. SPORE-FORMATION. 309 fi Fig. bS.—Saccharomi/ccx with ascospores (after Hansen).— 1, Sacch. cerevisue I.; 2, Sacch. Pattorianus I.; 3, Sacch. Pott. II.; 4, Sacch. Pant. I [I.; 5, Sacch. ellipsoideux I.; 6, Sacch. fllipis. 11.; a, cells with partition-wall formation ; b, cells containing a larger number of spore* than usual ; c, cells showing distinct rudiments of spores. 310 MICRO-ORGANISMS AND FERMENTATION. They sink from the ordinates of the lowest temperature towards the axes of the abscissae, and then rise. At the same time, however, these curves indicate that the cardinal points deter- mined, more especially for the highest and lowest temperatures, give characteristic distinctions for the different species — i.e., that the limits of temperature within which the formation of spores can take place differ for the various species (see- systematic description). In a course of years a number of investigators have carried out similar researches, including Holm, Will, Aderhold, Kayser, Seyffert, Marx, Schionning, and the author. The following observations were made regarding the time required for the appearance of the first indications of spores in the six species maintained at the same temperature. At the highest temperature thirty hours were required for the- development of each species ; at 25° there was again no great difference in the time required ; at the lower temperature, however, marked differences occurred. Thus, in the case of S. cerevisice I., the first indications of spore-formation appear at 11-5° C. after ten days, but in the case of S. Pastorianus II. t they appear within seventy-seven hours. In all such determinations a considerable influence is exerted by the state of the cells, and the results vary with the temperature at which they have been grown, with their age, vigour, etc. (compare section on Variation of yeast cells). It follows that the composition of the nutrient fluid also exercises an influence. Thus in methodical, comparative experiments of this nature, it is a necessary condition that the previous culti- vation of the cells should always be carried out in the same manner. If these external conditions are varied, the limits for the reactions of the species must be determined in each case. By these experiments Hansen has established an important character for the determination of the Saccharomycetes. It is also of great interest to note that the spore-formation has a lower temperature maximum than budding, but a higher temperature minimum ; in other words, spore-formation takes place within a narrower range of temperature than budding. The method given below for the practical analysis of low brewery yeast was based by Hansen on the temperature curves ANALYSIS OF YEAST, 311 for the development of spores. Thus, it was found that at certain temperatures the species employed in the brewery, the culture yeasts, develop their spores later than the wild yeasts, several species of which occur as disease germs in the brewery. It is also important to note that the structure of the spores in these two groups is usuaUy different. The young spore of culture yeast has a distinct wall or membrane ; the contents are not homogeneous, but are granular, and exhibit vacuoles. In the case of wild yeast, on the other hand, the wall of the young spore is usually indistinct, the contents are homogeneous and strongly refractive. It should also be added that the spores of culture yeasts are usually larger than those of wild yeasts. 1. For the continuous daily control of low brewery yeast, as regards contamination with wild yeast, the following very convenient method is made use of : — At the conclusion of the primary fermentation, a small sample of the liquid is transferred from the fermenting vessel to a sterilised flask; this is set aside for some hours until the yeast has settled to the bottom, when the sediment is transferred to a gypsum block. It is then placed in a thermostat at a temperature of either 25° C. or 15° C. It was shown that the species of culture yeasts employed in low-fermentation breweries can be divided into two groups. This has subsequently been confirmed by the elaborate in- vestigations of Holm and Poulsen. At 25° C., one group yields spores at a later period than wild yeast, the other group produces spores in about the same time as wild yeast, but at a temperature of 15° C. the cells of wild yeast show spore-formation much sooner than the cells of either group of culture yeasts. The cultures maintained at 25° C. are examined after an interval of forty hours, and those maintained at 15° C. after an interval of three days. The author has shown that high brewery yeasts can be analysed in a similar manner. In the case of some species, however, the analysis is best made at 10°- 12° C., because a well-marked difference of time between the beginning of spore-formation in culture yeast on the one hand and wild yeast on the other can only be observed at this temperature. 312 MICRO-ORGANISMS AND FERMENTATION. According to the author's researches, distillers' yeast may be analysed in the same way. Lower temperatures are to be preferred for this analysis. Often, however, the investi- gation into the construction of the spore in the selected yeast- type must form the chief part of the analysis, the difference of time for spore-formation in culture yeast and wild yeast frequently proving inadequate. Aderhold has established the fact that wine yeast, like beer yeast, may be analysed by Hansen's method. By means of experiments undertaken to determine to what extent Hansen's analytical method can1 be relied on for tech- nical purposes in low-fermentation breweries, Holm and Poulsen concluded that a very small admixture of wild yeast, about ^ihyth of the entire mass (Carlsberg bottom -yeast No. 1), can be detected with certainty. Hansen's previous researches had shown that when the two species, Sacch. Pas- torianus III. and Sacch. ellipsoideus II. — which are capable of producing yeast-turbidity in beer — are present to the extent of only 1 part in 41 of the pitching yeast, the disease is not developed, provided that the normal conditions of fermentation and storage have been maintained. Further, Sacch. Pastorianus I., which imparts to beer a disagreeable odour and an unpleasant bitter taste,, can scarcely exert its injurious influence under the same conditions when the admixture of this yeast amounts to less than 1 part in 22 of the pitching yeast. Consequently, Hansen's method for the analysis of yeast by means of ascospore formation is sufficient to establish its purity.* When the object of the analysis is to characterise the different species present in the sample with greater accuracy, a number of cells are isolated by fractionation, and each of the growths obtained is separately examined. In an investigation of bottom yeast in the different stages * It is obvious that such an analysis from the vat does not enable direct conclusions to be drawn regarding the predominant biological conditions existing during secondary fermentation in the cask. If, for instance, the beer is run off with a very small quantity of yeast, even if the infection is a small one, the wild yeast will chiefly be found floating in the liquid, and will be carried over into the cask, whilst the greater part of the culture yeasts will have sunk to the bottom of the vat. ANALYSIS OF YEAST. 313 of the primary fermentation, published by Hansen in 1883, it was shown that the young cells of wild yeasts are present in largest amount during the last stages of primary fermenta- tion and in the upper layers of the liquid. The samples taken from the fermenting vessel for the analysis of yeast must, therefore, be taken during the last few days of the primary fermentation. If a dried or partially dried sample of yeast is to be examined, it must first be transferred to wort, and one or more fermentations must be completely carried out with it. The rule that wild yeasts develop only in the more advanced stages of fermentation applies also to top-fermentation yeast as used in breweries. This was shown by numerous analyses of beer from Danish, English, French, and German breweries carried out in the author's laboratory. As is well known, it was this very appearance of wild yeast in English top-fer- mentation breweries which gave rise to the erroneous view that such species are necessary for conducting a normal secondary fermentation. 2. The analysis of the yeast in the propagating apparatus, which must be absolutely pure, is carried out as follows : — At the conclusion of fermentation, samples are withdrawn, with every precaution, into Pasteur flasks or into the Hansen flasks employed for sending out yeast samples ; from these, small quantities are introduced into flasks containing neutral or slightly alkaline yeast water or yeast- water dextrose, and maintained at a temperature of 25° C., the object being to test the yeast for bacteria. The remainder is set aside to allow the yeast to settle, the beer is decanted, and an average sample of the sediment is introduced into a cane-sugar solution containing 1 to 4 per cent, of tartaric acid. After three or four cultivations in such a solution it is further culti- vated a few times in beer-wort, and then tested for spore- formation. The smallest traces of wild yeast in the apparatus are brought into a state of vigorous development by this treatment.* * It is evident that this method is not available for the analysis of ordinary yeast, because the cultivation in the tartaric solution will cause the wild yeast •cells to increase very considerably in number, and consequently render it im- possible for the analyst to judge of the degree of contamination. 314 MICRO-ORGANISMS AND FERMENTATION. I. SACCHAROMYCES. The name Saccharomyces is used to distinguish budding- fungi with endogenous spore-formation. The great majority of species are only known in this form, but a few can develop a mycelium. In the case of one particular group of Schizo- saccharomyces division of the cell takes place instead of budding, exactly in the same way as with certain of the mould fungi. In addition to these fungi many other kinds of budding species occur in nature which do not display endogenous spore-formation. Thanks to investigations by de Bary, Zopf, Brefeld, and others, it is now known that certain of these are developed from the higher fungi Vstilaginece (smut-fungi), Basidiomycetes, etc. A glance at the following figures shows that the Saccharo- mycetes may develop mycelial cells in their films. Thus cultures of S. Marxianus may occur with typical branched mycelium. Such formations may probably be regarded as tending to show that if these fungi are afforded more favourable conditions of development in nature than those obtaining when they are artificially cultivated in a laboratory, they are likely to develop as typical moulds. The following observations of the author appears to favour this view : — On dried grapes, growths of Dematium-like moulds have been observed with a rich formation of spores (see Fig. 45).* If such growths are cultivated either in or upon a sub- stratum in flasks, their spores develop only budding cells with endogenous spore-formation. In the same way vigorous growths of mould have been found on slices of Agave stems from Mexico, which at first suggest Manilla, and give a strong formation of spores. By cultivation in nutritive liquids and on gelatine only Saccharomyces cells are produced. On saccharine material received from Jamaica, growths of moulds were found resembling Oidium, but the cells also exhibit spore-formation, and by further development * The fungi do not possess the characteristic coloured and thick- walled resting cells of Dematium puttulans. SACCHAROMYCES. 315- .) of the mould in and upon sterile substrata nothing but a growth of Schizosaccharomyces is obtained, and no- mycelium.* In all three cases such substrata were utilised as had otherwise proved favourable for the growth of moulds. But it was impossible, under laboratory conditions, to reproduce the natural conditions which favour the formation of these- Dematium, Monilia, and Oidium-Uke fungi. Further investigation will determine how far such condi- tions are to be found in nature. These observations, at any rate, show that there are cases where the natural conditions allow of a development which cannot be substantiated by artificial conditions in the laboratory, and the conclusion appears to be warranted that other fungi, including higher fungi, may behave in the same way, like the Ustilaginece and other forms which regularly reproduce budding growths, which are incapable of forming endospores. An excellent but isolated example of the development of Saccharomyces cells from a fungus, Glososporium, belonging to a higher system, has been recorded by Vialla and Pacottet.f The basis of a scientific system of classification was suggested! by Hansen in 1904 as follows : — Family — Saccharomycetes. Budding fungi with endospores and vigorous formation of yeast cells. Typical mycelium only occurs in few cases. Every cell may occur as the mother-cell of a spore. Spores- unicellular. Number of spores in each mother-cell usually from one to four, seldom, up to twelve. * As stated in the section on Variation, Lepeschkin observed a similar weak formation of mycelium in individual cells of S. mellacei. •f Whatever may be the much-discussed genetic connection between Asper- gillua Oryzce and Saccharomyces, it is certain that the conidia of many individuals have been observed to bud. How far a mistake may have occurred in transferring: these budding growths to gypsum blocks, where they showed endogenous spore- formation, it is no longer possible to say. All subsequent experiments with growth* of Aspergillus species have given negative results. 316 MICRO-ORGANISMS AND FERMENTATION. A. TRUE SACCHAROMYCETES. 1st Group. The cells immediately form sedimentary yeast in saccharine nutritive liquids, and only at a much later stage form a film with slimy growth and without inclusion of air. Spores smooth round, or oval, with one or two membranes. Germination by budding or by the formation of germinal tubes (promycelium). All, or at any rate the greater number, of this group bring about alcoholic fermentation. GENUS I. — Saccharomyces Meyen. The spores provided with one membrane germinate by budding. In addition to formation of the yeast cells a few give mycelium with distinct transverse walls. (To this genus belong the culture yeasts and the great majority of wild yeasts.) GENUS II. — Zygosaccharomyces Barker. Distinguished by the copulation of cells. In other respects identical with the preceding genus. GENUS III. — Saccharomycodes E. C. Hansen. By germination of the spores, possessing one membrane, form a promycelium. From these, as well as from the vege- tative cells, budding takes place with incomplete separation. Formation of mycelium with distinct transverse walls. GENUS IV. — Saccharomycopsis Schionning. The spores possess two membranes, otherwise the characters, «o far as they are known, are identical with those of Saccharo- myces. 2nd Group. The cells immediately form a film in saccharine nutritive liquid, which is dry and opaque en account of the inclusion of air, and can readily be distinguished from the film-formation of the first group. The spores are hemispherical, angular. BREWERY YEASTS. 317 hat-shaped or lemon-shaped ; in the last two cases provided with a distinct projecting rim ; otherwise smooth. They have only one membrane ; germination takes place by budding. The majority of species are distinguished by the formation of esters ; a few do not bring about fermentation. GENUS V. — Pichia E. C. Hansen. The spores hemispherical or irregular and angular. No- fermentation ; strong growth of mycelia. GENUS VI. — Willia E. C. Hansen. Spores hat-shaped or lemon-shaped with distinctly pro- jecting rims. The majority produce esters vigorously ; a few produce no fermentation. B. DOUBTFUL SACCHAROMYCETES. Monospora. Nematospora. The genus Schizosaccharowyces cannot be included in the family of Saccharomycetes. 1. THE SPECIES USED INDUSTRIALLY (CULTURE YEASTS). (a) Brewery Yeasts. According to the physical phenomena of fermentation, a distinction is made between low- and high-fermentation yeasts, both in the brewery and elsewhere. The low-fermentation yeasts gradually collect during fermentation to form a deposit in the fermenting liquid, whereas the top-fermentation yeasts, in the normal course of fermentation, partly form a layer on the surface of the liquid, differing in character and thickness according to the race, and partly form a deposit. The two kinds of yeasts in the brewery impart a different character to the fermented liquor. This has been established by parallel experiments with wort of identical composition. The two groups of yeast may, therefore, be said to exhibit a different form of chemical activity. Bau has proved that most of the known species of low-fermentation yeasts ferment melibiose. whilst some of the top yeasts are incapable of fermenting it. 318 MICRO-ORGANISMS AND FERMENTATION. According to Bau and Fischer, melibiase, the enzyme that ferments melibiose, could only be detected in low-fermentation yeasts, and not in those top yeasts that are unable to ferment melibiose. According to Hansen's recent work (referred to in the section on Variation), individuals may occur in old cultures of bottom yeasts which exhibit top-fermentation phenomena, and similarly, if in smaller numbers, individuals in top yeasts which behave like bottom yeasts. After Hansen had introduced a pure culture of bottom yeast into the Carlsberg brewery in Copenhagen it was possible to discover how extensive and how deep-seated the differences are which distinguish the various bottom-fermentation brewery yeasts. With this object in view the writer undertook a long series of comparative experiments with pure cultures of top and bottom yeasts from every part of the world, noting in particular the degree of fermentation, the clarifying power of the liquid, the physical phenomena of fermentation, and the stability of the fermented liquor. As early as 1886, in the first edition of this book, he set forth a classification of typical species or races, the correctness of which has been confirmed by subsequent workers in this field. A. BOTTOM-FERMENTATION SPECIES. 1. Species which clarify very quickly and give a feeble fermentation ; the beer holds a strong head. The beer, if kept long, is liable to yeast-turbidity. Such yeasts are only suitable for draught beer. 2. Species which clarify fairly quickly and do not give a vigorous fermentation ; the beer holds a strong head ; high foam : the yeast settles to a firm layer in the fermenting vessel. The beer is not particularly stable as regards yeast-turbidity. These yeasts are suitable for draught beer and some for lager beer. 3. Species which clarify slowly and attenuate more strongly. The beer is very stable to yeast-turbidity. These yeasts are suitable for lager beer, and especially for export beers. BREWERY YEASTS. 319 B. TOP-FERMENTATION SPECIES. 1. Species which attenuate slightly and clarify quickly. The beer has a sweet taste. 2. Species which attenuate strongly and clarify quickly. Taste of beer more pronounced. 3. Species which attenuate strongly and often clarify slowly. The beer is stable to yeast-turbidity. By far the greater number of high-fermentation yeasts examined in this respect are able to carry through a secondary fermentation. In class 2, and especially 3, the secondary fermentation is very vigorous and long continued. Before the results of these comparative experiments had been published, both Hansen and the author had had the opportunity, as will be seen, of demonstrating that many of the species so characterised appeared as strongly marked types when applied in the form of mass cultures in practice, and that both in the above and other respects typical differences made their appearance between the individual races or species which found application as culture yeasts. The experience gained during the years that have since elapsed goes to prove that by a methodical selection of a race, an element of cer- tainty is introduced into the fermenting conditions, which was impossible when a mixed yeast of unknown composition was employed. In 1884 Hansen made the following pronouncement : — " We find by closer investigation that differences exist amongst the kinds of yeast which must be described as good from the standpoint of the brewer. Thus, under similar con- ditions, some give a quicker and more complete clarification in the primary fermentation and a more feeble attenuation than others. Again, differences are found in respect to flavour. If my method is followed, it is possible, nevertheless, to select with care and quite methodically, that species, which is best suited for the particular work. This phase of the question has been practically solved at Old Carlsberg, where a yeast has been selected, in addition to that previously described, which is better suited for making lager beer, whilst the former is better for export beer. Where the fermentation industry 320 MICRO-ORGANISMS AND FERMENTATION. formerly groped blindfold, and everything was a matter rational of guess work, a path has now been opened to a technique." Hansen is here referring to the two races of yeast that were first isolated and described. To what extent individual types display a pronounced character in their practical application depends largely on the nature of the treatment. Thus the degree of fermentation is determined both by the composition of the liquid and by the other conditions of fermentation. A race which gives a vigorous fermentation, can obviously only display this property under certain conditions. On the other hand, typical characters exist which may become noticeable under very different external conditions. Thus it was shown by the author, in the earliest stages of the development of this important reform, that top-fermentation species from the brewery, which have a definite influence on the odour and flavour of the fermented liquor, can be recognised by this fact when they are used in breweries in distant countries, where both the raw materials and the methods of working may be entirely different. The same applies when such species are introduced in absolutely pure cultures, which, beginning on the small scale with one or two litres of thin yeast liquor, are propagated by degrees in brewery wort, and thus adapted to it. The two first races obtained as pure cultures, referred to- by Hansen in the above quotation, were — Carlsberg No. 1, a yeast applied for many years in the Carlsberg brewery in Copenhagen, and Carlsberg No. 2, which was introduced from a German into a Copenhagen brewery, the fermentation being under the control of the author. After he had drawn attention, to the remarkable fermentation phenomena observed with this yeast, which differ widely from that of Carlsberg No. ly it was introduced into the Carlsberg brewery and isolated as a pure culture by Hansen and the author. In 1885 the author had the opportunity of answering the question wrhether different races or species of 8. cerevisice exist, the answer being based upon his own investigations of these two species. LOW-FERMENTATION YEASTS. 321 The first race chiefly exhibits slightly elongated cells, amongst which somewhat smaller pointed individuals dis- tinguished by granular contents are not infrequently found. If the yeast is taken from the fermenting vessel, washed with water, and placed for a short time on ice, it will be observed that the whole cell content rapidly changes to a granular structure, and if maintained under these conditions for several days, it will be found that the number of dead cells rapidly increases. The second race behaves in quite a different way. The cells are short and oval, or almost spherical, under normal conditions in the fermenting vessel ; only a few bent indi- viduals are observed, and in a washed condition the cells retain their clear or slightly granular contents for a long time ; very few dead cells are observed even after long preservation in this condition. If each of the growths is placed on moist gypsum blocks, maintained at the same temperature, and their further develop- ment observed from day to day, it will be seen that the two races behave quite differently, assuming that the temperature lies within the limits for the growth of spores. Race II. forms fully ripe spores at a time when Race I. does not show a trace of these organs of propagation. The following distinctions are of value in practice in determining the two races : — In order to obtain the normal course of the primary fer- mentation it is essential that Race I. should be introduced at a somewhat higher temperature (7-5° C.) than Race II. Larger quantities of /. than of //. must be used for pitching, in the proportion of 66 to 58. The time of setting and of frothing naturally differs. Both phenomena appear to occur somewhat earlier with Race II. than with Race I. The nature of the frothing and the coating of yeast differ greatly. //. gives a strong high head and a dense coherent cover ; /. a low head, and the liquid often shows bald patches. Moreover, Race I. gives a very lasting fermentation, and, as a consequence, a slower clarification than Race II., which when pure gives a particularly bright clarification. The sedimentary yeast in the vat lies more compactly, and the colour of //. is somewhat lighter than that of /. 21 322 MICRO-ORGANISMS AND FERMENTATION. The attenuation during the primary and secondary fer- mentation with normal wort and in the same brewery is stronger with Race I. than //. With regard to the finished beer, similar differences are noted, particularly regarding flavour and resistance to tur- bidity. Most experts prefer //. for flavour, but some difference of opinion exists. It is otherwise regarding the stability of the beer, especially with regard to yeast turbidity. In this respect the difference is very marked. /. gives a quite ex- ceptionally stable beer, and is specially suitable for export beer, which when fermented with this yeast remains unaltered for about a month without any further treatment, and by mild pasteurisation is rendered stable for much longer periods. Race II., although it displays much finer phenomena during the primary fermentation, is unable to produce completely stable beer (about ten days in bottles at room temperature), and it is also noteworthy that this race is much less resistant to wild yeast than /. On account of the rapid clarification and quick fermentation of the liquor this race is adapted for beers which are to be stored for a short time, and are to be consumed immediately. In general, it may be stated that the whole of the differences indicated have been observed for years in different breweries, and that they are so sharply defined that every brewer could at once distinguish the two yeasts with certainty when they have been put into the fermenting vessel, and could foretell the nature of primary and secondary fermentation. In fact, no one could be in doubt that we are dealing with two truly distinct races or species. In the detailed descriptions of these two races (see Figs. 59 and 60), published by Hansen in 1888 (which might equally well have appeared before the author's publications, as will be seen from the preceding historical description), the characteristic distinctions between these two species are further emphasised. Amongst other observations, reference is made to the giant cells, remarkable and abnormal large round cells which suggest the cells of Mucor yeasts. In 1908 Hansen described further typical differences, and gave the species the names 8. Carlsbergensis and S. Monacensis. LOW-FERMENTATION YEASTS. 323 S. Carlsbergensis ( = No. 1) has temperature limits for budding in wort at about 33-5° C. and 0° C. At the maximum temperature the cells are considerably larger than at the outset, but have approximately the same shape ; giant cells are numerous. At the minimum and up to 9° C. many of the cells .assume the sausage-shape, and form large mycelial colonies. In the films elliptical and round cells principally occur. The giant colonies are rosette-shaped with a depression, and less frequently a distinct knot in the middle, with concentric rings and radial streamers ; they have a smooth or scaly surface .and wavy outline. The colonies in the usual plate-cultures Fig. 59.— Carlsberg low-fermentation yeast No. 1 (after Hansen). fQ& Fig. 60.— Carlsberg low-fermentation yeast No. 2— a few cells with spores (after Hansen). built up as small pin heads with a light greyish-yellow and waxy appearance. S. Monacensis ( = No. 2) has temperature limits for budding at about 33° and 1°. At the maximum the cells are larger, and especially longer, than at the outset, and at the minimum and up to 9° C., in contrast to the former species, it develops colonies consisting principally of spherical and elliptical cells. In dextrose-yeast-water, the giant cells may assume huge dimensions. In the film, the cells are spherical and elliptical. The giant colonies and the small colonies agree in appearance with those of the former species. 324 MICRO-ORGANISMS AND FERMENTATION. As an admirable example of the application of Hansen's- biological methods to the differentiation of yeast species, we give the comparison of four low-fermentation brewery yeasts carried out by Will. This, again, emphasises the fact that, within this group, just as clearly distinguished species occur as in those groups of Saccharomycetes which have not yet found an industrial application. Will began his characterisa- tion by adopting the classification of brewery-yeast types published by the author in 1886. He classed Races 93 and 2 as high-fermenting, Race 7 as a low-fermenting type, and Race 6 as a yeast of intermediate fermentation. The four yeasts can be distinguished as follows : — Race 2 has large roundish or oval cells ; the colonies on> gelatine are spherical or lenticular ; the spores are formed easily and freely ; spore-formation takes place between 31° and 11° ; the optimum is 25°- 26° ; film-formation occurs between 31° and 7° ; very slow. Race 6 ; oval cells predominate, but the species has a great tendency to form sausage-shaped cells ; colonies on gelatine are spherical or lenticular ; the spores form easily and freely; spore-formation occurs between 31° and 11° C. ; the optimum is 28° ; film-formation between 31° and 7°,. occurring later than with Race 7. The cells of Race 93 are typically oval with a tendency to- assume a roundish shape. . The colonies in gelatine are spherical or lenticular ; spores are freely and easily formed ; spore- formation occurs between 30° and 10° C. ; optimum 28° ; film-formation between 31° and 4° ; very feeble and slow ;. resting cells occur freely in the film. Race 7 has oval cells which closely approach the spherical shape ; giant cells regularly occur, and at the end of the fermentation large budding colonies with small oval cells frequently occur ; the young colonies on gelatine are irregular with a marked wavy and fringed outline ; the species develops spores with great difficulty ; spore-formation occurs between 30° and 13° ; optimum 25° to 26° ; film-formation between 28° and 4° ; appears earlier than with the other species ;. resting cells are to be found only in small numbers in film. LOW-FERMENTATION YEASTS. 325 P. Lindner distinguished two species of low-fermentation yeast in 1889, which he called " Saaz " (weakly fermenting) -and " Frohberg " (strongly fermenting). These names have been adopted in the literature as a description of weakly and strongly fermenting yeast types in general. The investigation of these groups was subsequently undertaken by Delbriick, Reinke, Irmisch, and others. A thorough description of two low-fermentation yeasts of the Frohberg type (D and K) has been given by Schonfeld and Rommel. D gives longish, almost sausage-shaped cells, K predominantly spherical and oval cells. I) is more inclined to form spores than K. In hanging drops, differences in the shape and size of the cells can be remarked. K forms budding colonies more rapidly and in greater number. In the growth of giant colonies similar differences have been observed. The content of albumen, the percentage of ash and phosphoric acid are higher in K than in D, and K has a higher specific gravity than D. Auto-digestion occurs more rapidly with K. K has a higher " raising power," and is more sensitive to high temperatures. Fermentation sets in more rapidly with K. At temperatures above 30° the fermentative activity of K yeast is weakened to a much greater extent than that of D. The film growth of K is capable of fermenting more carbohydrate than the sedimentary yeast, whilst with D the difference is unimportant. In the brewery, K ferments 10 per cent, higher than D in the fermenting vessel, and gives a lighter coloured beer. The final fermentation is identical with the two species. In 1883-85 very detailed researches were carried out by the author in elaborating the principle laid down by Hansen, and introduced in the Carlsberg brewery, the application of methodically selected pure cultures derived from a single cell. The experiments were carried out with a view of securing practical conditions, and the results gained in the laboratory were applied on the large scale in breweries in many European countries. The reform found acceptance by prominent fermentation technologists (in the early stages, especially by Thausing, Lintner, and Aubry), and it was gradually incorporated into the courses of all zymotechno- logical institutes. 326 MICRO-ORGANISMS AND FERMENTATION. Saccharomyces cerevisiae or Saccharomyces cerevisiae L Hansen. This species, described in 1883, is an old English top- fermentation yeast which is in use in London and Edinburgh breweries. The young growth of sedimentary yeast developed in wort consists essentially of large round and oval cells ; truly elongated cells do not occur under these conditions. Ascospore-formation (Figs. 52 to 54, and 58, 1) : — * At 37-5° C. no ascospores are developed. 36-37 the first indications are seen after 29 hours. 35 „ „ 25 „ 33-5 ,, ,, 23 ,, 30 „ „ 20 „ 25 „ „ 23 „ 23 ,, ,, 27 ,, 17-5 „ „ 50 16-5 ,, ,, 65 ,, 11-12 „ ;, 10 days. 9 no ascospores are developed. Wall of spores very distinct. Size of spores 2-5 to 6 yu. Film-formation : — At 38° C. no film-formation occurs. 33-34 feebly-developed film specks are seen after . .9-18 days. 26-28 „ „ 7-11 „ 20-22 „ „ 7-10 „ 13-15 15-30 „ | 6- 7 „ „ 2- 3 months.] 5 no film-formation occurs. Microscopical appearance of the cells in the films : — At 20° to 34° C. ; colonies frequent ; sausage-shaped and curiously formed cells occur. < * The preparatory treatment of a Saccharomyces species for these investi- gations must be made in the following manner : — After the cells have been culti- vated for some time in ordinary wort (14° Ball.) at the ordinary room temperature, the vigorous young cells obtained are introduced into fresh wort, in which they are allowed to develop for about twenty-four hours at 25°-27° C. This growth is used for the gypsum-block culture. SACCH. CEREVISLE. 327 At 15° to 6° C. (Fig. 62).— The greater number of the cells resemble the original cells ; isolated abnormal forms occur. In old cultures of films all forms occur, including extremely elongated mycelial cells (Fig. 63). The temperature limits for budding in wort are 40° C. and 1° to 3° C. The species develops invertase and maltase ; it ferments saccharose, maltose, and dextrose, but not lactose. It produces a vigorous fermentation in beer- wort. The first series of pure top-fermentation species were pre- pared by the author in 1884 from material collected in many Fig. 61.— Saccharomyces cerevisice I. (Hansen).— Cell-forms of young sedimentary yeast (after Hansen). Fig. (V.—Saccharomyces cerevisice I. (Hansen).— Film-forms at 15° to 6" C. (after Hansen). European countries, with the object of introducing such pure, selected types, developed from single cells, into practice. It was soon seen that the typical differences between the species were much more pronounced than is the case with low-fermen- tation yeast. It was found that one group of the species used in breweries had an extraordinarily weak fermentative activity. The fermentation ceased, under the conditions existing in the breweries, when 1 to 2 per cent, of alcohol had been formed in beer-wort ; the main mass of the yeast usually spread out over the surface of the liquid to form a coherent layer. Fig. 63.—Saccharomyces cerevisice I. (Hansen).— Cell-forms in old cultures of films (after Hansen). TOP-FERMENTATION YEASTS. 329 Species of the second group behave quite differently. Under similar conditions fermentation can be carried on for a much longer time, clarification goes on slowly, and when the primary fermentation is at an end the beer is decanted from the yeast, which to a large extent is sedimentary yeast ; a secondary fermentation takes place, the duration varying according to the species. As representatives of the first group, the species chiefly used in Danish breweries may be mentioned, and of the second group, many of those applied in English breweries. The purely-cultivated Danish, top-fermentation species fall into two distinct types, according to their chemical activity. The first impart a decidedly mild flavour ; the fermentation is weak without noticeable secondary fermentation, and the layer of yeast forming on the surface of the liquid is loose and slimy. The second type gives a strongly pronounced flavour, the fermentation is stronger, with subsequent secondary fermentation, and the layer of yeast has a dense consistency. By long-continued use of both types the latter has proved to be more generally acceptable. The English species that have been subjected to examina- tion, and have been proved to bring about a distinct secondary fermentation, exhibit a great variety of form and various construction of spores. Many of these differences have been recorded, both in these respects and in relation to fermentation. According to the observations * of J. C. Holm and the author, the following facts have been established : — The formation of cells at the different stages of alcoholic fermentation was determined by growths which were first kept for a long time in a 10 per cent, sugar solution, then grown for several generations in beer-wort, and finally developed for twenty-four hours in Pasteur flasks at 25° C. The develop- ment of the films and their appearance to the naked eye were studied in growths in Erlenmeyer flasks at room temperature (about 20° C.). Growths in Pasteur flasks at room temperature were made use of for determining the physical character of the sedimentary yeast. The fermentation experiments were * Published for the first time in Micro-Organisms and Fermentation, 3rd •edition, 1900. 330 MICRO-ORGANISMS AND FERMENTATION. carried out at room temperature in sterilised, hopped wort contained in tall cylindrical glasses covered with several layers of filter paper. After the primary fermentation was completed, the liquids were poured into sterile flasks and allowed to stand at low temperatures. The amount of alcohol was determined at the completion of the primary fermentation and again after the first fortnight of the secondary fermentation, and, lastly, after the following fortnight. The primary fer- mentation was interrupted when the appearance of the cells showed that the first vigorous development had ceased. In this comparison no attempt was made to decide what quantity of alcohol could be produced by the species during primary and secondary fermentation. The object was simply to in- stitute a comparison. The flavour of the fermented liquor was recorded after the beer had undergone a secondary fermentation at a low tem- perature in flasks closed at first with cotton-wool and after- wards with ground-glass stoppers. 1. (Fig. 64, a, and Fig. 65, a.) The cells during fermentation are comparatively small, oval, and linked in chains ; among them occur big, round and grotesque forms. The yeast lies rather loose in the flask ; if shaken it does not distribute itself equally in the wort, but separates into clots. Film - formation : After a lapse of 31 to 32 days a very thin film covers almost the whole surface of the liquid. The cells of the film are of about the same size as those seen during the primary fermentation ; some cells much elongated. The spores, if developed at a low temperature, are small, full of vacuoles, and slightly granulated ; as a rule, only one or two in each cell. At 11° to 12° C. a few spores make their appearance on the seventh day ; at 25° C. abundant development of spores in forty hours. TOP-FERMENTATION YEASTS. 331 Q 0 O x O 5 Fig. 64.— a-d, Young growths of English top-yeasts (Holm). 332 MICRO-ORGANISMS AND FERMENTATION. Fig. 65.— a-d, Film-formations of English top-yeasts (Jorgeusen). TOP-FERMENTATION YEASTS. 33S When the principal fermentation was broken off, the liquid contained 2-49 per cent, by vol. of alcohol ; during the two- following periods (see above) 0-31 and 0-57 per cent, by vol. were produced. Production of acid, after expulsion of CO0! corresponds to 5 c.c. of decinormal caustic soda solution. The fermented liquid has an agreeable smell and a fine- aromatic taste. 2. (Fig. 64, b, and Fig. 65, 6.) During fermentation most cells are free, medium-sized, round and oval ; among them there occur round and oval giant cells. The yeast lies loose in flask ; if shaken slightly, it is dis- tributed like a cloud throughout the liquid. Film-formation : After 31 to 32 days, a few large patches. The cells of the film are smaller than those seen during- primary fermentation ; ellipsoidal and slightly lengthened. The spores are big, if developed at a low temperature ; formation of partition walls readily occurs. At 11° to 12° C. very few spores occur on the seventh day \ at 25° C. rather abundant spore-formation in forty hours. When the principal fermentation was broken off, the liquid contained 2-3 per cent, by vol. of alcohol ; in the two fol- lowing periods 1 and 0-46 per cent, by vol. were formed. Acid-production : 6. Disagreeable smell and taste. 3. (Fig. 64, c, and Fig. 65, c.) During fermentation the growth shows free cells and small chain-formations of oval forms ; a few globular giant cells. The yeast lies very compact in flask ; it partially rises in the liquid only when violently shaken. Film-formation : In 31 to 32 days the growth forms a very thin film, which does not cover the entire surface of the liquid. Some of the cells of the film have the same size and shape as those seen during primary fermentation ; others are slightly lengthened. 334 MICRO-ORGANISMS AND FERMENTATION. If developed at a low temperature, the spores are of very varied size, with comparatively feeble refractivity, and without distinct vacuoles. Partition- wall formations occur. At 11° to 12° C., in seven days, only rudiments of spores appear ; at 25° C., in forty hours, spores are very freely formed. When the principal fermentation was broken off, the liquid •contained 2-26 per cent, by vol. of alcohol ; during the fol- lowing two periods 0-79 and 0-00 per cent, by vol. were formed. Acid-production : 5-5. Disagreeable smell and taste. 4. (Fig. 64, d, and Fig. 65, d.) During fermentation, colonies consisting of many small spherical cells occur, and among these spherical giant cells. The yeast lies loose in flask ; if slightly shaken, it is dis- tributed like a cloud throughout the whole liquid. Film-formation : After 31 to 32 days, only the merest trace. The cells of the ring-growth occur in colonies, which some- times contain upwards of a hundred cells, all derived from a single cell ; the youngest growths are elongated and very narrow. The spores, if developed at a low temperature, are small and vacuolised. At 11° to 12° C., even after a fortnight, no spore-formation ; at 25° C., for forty hours, a very scanty •development of spores. When the principal fermentation was broken off, the liquid •contained 1-8 per cent, by vol. of alcohol ; during the following two periods 1 and 0-82 per cent, by vol. were formed. Acid-production : 5-5. Disagreeable smell and taste. 5. (Fig. 66, a, and Fig. 67, a.) During fermentation most cells are free, medium-sized, and oval. The yeast lies rather loose in flask ; if shaken, it is not distributed equally in the wort, but separates into clots. Film-formation : After 31 to 32 days a distinct film, which, however, does not cover the whole surface, and subsequently develops slowly. TOP-FERMENTATION YEASTS. 335 A 0 Fig. 66.— a-«, Young growth! of English top-yeasts (Holm). 336 MICRO-ORGANISMS AND FERMENTATION. Fig. 07.— a-e, Film-formations of English top-yeasta (Jorgensen). TOP-FERMEXTATION YEASTS. 337 The cells of the film have a very different appearance from those seen in the fermentation-stage. Many of them are much lengthened and irregularly twined ; some have developed a ramified mycelium. If developed at a low temperature, the spores are small, coherent, granulated. At 11° to 12° C. no spores appear within a fortnight ; at 25° C., a very scanty spore-formation takes place in forty hours. When the principal fermentation was broken off, the liquid contained 2-49 per cent, by vol. of alcohol ; in the following two periods 0-86 and 0-12 per cent, by vol. were formed. Acid-production : 5-2. Agreeable smell and fine aromatic taste. 6. ^(Fig. 66. 6, and Fig. 67, 6.) The cells are round, oval, and elongated during fermenta- tion, all forms occurring in chains ; isolated round giant cells occur. The yeast lies rather compact in flask ; it requires strong shaking to distribute the cells equally throughout the liquid. Film-formation : After 26 days the surface growth forms a ring of yeast cells on the wall of the flask ; only slight indi- cations of film-formation. After 31 to 32 days the film had not developed further. The cells of the ring-growth cannot be distinguished from those occurring during alcoholic fermentation. If developed at a low temperature, the spores are com- paratively small, granulated, with no distinct vacuoles. At 11° to 12° C., for seven days, very few spores are formed ; at 25° C., for forty hours, a scanty spore-formation takes place. When the principal fermentation was broken off, the liquid contained 1-85 per cent, by vol. of alcohol ; during the following two periods 0-65 and 0-2 per cent, by vol. were formed. Acid-production : 6. Agreeable smell and slightly aromatic taste. 7. (Fig. 66, c, and Fig. 67, c.) During fermentation round and oval cells, some free, others linked in short chains. 22 338 MICRO-ORGANISMS AND FERMENTATION. The yeast lies rather compact in flask ; violent shaking is required to distribute the cells equally throughout the liquid. Film-formation : After 26 days a thin, almost continuous film appears, which in the course of the next five to six days forms a conspicuous covering extending over the whole surface of the liquid. The cells of the film have in the main the same shape as those seen during fermentation ; only the youngest generations are elongated and narrow. If developed at a low temperature, the spores are medium sized, with no distinct vacuoles. At 11° to 12° C., after nine days, fully developed spores appear ; at 25° C., for forty hours, spores are formed freely. When the principal fermentation was broken off, the liquid contained 2-4 per cent, by vol. of alcohol ; during the following two periods 0-95 and 0-00 per cent, by vol. were formed. Acid-production : 6-5. Agreeable smell and slightly aromatic taste. 8. (Fig. 66, d, and Fig. 67, d.) During fermentation, round, oval, and elongated cells, both free and linked together. The yeast lies rather compact in flask ; on violent shaking the cells are distributed equally throughout the liquid. Film-formation : After 31 to 32 days very slight isolated patches of a film on the surface, and a slight ring of yeast-cells on the glass, round the edge of the liquid. The cells of the film have assumed quite different shapes from those of the fermentation-stage ; they are very much lengthened, mycelial, and irregular. If developed at a low temperature, the spores are medium- sized, with no distinct vacuoles. At 11° to 12° C. spores are formed pretty freely on the ninth day ; at 25° C., they are formed freely in forty hours. When the principal fermentation was broken off, the liquid contained 2-77 per cent, by vol. of alcohol ; during the fol- lowing two periods 0-98 and 0-00 per cent, by vol. were formed. TOP-FERMENTATION YEASTS. . 339 Acid-production : 6-5. Odour good, but bitter, persistent taste. 9. (Fig. 66, e, and Fig. 67, e.) During fermentation a very uniform growth of big, round and oval cells. The yeast lies rather loose in flask ; on shaking, the cells are distributed equally throughout the liquid. Film-formation : In 31 to 32 days very slight, isolated patches on the surface, and a slender ring of yeast cells on the glass, round the edge of the liquid. The cells of the film differ but little from those of the fermentation. If developed at a low temperature, the spores are medium in size and granulated. At 11° to 12° C. spore-formation sets in on the ninth day ; at 25° C., for forty hours, a somewhat scanty spore-formation takes place, accompanied by a con- siderable formation of net-work. When the principal fermentation was broken off, the liquid contained 2-96 per cent, by vol. of alcohol ; during the following two periods 1-19 and 0-00 per cent, by vol. were formed. Acid-production : 7. Odour good, pronounced vinous taste. Regensburger has since undertaken very detailed com- parative experiments with regard to three species of top- fermentation yeasts, which, like the examples just referred to, display characteristic differences in the general appearance of the young growths. Spore-formation occurs within differing periods, and conforms to the rule laid down by the author many years ago that the development of spores usually takes place more rapidly with top yeasts than with bottom yeasts. Distinct points of difference can also be observed in the visible course of fermentation, in the cardinal points for skin formation and in the development on solid substrata. I At the time, the author's argument that purely-cultivated top yeasts would quickly become impure on account of the prevailing high temperatures was subjected to criticism. Ex- perience has shown that these criticisms have no weight, 340 MICRO-ORGANISMS AND FERMENTATION. and that great progress .may be made in this field, and con- siderable advantages may be derived by the use of a single selected type. A further objection raised was that it is im- possible by means of a single species to obtain a stable secon- dary fermentation, a wrong assumption previously made regarding a low-fermentation yeast. Van Laer strongly em- phasised this view, and while he freely admitted that low yeast types exist, capable of carrying through a normal secondary fermentation, he believed that the author was wrong in ascrib- ing the same properties to top yeasts. Notwithstanding the practical results attained by exact experiments carried out with selected types, even when due allowance was made for the special English conditions referred to by van Laer, and in spite- of the fact that no exact proof was forthcoming to warrant the opposite view, the author's experience was ignored, and van Laer prepared mixtures of top-yeast species which were distributed for use in breweries. They were designed to satisfy practical demands, the intention being that one species should carry through the primary fermentation, the other the secondary fermentation. It is true that the possibility is not excluded that such a composite yeast could be prepared, but even when van Laer's preparations gave good results in practice it could not be proved that it was due to the activity of the composite yeast as such. It must first be demonstrated that this new yeast really reacted as a composite yeast — i.e., that the different constituents are really capable of acting together. In con- junction with J. C. Holm, the author investigated many of the preparations distributed in the industry, and it was shown that even during the primary fermentation one of the species very strongly predominated, whilst in the secondary fermenta- tion the other species disappeared. Thus the problem of preparing a truly composite yeast had not been solved. The experience of subsequent years has always confirmed the cor- rectness of the first results, even in fermentations carried out on the English system. It is possible in both top and bottom fermentations to carry out the whole primary and secondary fermentation with a single selected species. Quite recently the question of applying two species in English top-fermentation has been re-opened. It has been TOP-FERMENTATION YEASTS. 341 supposed that the secondary fermentation of stout is brought about by certain species of Torula (two are described in the systematic part of this chapter), and that it is due to their activity that this variety of beer acquires its peculiar sourish taste. Here, however, two separate facts have been confused. The true secondary fermentation is carried out by the properly selected type of yeast, and can be regulated like any other secondary fermentation. In this connection the activity of the Torulas is unnecessary, as may plainly be seen from the fact that in every European country, and in other parts of the world, as shown by the author, stout and similar kinds of beer can be prepared from one -of certain selected types of yeast. These species of Torula are to be reckoned in the same category as the lactic - acid bacteria, acetic - acid bacteria, Sarcina, etc., which also impart a special taste to a fermented wort. If such a taste is desired, it is of importance to regulate the activity of the organism in question, so that the quantity of the peculiar fermentation or assimilation product may stop short of a given limit. If this is exceeded the liquid becomes undrinkable. It has frequently been suggested within recent times that all the races of culture yeast present in an impure top- fermentation yeast should be isolated and then again mixed in the same proportion, with a view of using this culture mixture in a brewery, where it would reproduce the original yeast, freed from bacteria and wild yeasts. Such a process, however, oould not be carried out, at any rate, in this world. After the author had introduced pure cultures into practice in many European countries, the reform met with general agreement. The work was taken up in the early stages by Kokosinsky, de Bavay, M'Cartie, W. R. Wilson, A. Miller, and R. Grey, to give but a few names. At a later stage, J. Schon- feld attacked the problem, and selected types of top yeasts were introduced into many German breweries, though chiefly as pitching yeast and not as pure culture. In the case of top-fermentation lactic-acid beers, like German "Weissbier," the rational treatment must consist in the main in first carrying through a lactic-acid fermentation, and then applying the pure-cultivated top yeast. 342 MICRO-ORGANISMS AND FERMENTATION. (b) Distillery and Pressed Yeasts. To solve the problem whether distillery and pressed yeasts are capable of forming endogenous spores, a possibility denied by Wiesner and Brefeld, the author, in 1884, undertook an exact examination of a number of samples of such yeasts, and in the same year, together with Hansen, published the results in Dingler's Polytechnic Journal, showing that there ia no possible difficulty in obtaining an abundant and rapid spore-formation from these species. At the same time the author was able to arrive at certain conclusions regarding the composition of such yeast. By the help of fractional cultures, it was found that both top and bottom fermentation species occur in ordinary distillery and pressed yeast. Further investigations showed that in one and the same mass of yeast two morphologically different types may frequently occur, one chiefly giving isolated cells in a fully-developed state, the other, budding colonies of many cells. It was impossible, therefore, to trace any connection between this and the fer- mentation phenomena brought about by the two species. The two morphological types remain unaltered after being preserved for years. Detailed researches further showed that both pressed yeast and the top yeast used in distilleries include a multitude of clearly distinguishable types, and a few years later pure selected races from the author's laboratory were first intro- duced into yeast factories, and then into the distilleries of Northern Europe and into Molasses factories. Owing to the physiological state of the species, due to the dissimilar composition of the nutritive fluids, very important differences are exhibited with respect to propagating power, yield of alcohol, character of the alcohol, etc. These are retained throughout many years, so that it is necessary in many cases to instal an absolutely pure culture of a suitable yeast in each individual factory. By expert applica- tion of such cultures, and particularly by a rational lactic acidification, which arrests the development of foreign organ- isms found in the mash, it is possible to secure a higher yield and a better quality both of alcohol and of yeast. Rayman. WINE YEASTS. . 343 and Kruis have undertaken elaborate investigations with regard to the character of the distillate obtained by the use of different species. In 1890 the Berlin Experimental Station sent out the first yeast species cultivated from this group by P. Lindner. It was described as Race 2. More recently another species, Race 12, has been brought into practical use, and this appears to be preferred, according to Lindner's communications, both in potato distilleries and in pressed yeast factories. The yeast is grown in ordinary large fermenting vats, and is supplied in a pressed state in kilogramme lots. Henneberg has given a detailed description of both species, from which it may be noted that the giant colonies differ in appearance. In Race 2 they have an almost smooth surface, scored by a few shallow, concentric and radial furrows ; the outline is fairly straight. Race 12 has a very uneven surface, scored by deep, irregularly radial furrows. The ridges so formed constitute an extremely delicate concentric pattern, and the outline is formed erratically by the termination of the ridges at varying distances from the centre. The small colonies in plate-cultures are similar. Race 2 has feebly- developed budding colonies, whilst Race 12 forms large and dense clusters. The cells of 2 are an elongated oval ; those of 12 are roundish and oval. (c) Wine Yeasts. When a number of pure growths are isolated from the usual elliptical wine yeasts, it will be readily seen that they vary greatly in morphological character, under similar con- ditions of cultivation, especially if the general appearance is taken into account. Species with both large and small cells and every intermediate form are met with, from elongated and elliptical, to oval and almost spherical cells. Pastorianus forms of yeast also exist. Before 1890 a series of such types, exhibiting stable morphological characters, and displaying characteristic differences in spore-formation, had been isolated in the author's institute. Hansen had published further information regarding the individual species in his description of S. ellipsoideus I. (see 344 MICRO-ORGANISMS AND FERMENTATION. following section), and subsequently in his notes regarding Johannisberg II. He found that the temperature limits for budding in wort are 37° to 38° C. and 0-5° C. ; for spore-for- mation on gypsum blocks 33° to 34-5° C. and 2° to 3° C. Further publications we owe to Aderhold, Hotter, Kayser, Marx, Miiller-Thurgau, Seifert, Wortmann, and others. As examples of the different biological characters observed in wine yeast, we will discuss more closely a few of the species described by Aderhold. Johannisberg I. has round or oval-pointed cells ; in the young film produced at 26° to 27° C.* the cells are oval ; spores appear in 28 to 30 hours at 25° to 26-5° C * Johannisberg II. has large oval cells, characterised by longish but blunt ends. The film cells are round, oval, and sausage-shaped ; spores are formed within 23 to 24 hours. Kreuznach has the same cell-formation as the previous species, but somewhat smaller ; film cells like Johannisberg I. ; spores in 30 hours. Mulheim has broad, oval, and, less frequently, round cells, with short pointed ends smaller than the previous species ; only round and oval film cells ; spores within two to three days. Walporzheim I. has round cells, the oval forms scarcely pointed ; often budding colonies in the film, elongated links forming an axis for the colony surrounded by round cells ; spores in 80 hours. Piesport ; predominantly elliptical cells without pointed ends ; only spherical cells in the film ; spores in 23 to 24 hours. Grown on solid substrata, differences can be observed in the development of colonies. Pure selected races have gradually been introduced in large numbers into the wine fermentation by Miiller-Thurgau. Wortmann, Kayser, Jacquemin, the author, and others. Miiller-Thurgau and Wortmann, amongst others, have indi- cated and proved the importance of these pure cultures, as not only the must but also the fermented product, the wine, is dominated by the pure yeast throughout every stage of its development, extending, it may be, through many years. f * Similar temperatures hold good for the following species. . "j" See the section on the behaviour of Saccharomycetes with sugars, etc. SACCH. PASTORIANUS I. 345 2. YEASTS NOT YET APPLIED INDUSTRIALLY. Saccharomyces Pastorianus or Saccharomyces Pastorianus I. Hansen (Figs. 68, 69). Bottom-fermentation yeast. Sedimentary forms developed in yeast : — Predominantly elongated and sausage-shaped, also large and small oval and round cells (Fig. 68). When this species is cultivated in wort near its maximum temperature for growth, its vegetation consists of sausage-shaped and elongated cells. The tempera- ture limits for budding in wort are 34° and 0-5° C. It frequently occurs in the air of fermenting rooms, and is also found in diseased beers. It imparts to beer a disagreeable bitter taste and unpleasant odour ; it may also produce turbidity, and interfere with the clarification of beer in the fermenting vessel. According to the investigations of Mach and Portele, this species may also be successfully used in wine fermentation. Saccharose, dextrose, Isevulose, and maltose are fermented, but not lactose. Ascospore-formation (Fig. 58, 2) : — At 31-5° C. no ascospores are developed. 29-5-30-5 the first indications are seen after 30 hours. 29 ,, ,, 27 ,, 27-5 „ „ 24 „ 23-5 „ „ 26 „ 18 ,, ,, 35 ,, 15 „ „ 50 „ 10 ,, „ 89 ,, 8-5 „ „ 5 days. 7 7 5> » J> 3-4 „ „ 14 „ 0-5 no ascospores are developed. Size of spores 1-5 to 5 p. 346 MICRO-ORGANISMS AND FERMENTATION. Fig. 6S.—Saccharomyces Pastorianus I. (Hansen).— Cell-forms of young sedimentary yeast (after Hansen). Fig 69.—Sa.ccharomycefi Pastoriamts I. (Hansen).— Film-forms at 13° to 15° C., from Holm's drawing in Hansen's Memoir. SACCH. PASTORIANUS II. 347 Film-formation : — At 34° C. no film-formation occurs. 26-28 feebly-developed film-specks are seen after 7-10 days. 20-22 13-15 6- 7 3- 5 . 69.) ,, ,, 1- 2 months. 5- 6 „ like Fig. 69, but without the large colonies. 2- 3 no film-formation occurs. Microscopic appearance of the cells in the films :— At 20° to 28° C. almost the same forms occur as in the sedimentary yeast. At 13° to 15° C. strongly-developed mycelial colonies of very elongated, sausage-shaped cells are fairly frequent (Fig. 69). In old cultures of films the cells are smaller than in the sediment ; very irregular and sometimes almost thread-like cells are found. Saccharomyces intermedius or Saccharomyces Pastorianus II. Hansen. (Figs. 70, 71.) Feeble top-fermentation yeast. Fig. 70.—Saccharomycfs Pastorianus II. (Hansen).- Cell-forms of young sedimentary yeast (after Hansen). Sedimentary forms grown in wort : — Mainly elongated, sausage-shaped cells, but also large and small, oval, and round cells (Fig. 70). 348 MICRO-ORGANISMS AND FERMENTATION. When this species is cultivated in wort near the maximum temperature for growth its vegetation consists of round and oval cells. The temperature limits for budding in wort are 40° and 0-5° C. It frequently occurred in Hansen's analyses of air in the brewery ; it appears to belong to the species which do not cause diseases in beer. Saccharose, dextrose, laevulose, and maltose are fermented, but not lactose. Fig. 7L—Saccharomyces Pastorianus II. (Hansen).— Film-forms at 15° to 3° C. (after Holm's drawing in Hansen's Memoir). Ascospore formation (Fig. 58, 3) : — At 29° C. no ascospores are developed. 27-28 the first indications are seen after 34 hours. 25 ,, ,, 25 ,, 23 „ „ 27 „ 17 ,. ,, 36 ,, 15 „ „ 48 „ H-5 „ „ 77 „ 7 „ ,,7 days. 3-4 „ „ 17 „ 0-5 no ascospores are developed. Size of the spores 2 to 5 /K. SACCH. PASTORIANUS III. Film-formation : — At 34° C. no film-formation occurs. 26-28 feebly-developed film-specks are seen after . 7-10 days. 20-22 „ „ 8-15 „ * 13-15 „ „ 10-25 „ \ 6- 7 „ „ 1- 2 months. WFig. 71. > 3- 5 „ „ 5- 6 „ j 2- 3 no film-formation occurs. Microscopic appearance of the cells in the films : — At 20° to 28° C., almost the same forms as in the sedi- mentary yeast ; also irregular sausage-shaped cells. At 15° to 3° C., mostly oval and round cells. In old cultures of films the cells are smaller than in the sediment ; very irregular and sometimes almost thread-like cells are found. Streak cultures of this species in yeast-water gelatine give growths with comparatively smooth edges after sixteen days at 15° C., and in this respect it also differs from the following species. Saccharomyces validus or Saccharomyces Pastorianus III. Hansen. (Figs. 72, 73.) Top-fermentation yeast. Sedimentary forms grown in wort : — Mostly elongated, sausage-shaped, but also large and small, oval, and round cells (Fig. 72). When this species is cultivated in wort near the temperature maximum for growth, the vegetation consists of round and oval cells. The temperature limits for budding in wort are 39° to 40° C. and 0-5° C. It was separated from a bottom-fermentation beer which showed yeast-turbidity, and has been proved by Hansen to- be one of the species which produce this disease. Recent experiments of Hansen show that this disease-yeast possesses another peculiar property — its addition will in certain cases effect a clarification when the fermenting wort has an opal- escent appearance. According to investigations made by the author, a strong infection of low-fermentation yeast with this species may in MICRO-ORGANISMS AND FERMENTATION. certain cases effect an excellent clarification and good " break- ing " in both fermentation vessel and cask. Saccharose, •dextrose, laevulose, and maltose are fermented, but not lactose Ascospore-formation (Fig. 58, 4) : — At 29° C. no ascospores are developed. 27-28 the first indications are seen after 35 hours. 26-5 „ „ 30 „ 25 „ „ 28 „ 22 „ „ 29 „ 17 „ „ 44 „ 16 „ ,, 53 ,, 10-5 „ „ 7 days. 8-5 ,, ,, 9 ,, 4 no ascospores are developed. Size of the spores 2 to 5 ju. Fig. 72.—Saccharomyces Pastorianug III. (Hansen).— Cell-forms of young sedimentary yeast (after Hansen). Film-formation : — At 34° C. no film-formation occurs. 26-28 feebly-developed film-specks are seen after . 7-10 days. 20-22 „ „ 9-12 „ 13-15 „ „ 10-20 „ ) 6- 7 „ „ 1- 2 months. V(Fig. 73.) 3 5 „ „ 5-6 „ j 23 no film-formation occurs. SACCH. ELLIPSOIDEUS I. 351 Microscopic appearance of the cells in the films : — At 20° to 28° C. : Almost the same forms as in the sedi- mentary yeast. At 15° to 3° C. : Strongly-developed colonies of elongated, sausage-shaped or thread-like cells, which closely resemble a mycelium in appearance (Fig. 73). In old cultures of films, the cells have the same forms as at 15° to 3° C., but are often still thinner and more thread- like. Fig. 73. — Saccharomyces Pastorianus 111. (Hansen).— Film -forms at 15° to 3° C. (after HunseiiV Streak cultures of this species in yeast-water gelatine, after sixteen days at 15° C., give growths with distinctly hairy outline. Saccharomyces ellipsoideus or Saccharomyces ellipsoideus I. Hansen. (Figs. 74, 75.) Bottom-fermentation yeast. Sedimentary forms grown in wort : — Mostly oval and round cells ; sausage-shaped cells are rare (Fig. 74). If this species is cultivated in wort near the maximum temperature for growth, the vegetation consists of round and oval cells. The temperature limits for budding in wort are 40° to 41° C. and 0-5° C. Saccharose, dextrose, Isevulose, and maltose are fermented, but not lactose. Occurs on the surface of ripe grapes. 352 MICRO-ORGANISMS AND FERMENTATION. Ascospore-formation (Fig. 58, 5) : — At 32-5° C. no ascospores are developed. 30-5-31-5 the first indications are seen after 36 hours. 29-5 25 18 15 10-5 7-5 4 23 ^1 33 45 4 days, no ascospores are developed. Size of the spores 2 to 4 /u. Film-formation : — At 38° C. no film-formation occurs. feebly-developed film-specks are seen after . 8-12 days. 9-16 „ 10-17 „ 15-30 „ (Fig. 75.) ,, ,, 2- 3 months, no film-formation occurs. 33-34 26-28 20-22 13-15 6- 7 5 Microscopic appearance of the cells in the films c — At 20° to 34° C. and 6° to 7° C., the cells are smaller and more sausage-shaped than in the sedimentary yeast. At 13° to 15° C., freely- branched and strongly- developed colonies of long or short sausage - shaped cells, often with verticillated branches (Fig. 75). In old cultures of films, the cell forms are the same as at 13° to 15° C. Streak cultures of this species in wort-gelatine (wort with the addition of about 5-5 per cent, of gelatine), in the course Fig. 74. — Saccharomyees ettipcoideus I. (Han- sen). — Cell-forms of young sedimentary yeast (after Hansen). SACCHAROMYCES. 353 of eleven to fourteen days at 25° C., give — in contradistinction to the other five species — a characteristic net-like structure, by Fig. 7S.—Saccharomyces ettipsoidevt I. Hansen.— Film-forms at 13° to 15" C. (from Holm's drawing in Hansen's Memoir). means of which it can be distinguished by the naked eye from other species. 23 354 MICRO-ORGANISMS AND FERMENTATION. Saccharomyces turbidans or Saccharomyces ellipsoideus II. Hansen. (Figs. 76, 77.) Usually a bottom-fermentation yeast. Sedimentary forms grown in wort : — Oval and round cells predominate ; sausage-shaped cells are rare (Fig. 76). It was separated from beers which showed yeast-turbidity ; is a species which causes yeast-turbidity, and is more dangerous than Sacch. Pastorianus III. If this species is cultivated in wort near the maximum temperature for growth, the vegeta- tion consists of round and oval cells. The temperature limits for budding in wort are 40° C. and 0-5° C. Saccharose, dextrose, laevulose, and maltose are fermented, but not lactose. Fig. 76. — Saccharomyces ellipsoideus II. Han- sen. — Cell - forms of young sedimentary yeast (after Hausen). Fig. 77. — Saccharomyces ellipsoideus II. Hansen.— Film- forms at 28" to 3" (after Hansen). Ascospore-formation (Fig. 58, 6) :— At 35° C. no ascospores are developed. 33-34 the first indications are seen after 31 hours. 33 „ „ 27 „ 31-5 ,, ,, 23 ,, 29 „ „ 22 „ 25 „ „ 27 „ 18 „ „ 42 „ 11 „ „ 5|days. " 55 55 " 55 4 no ascospores are developed. Size of spores 2 to 5 ju. SACCHAROMYCES. 355 Film-formation : — At 40° C. no film-formation occurs. 36-38 feebly-developed film-specks are seen after . 8-12 days. 33-34 „ „ 3- 4 „ 26-28 „ „ 4- 5 „ 20-22 „ „ 4- 6 „ 13-15 8-10 „ \ (Fig. 77.) 6-7 „ „ 1-2 months. 3-5 ,, ,,5-6 „ 2- 3 no film-formation occurs. Microscopic appearance of the cells in the films : — At all temperatures, the same forms as in the sediment ; at a,nd below 15° C., the cells are only slightly more elongated Fig. 91.— Torula (after Hansen).— Same species as Fig. 90. Film-formation on a wort- culture ten months old. cells of the films are very irregular in shape. This Torula produces only 1 per cent, by volume of alcohol in wort, does not ferment maltose, and neither ferments nor inverts cane- sugar. In 10 per cent, and 15 per cent, solutions of dextrose in yeast- water it gave 4-6 and 4-5 per cent, by volume of alcohol in 15 days at 25° C., and 4-8 and 4-7 per cent, in 28 days. In two other flasks 4-8 and 5«3 per cent, of alcohol were produced after long standing. Hansen assumes that this 388 MICRO-ORGANISMS AND FERMENTATION. species takes part in vinous fermentation, and considers it probable that species such as the sixth and seventh, which pro- duce a vigorous fermentation in dextrose solutions, take part in the fermentation of grape and other fruit juices. On the other hand, they have probably little importance in breweries- and distilleries, since they are unable to ferment maltose. Another species of Torula (Torula Novae Carlsbergice), the cells of which exhibit very different forms, has been described by Gronlund. It imparts a disagreeable bitter taste to wort. According to Schjerning's investigations it inverts cane-sugar, and induces alcoholic fermentation in solutions of cane-sugar, dextrose, and maltose. In ordinary brewTery-wort it can produce about 4-7 per cent, by volume of alcohol. The following species of Torula r together with the two rose-coloured Q Q £ budding fungi described under the fi e© CL 0 * e at i * j© tP ft ^fe name of Torula o and c, were pre- ff $ <£© ^ pared in a pure state in the author 'a "8 B^t« ft ^ ^» laboratory, and are used in the £ ex £> ft « course of instruction given in his- ^S% & laboratory. $ * . •* J. C. Holm was the first to give Fig. ^.-Torma a. Young an exact description of these three culture (Brask). X 560. SPCCieS Torula a — Torula Holmii n.sp- The culture of the young sedimentary growth, consists of small oval cells (Fig. 92). Here and there single larger cells occur both oval and round. The length of the cells varies from 3-5 to 5-5 JJL, the breadth from 1-4 to 2-1 /u. It gives a feeble fermentation in wort, yielding about 0-32 per cent, by weight of alcohol. It inverts saccharose and raffinose, and ferments the inverted sugars. It ferments dextrose, but not maltose, lactose, and dextrin (d. puriss. Kahlbaum). A film-formation takes place in wort in three to five days at 25° C. The cells of the film are round and oval in wort, whereas in dextrose-yeast water they assume Pastorianus or irregular forms. The surface colonies on wort-gelatine (10 per cent.) are round, white, lustrous, and slightly raised. The edge of the colonies is smooth. TORULA. 389 Schionning submitted the Torula found by Claussen in English beer to a close examination, during which he isolated from different English beers some other species belonging to the same group. He found that they fall naturally into two o _^ formation is influenced by the pro- & j? £ ^Jo ^n a Pure dextrose solution the de- <*& formed, only a minute sediment. By ^ ^ the addition of peptone the ring Fig. w.-Toruia b.— Young culture formation is restored. When the (Brask). x 560. , •_• • • ± quantity of sugar remains constant (10 per cent, dextrose), while that of peptone rises from 0-1 to 0-2, 0-5, and 1 per cent., the ring formation is favourably influenced. With a constant quantity of peptone (0-5 per cent.) and increasing quantities of dextrose (5-10-20 per cent.) the slimy ring formation is reduced with increasing quantity of sugar. This shows that the slime formation de- pends upon the presence of albuminoids, and not upon that of sugar. The surface colonies on wort - gelatine (10 per cent.) are round, moist, and glistening, pale pink in colour, and slightly arched. The young colonies have smooth edges, the older show a depression in the middle and slight transverse furrows at the edge. Torula c = Torula cinnabarina n.sp. The cells are pre- dominantly of an elongated and oval shape, often provided TORULA. 39T with promycelium. The length varies from 7-7 to 10-5 /LI, breadth from 3-5 to 5-0 /u. Giant cells often occur, some- times with rather elongated form, 14-6 /u in length, sometimes almost spherical and 9-5 /u in diameter (Figs. 94 and 95). When sown in wort or in various sugar solutions, it first forms a smooth and afterwards a dry wrinkled film with intense crimson-lake colour. The liquid under the film is- clear. No sedimentary yeast is formed, and no fermentation phenomena can be observed. The wort undergoes a remark- able bleaching effect in older cultures. At 25° isolated islands of film appear on the surface in sixty hours, and few cells show indications of promycelium. The formation of pro- mycelium takes place freely in eighty hours. The formation Fig. 94.— Tfinila c.— Wmng culture (Brask). x 5«0. Fig. 95.— Torula c. — Film-formation, old culture (Brask). x 560 of buds takes place both on the promycelium and on the cells. There is no fermentation in dextrose, maltose, lactose, sac- charose, raffinose, or dextrin, but saccharose and raflfinose solutions are inverted. In wort with 1 to 2 per cent, of alcohol a feeble fermenta- tion is visible. With higher percentages of alcohol, no develop- ment takes place. The surface colonies on wort-gelatine (10 per cent.) are round, pale pink in colour and opaque. At a later stage the surface is warty, the edge jagged. The old colonies are dry, and display a network of furrows and a finely fringed edge. Janssen and Mertens described a red form appearing in English beer, the cells of which develop many buds at one 398 MICRO-ORGANISMS AND FERMENTATION. .and the same place on the mother - cell — Will's " crown " formation. They form promycelium in the film where long mycelial threads are also to be found. L. van den Hulle and H. van Laer found a red Torula in Lambic (a Belgian beer), which decolourised wort, and imparted a bitter taste to it. Will found a red species on green malt. When dried the malt .assumed a dirty-brown colour. Kilned malt was discoloured and not presentable. The infection was derived from steeping water. Fischer and Bre beck's Blastoderma salmonicolor has a promycelium which divides off into pear, plum, and kidney- shaped cells. This species forms a tough and very wrinkled film ; it is found in sea water. Lasche has described two red Torulas under the names Mycoderma humuli and M . rubrum. The first is found on hop leaves, and forms a film on nutritive liquids. Oval, sausage- shaped, or, to a great extent, irregular cells frequently form a promycelium, from which the buds divide. Gelatine is rapidly liquefied. This species gives no fermentation, and •cannot develop in beer. M. rubrum, derived from a chance infection on a gelatine plate, has oval or sausage-shaped cells, often linked in short chains. Promycelium formation occurs more frequently, and this species develops longer in wort, whilst the colour of the film is a lighter red. It gives no fermentation, and does not develop in beer. Bed Torula species occur in milk and cream, where they often form red specks on the surface (air infection), as well .as in butter and cheese. Demme declares that the species occurring in milk and cheese is the cause of catarrh of the stomach in children. Kramer found a top-fermentation Torula in must which pro- duces a red soluble colouring matter. It ferments dextrose, and produces 4-5 per cent, by volume of alcohol in a 10 per cent, .solution. Saccharose is inverted by this species, and maltose is directly fermented. Lactose, on the other hand, is not affected. The " black yeasts " are probably related to Dematium, Cladosporium, or Fumago. They have been found and de- scribed by Marpmann, Grotenfelt (on cheese), and Guillier- mond (on carrots). TORULA. 399 Torula Yeasts fermenting Lactose. Duclaux found a yeast-fungus in milk which induces alcoholic fermentation in a solution of lactose. This fungus appears to be most closely related to the Torula species. The cells are 1-5 to 2-5 //. in diameter, and almost spherical. According to Duclaux's experiments, this yeast is more aerobic than the ordinary alcoholic yeasts. Even with strong aeration of the liquid, the whole of the lactose is used up in the alcoholic fermentation. In a 5 per cent, solution of lactose 2-5 per cent, of alcohol was formed in eleven days at 25° C. The most favourable temperature for the fermenta- tion of a neutral solution is 25° to 32° C., whilst at 37° to 40° C. the fermentation ceases. Small quantities of acid have a retarding influence on the fermentative activity of this yeast. Adametz likewise describes a budding-fungus which fer- ments lactose (" Saccharomyces lactis "). Since this fungus does not yield endogenous spores, it must be classed in the group of non-Saccharomycetes. The cells are of about the same size as those of brewery yeasts, and are spherical and elliptical. The colonies grown on peptone-gelatine are round, with slightly jagged borders, and are of a dark brown colour. A stab-culture in wort-gelatine yields a dull, flat mass on the surface and a vigorous growth in the puncture channel, and from this numerous offshoots penetrate into the gelatine. In sterilised milk this fungus induces fermentation phenomena within twenty-four hours at 50° C., in forty-eight hours at 38° C., and in about four days at 25° C. In this fermentation the lactose alone is decomposed. Both of the species mentioned above have been more closely investigated by Kayser, together with a new species, which likewise ferments lactose, and belongs to the non-Saccharomy- cetes. All three yield colonies on gelatine, which are more •widely spread than those of beer- and wine-yeasts ; in the middle of the colonies there is a thick portion, while the border resembles mycelium. In milk and in neutral liquids, when sufficiently aerated, they induce an appreciable fermentation at 25° to 30° C. The milk does not coagulate or become viscous during the alcoholic fermentation. All three species 400 MICRO-ORGANISMS AND FERMENTATION. ferment lactose, galactose, cane-sugar, glucose, invert-sugar, and finally maltose, but the last only with great difficulty. In the fermentation of milk-sugar with these yeasts, the resulting liquids are as rich in alcohol as the strongest beers. Kayser remarks that it may, perhaps, be possible to make practical use of this observation and by means of these fungi convert the large quantities of whey, obtained in the manu- facture of cheese, into an alcoholic liquor. Beijerinck has described two yeasts which also ferment lactose, and which must be provisionally regarded as non- Saccharomycetes ; these are Saccharomyces Kephyr, which occurs in kephir-grains and consists of longish cells of varied shape, and forms slightly jagged colonies liquefying gelatine ; and Saccharomyces Tyrocola (from Edam cheese), which consists of small roundish cells, and forms snow-white colonies on gelatine. Beijerinck found that these two species secrete a particular invertive ferment (lactase) which inverts not only cane-sugar but also lactose ; it does not, however, invert maltose. It is stated that lactase may be prepared as follows : — A 5 per cent, solution of lactose, containing nutrient salts and asparagin, is fermented with kephir-yeast ; the product is filtered and the ferment is precipitated from the filtrate by the addition of alcohol. According to Schuurmans Steckhoven, however, the enzyme of Beijerinck's kephir-yeast does not invert lactose. In Lombardy Grana cheese a unilaterally budding top- fermentation yeast was discovered by Bochiccio, which is called Lactomyces inflans caseigrana. The growth consists of round, ellipsoidal, and oblong cells, and forms whitish colonies on gelatine, with smooth edges. No spore-formation was observed. In lactose-broth it produces a vigorous fermenta- tion at 25° to 40° C. ; the best temperature for the develop- ment is about 30° C., the limit of existence at about 60° C. Whey infected with this species is converted into a foaming beverage of a somewhat agreeable taste. Weigmann has isolated a pure culture of Torula from a defective butter. By fermentation in milk 51-2 per cent, by weight of alcohol and 34-4 per cent, of carbon dioxide, together with 3-6 per cent, of butyric acid were produced. Orla Jensen TORULA. 401 has also isolated a Torula from butter which fermented maltose in addition to lactose. P. Maze found ten different Torulas in soft cheese, one of which fermented lactose only ; the others, in addition, fermented dextrose, laevulose, maltose, and saccharose. The fermentations are more rapidly carried to an end, and a higher yield of alcohol is obtained if they are carried out in an alkaline liquor. Martin bouillon makes a good substratum with 0-088 per cent, of sodium carbonate. Maze believes it to be probable that these species produce aromatic bodies in soft cheese. In American cheese and milk a Torula occurred at one time, producing a bitter taste. Harrison proved that the infection was derived from milk cans, which, in their turn, had been infected by exposure under maple trees to dry air. The yeast-fungus, named by Harrison Torula amara, gives a strong and unpleasantly bitter taste to milk in fourteen hours at 37° C. : fermentation is brought about, and an odour developed resembling that of plum kernels ; the flavour becomes more astringent. At a later stage the milk curdles somewhat, and possesses a slightly acid and ethereal aroma. Lactose, glucose, and saccharose are easily fermented. In milk the last trace of sugar is fer- mented. The organism grows in broth containing 2 to 4 per cent, of lactic acid. A complete and comprehensive description of these lactose- fermenting Torulas (together with the lactose-fermenting Saccharomycetes) is given by Heinze and Cohn. They under- took a special and very detailed morphological and physiological investigation into Adametz' Sacch. lactis and Beijerinck's Tyrocola. Kalanthar detected three lactose-fermenting species in Mazun — viz., the greenish Mazun yeast with giant colonies, which are first greenish-grey and then plum-red, and two others, which, however, are declared by P. Lindner to be identical with the first. They ferment lactose, saccharose, trehalose, dextrose (feebly), but neither maltose nor a-methyl- glucoside ; they also produce acid. Torula species have also been detected in many defective butters and cheeses, which appear to be of more or less importance, thus Rogers found a Torula in a fishy and rancid 26 402 MICRO-ORGANISMS AND FERMENTATION. butter. It occurred several times in cases of preserved butter, and contains a fat-cleaving enzyme. Adametz has observed a Torula during the blistering of cheese, and it was also discovered by Bochiccio in Lombardy Grana cheese. The Lactomyces inftans caseigrana alluded to above brings about a marked blistering on the outer parts of hard cheese. It coagulates sterile milk, and partially liquefies the coagulum without noticeable formation of acid. It must, therefore, contain a clotting enzyme and a tryptic enzyme. Saccharomyces apiculatus Reess. (Fig. 96.) According to our present views, the name of this ferment is incorrect, for only those budding-fungi which yield endogenous spores are considered to belong to the Saccharomycetes, and the fungus in question does not possess this property. We will, however, provisionally retain the old generic name, as has been done by Hansen, until systematic classification has been further developed. This ferment was the subject of one of the finest and most thorough biological investigations of our time, for Hansen was enabled, after several years' work, to determine both its habitat in nature and its regular migrations at different seasons of the year (p. 272). The reason why this species was selected for the investigation was that, while other species occur in very varied and uncertain forms, making the study of their occur- rence in different localities very difficult, this ferment can be recognised with certainty, for it always occurs in cultures with lemon-shaped cells ; this is the typical form of the species. S. apiculatus occurs abundantly in wine fermentation, especially during the early stages, and also in spontaneously- fermented Belgian beer (Lambic, Faro, Mars, Krieckenbier), and, according to van Laer, imparts to it its peculiar taste and odour. In nature it is found on ripe, sweet, succulent fruits. If a little of such a growth is examined under the micro- scope in a drop of nutritive liquid, the development of the fungus can be followed. This is very characteristic (compare Fig. 96). It is seen that the buds formed from the typical SACCHAROMYCES APICULATUS. 403 lemon-shaped cells may be either lemon-shaped (a, b, c, e, /) or oval (a-c) ; it will also be noticed that the oval cells must first form one or more buds before they are able to assume the lemon-shape (e-/), and finally, that the lemon-shape of a cell attained by budding (k, k', k"} may be lost again on the Fig. 96. — Sa.ee/taruini/ces apiciUatug (after Hansen). — Budding cells : a-a", a cell which in the course of :ij hours developed a bud at its lower extremity ; b-b", a similar series, showing the development of a bud at the upper extremity of the mother-cell, whilst a bud had l>een previously formed at the opposite end ; c is a chain of cells, c' is the same three-quarters of an hour later ; the lowest bud had assumed the typical form of the species like those above it, but in the figure it is seen from the end, so that its longitudinal axis is at right angles to the plan« of the paper ; d-d", development during 1J houra ; e-e"', during 2J hours ; /•/"", during 3 hours : in e-/it is seen that the oval cells first develop a bud and only subsequently assume the typical lemon-shape ; g-m, abnormal cells : progressive development. formation of a new bud (k'"). Under other conditions the cells may assume quite different forms, sausage-shaped, crescent- shaped, like bacteria, etc. (g-m). Does any rule govern this apparent confusion ? It has just been shown that the fungus 404 MICRO-ORGANISMS AND FERMENTATION. can form two kinds of buds, and that the oval buds must develop one or more new buds before they can assume the typical form. The question then is : Under what conditions are those two kinds of buds developed ? It was shown by means of culture experiments that the lemon-shaped buds are developed especially during the early stages of the culture, but are afterwards crowded out by the oval forms. A further description of the fungus from a physiological and biological standpoint will now be given. Sacch. apiculatus is a bottom-fermentation yeast, capable- of exciting alcoholic fermentation in beer-wort ; the fermenta- tion in this liquid is, however, a feeble one, only 1 per cent. by volume of alcohol being produced, whilst a bottom-yeast under the same conditions gives 6 per cent. This arises from the fact that Sacch. apiculatus cannot ferment maltose. Hansen also found that it does not secrete invertase. On the other hand, it excites a vigorous fermentation in 15 per cent, and 10 per cent, solutions of dextrose in yeast-water, and in one experiment as much as 3 per cent, by volume of alcohol was formed. After three months the liquid still gave the sugar reaction whilst the amount of alcohol had not increased during the last six weeks. The fungus was thus unable to complete the fermentation. In another of Hansen's experiments as much as 4-3 per cent, by volume of alcohol was produced. According to Rohling, the application of oxygen increases the formation of alcohol by 5 to 8 per cent., both the life energy and the power of resistance of the cells being greatly increased. Without oxygen, alcohol formation amounted to- 2-3 to 3 per cent. The cells are very sensitive to chemical reagents. Sulphurous acid (0-025 per cent.) almost entirely prevents its fermentative activity (sulphuring casks), and alcohol acts very restrictively. On the other hand, tannin only acts at a strength of 0-5 per cent. It was found from experiments, in which a mixture of this fungus with Saccharomyces cerevisice was grown in beer-wort, that it was crowded out by the latter, being the weaker species, although it retarded the growth of Sacch. cerevisice to no small degree. In flasks with the same beer-wort, and at the same tempera- SACCHAROMYCES APICULATUS. 405 ture, each containing one species, Saccharomyces apiculatus will multiply to a greater extent than the brewery yeasts in a given interval of time. At the critical time of the year, the ferment, if present in the wort in considerable quantities, may exist for a length of time side by side with brewery yeasts, and will no doubt retard its action a little ; but when the beer is transferred to the lager cellar, the fungus remains inactive in the alcoholic liquid, and frequently perishes. According to Will, the fungus frequently occurs in slight traces in low-fermentation beer-yeast ; it may be caused to multiply more freely by treatment with tartaric acid. Will mentions a case in which the yeast in a brewery in Baden was so strongly infected with apiculatus that these could be directly detected by their characteristic form, and, indeed, were seen in large numbers. In the cask store and in the deposit from diseased beer, cells of S. apiculatus are occasionally found in considerable quantity in a living con- dition, along with wild yeasts, and the flavour of the beer may then be influenced. Miiller-Thurgau and Wortmann regard the fungus as injurious to wine, for it not only directly prejudices the quality of the wine and must, but also checks fermentation, and thus gives rise to disease. The organic non- volatile acids (tartaric and malic acid) present in fruit juices and grape must disappear by the culti- vation of S. apic. It is possible that they serve as sources of carbon for yeast, or else that they are decomposed in the fermentation process. As fast as the acid is consumed a fresh formation of acid takes place. Meissner has shown that 8. apic. can produce lactic acid ; succinic acid is also produced. According to P. Lindner, fruit ethers are formed. Will found that certain species cause the production of a bouquet resembling amyl ether : others give a fusty smell. According to Schander the pro- nounced " Bockser " taste occurring in wines may be produced by certain species of S. apic. Proteolytic enzymes are present, and gelatine is readily liquefied by the organism. Many varieties or races of S. apic. are known. This was 406 MICRO-ORGANISMS AND FERMENTATION. first observed by K. Amthor. One race produces more volatile acid or more alcohol and glycerine than another. Miiller-Thurgau subjected seven different races to exami- nation. They produced from 2-5 to 3-8 per cent, by weight of alcohol in grape juice ; two races gave as much as 6 per cent. Schander found cells of different shape and size in the apiculatus yeasts that he examined, some short, thick, and lemon-shaped, and others thin, elongated, and of a less distinct lemon-shape. Holding's different species exhibited slight physiological differences, and Will, who isolated a few species from wort, showed that they can be distinguished by the different aroma produced in wort. P. Lindner has described Sacch. apic., var. parasitic/us, living on wood-lice. One end of the cell is often drawn out to a point which penetrates their eggs and then forms a bud. In this way the eggs infect the mother insect, and the yeast is distributed by their offspring. They cannot be cultivated in fruit juice or in artificial nutritive liquids. We are dealing here with an obligatory parasite. The question how far 8. apic. is capable of forming endo- genous spores was thrashed out long ago ; in 1894 it was answered in the affirmative by Beijerinck. He stated : — " If a convincing proof is required of the property of spore- formation, it is only necessary to isolate this yeast when conveyed by air or dry dust on to fruit. In this way cultures are occasionally met with, containing individual cells swollen to asci, with from four to six ascospores." He did not, how- ever, observe a germination of the spores. P. Lindner examined cells of S. apic. from blossoms of Eobinia Pseudacacia in streak-cultures with wort contained in moist chambers, and found a development of spores in the culture. They show a distinct wall and a granulated pro- toplasm. According to the picture, each cell contains only one spore. Again, no germination of spores was observed. Descendants of this growth showed no tendency to form spores. Influenced by these experiments, Lindner classed the apiculatus yeast as a new genus, which he named Hansenia. A. Rohling worked with pure cultures of races derived MYCODERMA. 407 from samples of soil, and succeeded in bringing about spore- formation by cultivation on gypsum blocks for eight to ten days (the temperature is not mentioned). He also succeeded in causing the spores to germinate in an extract of horse dung with 5 per cent, of grape sugar. It may also be mentioned that J. C. Holm detected lemon-shaped cells, somewhat larger than the ordinary apiculatus cells in certain fermented ciders from England, in the author's laboratory in 1894. By trans- ference to gypsum blocks after a single cultivation in dextrose- yeast water, isolated cells were observed with one or two spores. On account of the extraordinarily small number of spore-forming cells, it was impossible to carry out germinating experiments. By prolonged cultivation and transference of cells to gypsum blocks, to nutritive gelatine, or to small quantities of sterile water, he never again succeeded in bring- ing about spore-formation. During budding, a septum was observed between the mother- and daughter-cell. J. C. Holm assumes that this form is closely related to one of the 8. Lud- wigii (Saccharomycodes Ludwigii), and that possibly the work just mentioned was carried out with such forms, and not with 8. apic. Reess. Mycoderma cerevisiae and vini. It is characteristic of these species that they very readily form films on various alcoholic liquids. Under these names are included a number of different species, some of which may excite a feeble alcoholic fermentation ; they behave differently towards lager beer, some causing disease whilst others do not. The Mycoderma cerevisice (Fig. 97) examined by Hansen, which is universally met with in Copenhagen breweries, forms variously-shaped cells. The cells are usually transparent and less refractive than the true Saccharomycetes ; in each cell there are generally one, two, or three highly refractive particles, which often have a quivering, rolling motion. This micro- organism forms a dull, greyish, wrinkled film on wort and beer, and does not excite alcoholic fermentation ; neither does it invert solutions of cane-sugar. The colonies on the surface of the gelatine are light grey, dull, and spread out like a film or hollowed like a shell. By 408 MICRO-ORGANISMS AND FERMENTATION. means of this macroscopic appearance Mycoderma is readily distinguished from the ordinary Saccharomycetes, which, on the same medium, form light greyish-yellow colonies with a dry or lustrous surface, and a more or less arched form. Sacch. membrancefaciens, which differs so markedly in its biological behaviour, and which very rapidly gives a strong film on the liquid, alone resembles Mycoderma in its behaviour on plate cultures. This kind of film-formation was noted by Hansen when lager beer had been exposed in open vessels at temperatures between 2° and 15° C. ; at 33° C. development still occurred, but at temperatures above 15° C. this species gave place more and more to competing forms. As low temperatures are favourable to its develop- ment, it will readily thrive in the storage cellar, especially as lager beer forms a much more favourable medium for its growth than wort. This is seen to be the case when traces of a pure film are introduced into lager beer and wrort, contained in open vessels, and then left to develop ; the culture in lager beer nearly always remains pure, while in wort various other species make their appearance. In Hansen's comprehensive experiments on Carlsberg beer, it was always found that both lager and export beers were attacked by this fungus ; but there was never the slightest indication that the beer had acquired any disease from this source. The fungus was widely distributed just at those periods when the beer was found to be particularly stable and of good flavour. This has also been confirmed by numerous experiments on lager and export beers conducted by Gronlund and A. Petersen, and those carried out in the author's labora- tory. It is self-evident that we are only speaking of beer which has been properly treated. In imperfectly closed bottles and casks, Mycoderma cerevisice will of course rapidly Fig. 97. — Mycoderma cerevisice from Copenhagen brewerie^ (drawn from nature by Holm). MYCODERMA. 409 develop a film, which is sufficient, unaided, to destroy the product. Belohoubek was the first to observe that, under certain conditions, Mycoderma may cause considerable injury in the brewery. Subsequently, Kukla described a curious cloudiness in lager beer, having the appearance of a cloud of fine dust in the liquid, which manifested itself either during storage or after tapping ; he attributes this disease to Mycoderma, and further assumes that it is weak wort, having certain peculi- arities in its composition, which specially favours the develop- ment of Mycoderma. It is to be hoped that further investiga- tions will throw more light on this subject. Hansen expressed the opinion that the name Mycoderma cerevisice denotes not one, but several different species, and Lasche's experiments subsequently confirmed this. The latter investigator describes four different species which he isolated from cloudy beers. They are distinguished from the species described by Hansen by the fact that they produce alcohol in beer- wort ; one yields 0-26 per cent, by volume, two yield 0-79 per cent., and the fourth produces 2-51 per cent. Lasche concludes from his experiments that these four species cause diseases in beer, both turbidity and changes in taste and odour ; in this respect they also differ from Hansen's Myco- derma. Lasche is inclined to assume that the chemical com- position of the wort has no influence on the disease caused by Mycoderma, for, in his experiments, the disease was pro- duced in worts of high extract and worts of low extract, in worts rich in sugar and worts poor in sugar. Winogradsky found that the Mycoderma occurring in wine, prepared in pure culture by Hansen's method, alters its shape with the composition of the nutritive solution ; he experi- mented both with solutions, the mineral constituents of which remained constant while the organic substances varied, and also with solutions in which the reverse was the case. Many experimenters have subjected Mycoderma to close investigation during the last few years, and especially Henne- berg, Heinze, Meissner, Seifert, and Will. Meissner's researches are particularly comprehensive, both morphologically and physiologically, and they concern twenty- three different species 410 MICRO-ORGANISMS AND FERMENTATION. of Mycoderma vini. Greater or less distinctions were noted in the shape of the cells, their content in glycogen, the presence of oil drops, and in their giant colonies. The film-formation also showed distinct differences with different species, both with regard to the time required for their appearance, their character, and colour (white, cream, yellowish-brown, and yellowish-olive-green). When covered with the film, the liquor remains clear in certain cases, whilst in others a turbidity takes place of a permanent or temporary character. Will believed that the decolorisation of the liquid may be referred to the formation of acid brought about by the film cells, whereas Heinze thought that it was caused by the removal of acid. Meissner confirmed the fact that a more or less marked decolorisation takes place, but showed that afterwards a. reversal of the colour tints may come about, so that must which had turned pale assumed by degrees a dark brown colour. Not only is the total amount of acid destroyed, but the must at last acquires an alkaline reaction. Meissner succeeded in proving experimentally both with large and small amounts of must, that one and the same race may appear, first as a producer, and then as a destroyer of acid. It is, therefore, necessary to forego the division of film yeasts, into acid-destroying and acid-forming species. Once the sugar in the must is destroyed, all so-called acid ferments will act as destroyers of acid. With regard to this question of the formation and destruction of acid, Meissner also states that when an increase of acid takes place it must be regarded as a- result of two simultaneous processes of construction and destruction of acid, and that production has exceeded destruc- tion. If, on the other hand, a reduction of acid takes place, the destructive action must be regarded as exceeding the constructive. Butyric acid is formed amongst others, and ammonium compounds are also produced. Meissner's experiments regarding the reaction of the Mycodermas with organic acids gave the following results : — Malic acid is only very slightly attacked by certain races, but strongly attacked by others. Tartaric acid is slightly decomposed. Lactic, citric, and succinic acids are in some MYCODERMA. . 41 1 cases strongly affected. Acetic acid reacts strongly with a few species ; in other cases the species cannot grow at all . in a solution containing acetic acid. Alcohol, the sugars, glycerine, and tannic acid are decom- posed. Alcohol is converted by an oxidation process into- carbon dioxide and water, but may also act as an organic foodstuff. As early as 1878 Schulz found that " the film fungus can produce within itself ready formed organic com- pounds, and requires nothing but ammonia and alcohol for the purpose." Schulz did not, however, work with pure cultures. Meissner utilised for his experiments both the nitrogenous- nutritive material used by Schulz (ammonium nitrate, aspar- agin, ammonium tartrate) and an artificial solution containing ammonium phosphate and ammonium chloride, together with the necessary mineral constituents. The vigorous growth of Mycoderma proves that these two solutions supply them with nitrogen. Consequently the alcohol of the nutritive solution is partially respired and partially utilised for the building up of the cells. With regard to the sugars, Meissner found that the Mycoderma species cultivated on sterile grape juice respired dextrose and laevulose to some extent, partially producing acids from them. On artificial nutritive solutions which contain dextrose or saccharose as the only organic substance in addition to the necessary mineral nutrients, the sugars are oxidised, part being utilised for the construction of new cells, and part for the fresh formation of acid. Glycerine is not only destroyed, but may be produced from other organic substances. This fact was confirmed by W. Seifert. The Mycodermas also produce various volatile acids. Thus Wortmann has drawn attention to the fact that many wines which have become filmy have an odour which resembles rancid butter to an extraordinary degree. Butyric acid has been formed in these cases. Lafar found in a cask store a Mycoderma which imparted a flavour to beer resembling wine ether. Non-volatile acids and esters are also formed. In finished wines the yeast has generally, but not always, finished its special work, but nevertheless wine may undergo 412 MICRO-ORGANISMS AND FERMENTATION. fundamental alterations caused by other micro-organisms, and amongst them, according to Wortmann, Mycodermas take the first place. By their agency, alcohol is converted into carbon dioxide and water, and they also influence the amount of acid and destroy the bouquet. A wine may become filmy, and may deteriorate in time without a visible covering of film appearing on its surface. The suspended cells of Mycoderma, which need not be numerous, may bring about in the course of years the same action which would occur more rapidly when the cells form a coherent mass on the surface. Mycodermas which often inhabit corks (like the moulds) may impart the well-known corked taste to wine. Will has isolated a Mycoderma from top-fermentation beer, which brings about a marked decolorisation of beer. This occurs within a short time at the high temperatures requisite for completing top-fermentation. The species develops a large amount of acid in beer, but under certain conditions a destruction of acid may also take place. It cannot bring about alcoholic fermentation. Will also made a series of observations concerning the duration of life in wort-cultures and in the dry state, and with regard to the power of resistance to heat in liquids. Seifert closely examined two Mycoderma species isolated from wine, which produced from 0-064 to 0-904 per cent, of acetic acid in an ordinary Austrian white wine, and reduced the amount of alcohol. The Mycoderma investigated by Heinze (If. cucumerina, Aderhold) was derived from a fermentation of sour cucumbers : he declares it to be a dangerous enemy of lactic acid fermentation. In beer the organism attacks alcohol strongly, and produces a bitter flavour. It is capable of producing alcoholic fermentation in 'dextrose solutions. In cider, with 10-62 grammes of sugar, it yielded 4-34 grammes of alcohol per 100 c.c. of fermented liquid, in five minutes, at 25° C. There is no fermentation with maltose, saccharose, and lactose. Heinze also closely investigated the question of acid production and acid destruction. Henneberg mentions two species of Mycoderma, which he frequently found in distillery and pressed yeast. The shape of the cells is very varied. The one species frequently MYCODEUMA. 413 forms mycelial chains, and the other Monilia-like chains. The difference between the two forms is specially marked in cultures on solid substrata (giant-cultures, streak-cultures, etc.). In dextrose and laevulose solutions bubbles of carbon dioxide form under the film, and a fairly vigorous fermentation is- produced by the cells, which sink to the bottom. Both speciea produce acetic ether. As is the case with S. anomalies, the optimum temperature for growth lies between 32° and 41° C. Dextrose and laevulose are readily fermented, galactose lesa readily, only traces of maltose and dextrin are fermented, whilst lactose, saccharose, raffinose, and inulin are not fer- mented at all. The two species can readily utilise lactic acid as food, and withstand up to 5 per cent, of the acid. Simi- larly they can withstand large quantities of alcohol (11 per cent.). The alcohol in this case is fairly quickly converted into carbon dioxide and water. These species are not, a& might be supposed, capable of withstanding large quantities of organic acids in general, as was proved by a few experiments with increasing quantities of acetic acid in beer. In Egyptian Leben (Leben raib), Hist and Khoury found a Mycoderma about which they say that it is improbable that it has any particular influence on the special flavour of the Leben, but in any case the rapid development of a sharp acid taste, which renders the beverage undrinkable in a few days, must be ascribed to this organism. It forms both non-volatile acids and acetic acid. It grows excellently in glucose and maltose, and gives a fermentation with the former, whilst it converts glucose into acid, and brings about the combustion of alcohoL In lactose solution it gives no fermentation, only film-for- mation. Although de Seynes, Reess, Engel, and Cienkowski claimed to have found ascospores in Mycoderma, it has since proved impossible to bring about their formation. It would appear from the drawings given that the fat globules, which occur in many unicellular fungi during the resting stage, had been mistaken for spores ; in some cases the mistake appears to have arisen through the presence of an admixture of true Sac- charomycetes. The old name Mycoderma is, therefore, more appropriate to this fungus than the new term Saccharomyces. 414 CHAPTER VI. THE PURE CULTURE OF YEAST ON A LARGE SCALE. Industrial Application. the industrial application of pure cultures of systematically selected yeasts inaugurated by Hansen and the author, it became possible to carry out fermentations with certainty in a way that was impossible so long as an unknown yeast- mass was used containing not only a mixture of culture yeasts, but also wild yeasts, bacteria, and even moulds in certain oases. Such selected races can be preserved by appropriate means for a long time as small cultures which can be developed afresh into mass cultures.* One very important result of the adoption of the process was to prove that the visible fermentation phenomena do not in general give any insight into the purity of the fermentation. On the other hand, these phenomena may sometimes give valuable information regarding the condition of the yeast, which is directly connected with the nature of the nutritive liquid. A real knowledge of the purity of fermentation can only be gained by a biological analysis combined with a micro- scopical examination. Pasteur demonstrated the harm that bacteria can do when they develop in alcoholic fermentation, and at the same time he emphasised the importance of the oxidation of the nutritive * The important point in preparing a pure culture is the selection of the right .species ; not the purely mechanical isolation of an individual cell. It demands insight into the particular branch of the industry concerned. The point to bear in mind in preparing such cultures is that a yeast actually in use in the industry must form the starting point. PURE CULTURES IN PRACTICE. 415 liquid for yeast activity. Hansen experimentally proved that some of the most dangerous diseases of beer are caused by wild yeasts.* When the absolutely pure culture developed in flasks in the laboratory has attained certain dimensions — e.g., 1 to 2 litres of fluid yeast — it is ready for practical application. In the vast majority of cases the pure culture is further de- veloped in a couple of small vats in which successive quantities of the nutritive medium are added in each case as soon as a vigorous development has taken place — e.g., from 5-50-100- 300 litres or from 5-50-500 litres, etc., depending on whether the yeast is a low- or a high-fermentation species. As a rule, the chief point to observe is that the mass of cells should be brought as quickly as possible to development, until the requisite quantity is secured for carrying out the normal fermentation on a large scale. In the preliminary stages the same nutritive liquid must be used as in the large fermenta- tions. It will be obvious that during such a rapid development the specific character of the race of yeast will not be brought out. If it is desired to observe this during the small fer- mentations, they must be carried through to completion. If a regular supply of absolutely pure yeast must be kept in stock, it is necessary to use the pure propagating apparatus designed by Hansen and by A. Kiihle. The apparatus (Fig. 98) consists of three chief parts and the necessary connecting tubes. First, the air apparatus, with air pump (A) and air holder (B), secondly, the fermenting- cylinder (C), and, thirdly, the wort-cylinder (D). The air, which has previously been partially purified, is pumped into * The " natural " selection of yeasts proposed by Delbriick must not be confounded with the preparation of a single pure race. His process consists in subjecting the whole impure yeast-mass to a treatment which may consist of the application of a higher fermentation temperature, or pumping into a new vat after the appearance of foam on the surface, or pitching with wort from the first stages of fermentation, etc. A summary process of this kind will always yield an uncertain result, because the impure mass contains elements of very different character, and, even in the most favourable case, if by good luck the disease germs are restricted, it is evidently impossible to depend on securing the best-selected type of culture yeast. It is essential to isolate the yeast species and then to select those which best fulfil the stated requirements. 416 MICRO-ORGANISMS AND FERMENTATION. the receiver, and thence may be passed into either the wort or the fermenting cylinder. In either case the air is sterilised by means of a cotton-wool filter (g, m). The wort cylinder is directly connected with the copper from which the boiling hopped wort is run in ; it is then aerated in the closed cylinder, and is cooled by spraying. Fig. 98. — Yeast-propagating apparatus devised by Hanseu and Kiilile. — A, air-pump; B, air- vessel ; C, fermenting -cylinder ; a, window ; It, b, b, stirrer ; c, c, doubly-bent tube ; d, vessel containing water ; I, outlet cock ; /, /, glass tube connected at e and h with the cylinder, and graduated for the measurement of fixed quantities of liquid ; tj, filter ; i, rubber connection for glass tubes ; j, tube with rubber connection for introducing the pure culture ; k, k, connection with the wort-cylinder D ; m, filter ; n, n, doubly-bent tube ; o, vessel containing water ; p, pt (•praying tube ; u, connection with cock * ; t, waste for cooling water. The wort is then forced into the fermenting-cylinder, which, like the wort-cylinder, is constructed on the same principle as the ordinary two-necked flask. It is fitted with a doubly-bent tube (c, d), which dips into a vessel containing water ; a vertical glass tube (/, i, /) for measuring the height of the liquid in the PURE CULTURES INT PRACTICE. 417 cylinder ; an appliance (b, b) for stirring up the deposited yeast, and a specially constructed cock (I) for drawing off the beer and the yeast. At about the middle of the cylinder there is a small side tube (j), fitted with india-rubber connection, pinch-cock, and glass-stopper. When a portion of the wort has been forced into the fermenting- vessel, the absolutely pure yeast — which is forwarded to the brewery in a flask specially constructed for this purpose — is introduced through the rubber tube at j ; this is again closed, and the remainder of the wort may then be added either at once or after the lapse of a few days, according to the quantity of yeast introduced. Where it is necessary to regulate the temperature during fermentation, the fermenting- vessel is surrounded by a water- jacket. By means of this simple apparatus it is possible to obtain, at short intervals, absolutely pure pitching yeast, sufficient for about eight hectolitres of wort, and when once started the apparatus works continuously. Another type of propagating apparatus has been described by Bergh and Jorgensen (Fig. 99). The filtered air passes through the three-way cocks at A, B, and C, into the two cylinders A and" B. The upper cylinder holds about 50, the lower cylinder 160 litres. A is provided with a stirrer E, and a tube (a) for introducing the yeast and withdrawing samples. The bent tube F is an outlet for carbon dioxide. The tube G P connects the two cylinders, and the connection can be made or broken by means of the cock O. H is the outlet for water used in cleaning A. The cylinder B is surrounded by a cast-iron jacket made in two parts ; the upper portion serves as a water-jacket for cooling the wort and for regulating the fermentation ; the lower portion is used as a steam-jacket, and is provided with a cock at O as an inlet for the steam, and another at 8 as an outlet. M is a ring-shaped tube provided with small holes ; this is connected with the cold-water main during the cooling of the wort ; the water is drawn off at N. The stirrer J is set in motion by means of toothed gear. The height of the liquid in the cylinder is indicated by means of a float, with pointer and arc L. A bent tube, K, projects from the top of 27 418 MICRO-ORGANISMS AND FERMENTATION. the cylinder. At the bottom is the cock Q, which is connected with the pipe b by cock T. Both the bent tubes dip into the vessel R, which is filled with water. The wort is introduced into the lower cylinder, where it is treated in the ordinary manner. The pure culture is intro- duced into the upper cylinder, and is then washed down into the lower cylinder by means of a little wort, which is forced from B into A, and then back again into B. When a vigorous multiplication of the yeast has set in, the liquid is stirred up, and a portion forced into A ; this is to be used to start the next fer- mentation. The cylinder B thus serves alternately as • fermenting- and wort- cylinder. A comprehensive intro- duction to the method of dealing with the apparatus used in the laboratory for the preparation of pure cultures (moist chambers and flasks) is to be found, along with the mode of operating the two types of propagating apparatus, in a small hand-book of the author's, entitled Practi- Fig. 99.— Yeast-propagatinp: apparatus devised by Cal Management of Pure Bergh and Jorgensen. -*7 , T j , nnn •»«• j- Yeast, London, 1903. Modi- fications of both forms of propagating apparatus have been described by Brown and Morris, Elion, Thausing, Van Laer, Pohl and Bauer, Wichmann, Fernbach, Jacquemin, and others. P. Lindner and Marx have constructed a somewhat different apparatus. PURE CULTURES IN PRACTICE. In order to be able to send the selected pure cultures in a liquid condition to a distance, special forms of flasks were devised by Hansen (Fig. 100) and by the author (Fig. 101). The yeast can be sent a great distance in these flasks, and there is no difficulty in safely transferring it from the flask to the fermenting-cylinder of the propagating apparatus. In sending small quantities of pure cultures, in such a manner that they may be safely and readily employed for further cultivation, the small Hansen flasks are employed Fig. 100. Kig. 101. (p. 37). They are connected, in the flame, with the Pasteur flask in which the pure culture has developed. A trace of the yeast is transferred to the cotton-wool, and the flask is again •closed in the flame with the asbestos stopper, which is then -coated with sealing-wax. When the culture is to be used, the flask is again connected with a Pasteur flask containing wort, and the yeast is rinsed into the latter. This process has made it possible to send large collections 420 MICRO-ORGANISMS AND FERMENTATION. of pure cultures to the most distant countries at a very small cost. It is of the greatest importance to note that, even after the lapse of years, the particular yeast once selected can always be procured again, a sample of the pure culture being preserved in the laboratory in a 10 per cent, solution of cane sugar, kept in the flasks described in Chap. i. (p. 37), devised by the author for the purpose. Culture yeasts may be kept alive in such a solution for years without any alteration in their properties. It is of importance, in order that the culture yeasts may be kept unaltered for a long time, that the layer of yeast deposited upon the bottom of the flask should not be frequently shaken. During the introduction of a few drops into a Pasteur flask shaking can only be avoided by the use of the flask depicted in Fig. 9. With any other variety it is necessary to maintain a number of flasks for each species of yeast, and each one will only serve for a few infections. On the other hand, no effect of temperature has been observed during storage, and the dilution of the liquid can be avoided by the use of the two flasks constructed by the author, and shown in Figs. 8 and 9. All physiological laboratories con- cerned with fermentation possess such collections of preserved growths. The author's collection of species which have been gradually introduced into practice dates back to the year 1884, and numbers many thousands of specimens. A few of these species have retained those properties which are of indus- trial value for more than ten years. According to Hansen's and the author's experiments yeasts may be kept alive under such conditions for a much longer period. Regarding the storage of dried yeasts on the large scale, A. Will has made extensive investigations, the results of which will be found in the technical literature. 421 BIBLIOGRAPHY. Adametz, L., Bakteriol. Unters. iiber den ReifungsprozeB d. Kase. Landw. Jahrb. xviii. 1889. Osterr. Molkereizeitung. vi. 1899. vii. 1900. — Saccharomyces lactis, eine neue Milchzucker vergarende Hefeart. Centralbl. f. Bakt. v. 1889. — Uber einen Erreger der schleimigen Milch, Bacillus lactis viscosus. Milch-Zeitung. 1889. — Die Bakterien normaler und abnormaler Milch. Osterr. Monats- schrift fiir Tierheilkunde und Tierzucht. xv. 1890. • — Untersuchungen iiber Bacillus lactis viscosus, einen weit verbreiteten milchwirtschaftlichen Schadling. Berliner landw. 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Brauw. x. 1887. - Neue Bemerkungen zu Foth's Abhandlung : " EinfluB der Kohlen- saure auf Garung und Hefebildung." Wochschr. f. Brau. 1887. - Methode zur Analyse des Brauwassers in Riicksicht auf Mikro- oiganismen. Zeitschr. f. d. ges. Brauw. xi. 1888. - Uber die zymotechnische Analyse der Mikroorganismen der Luft. Prager Brauer- und Hopfenzeitung. 1888. - Untersuchungen aus der Praxis der Garungsindustrie (Beitrage zur Lebensgeschichte der Mikroorganismen). 1. H. Miinchen, 1888. 2nd edit. 1890. 3rd edit. 1895. - Recherches sur la physiologic et la morphologie des ferments alcooliques. VII. Action des ferments alcooliques sur les diverses especes de succe. Compt. rend, du laborat. de Carls- berg. * ii. 5. H. 1888. - Recherches faites dans la pratique de Tindustrie de la fermentation. (Contribution a la biologic des microorganismes.) I. Introduc- tion. II. Culture pure de la levure au service de 1'industrie. 440 BIBLIOGRAPHY. III. Observations faites sur les levures de biere. IV. Sur 1'examen pratique, au point de vue de la conservation de la biere contenue dans les tonneaux des caves de garde. Compt. rend, du laborat. de Carlsberg. ii. 5. H. 1888. Hansen, E. Chr., Uber die im Schleimflusse lebender Baume beobach- teten Mikro-organismen. Centralbl. fur Bakt. v. 1889. — Some points in connection with practical brewing : I. On the bacteriological analysis of the water and the air for brewing purposes. II. On my system of pure yeast-culture and its application in breweries with top fermentation. Transac. of the Laboratory Club. London, 1889. — Production des varietes chez les Saccharomyces. Ann. de micro- graphic, ii. 1890. — Uber die Pilzstudien in der Zymotechnik. Hit besonderer Riick- sicht auf Prof. Dr. Zopf's neues Werk : " Die Pilze in mor- phologischer, physiologischer, biologischer und systematischer Beziehung." Zeitschr. f. d. ges. Brauw. xiii. 1890. • — Nouvelles recherches sur la circulation du Saccharomyces apiculatus dans la nature. Ann. des Sc. naturelles. Botanique. 7. ser. xi. 1890. Annales de Micrographie. 1890. — Qu'est-ce que la levure pure de M. Pasteur ? Une recherche experimental. Compt. rend, du laborat. de Carlsberg. iii. 1. H. 1891. — Recherches sur la physiologic et la morphologic des ferments alcooliques. VIII. Sur la germination des spores chez les Saccharomyces. Compt. rend, du laborat. de Carlsberg. iii. 1. H. 1891. - Neue Untersuchungen liber den EinfluB, welchen eine Behandlung mit Weinsaure auf die Brauereihefe ausiibt. Zeitschr. f. d. gee. Brauw. xv. 1892. - Kritische Unters. iiber eiuige von Ludwig und Brefeld beschriebene Oidium- und Hefenformen. Botan. Zeitung. 1892. — Recherches faites dans la pratique de Tindustrie de la fermentation. (Contributions a la biologic des microorganism.es.) V. Sur 1'analyse zymotechnique des microorganismes de Tair et de 1'eau. VI. Nouvelles recherches sur les maladies provoquees dans la biere par des ferments alcooliques. (Second Memoir.) VII. 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Brauw. xviii. 1895. — Practical Studies in Fermentation, being Contributions to the Life History of Micro-organisms. Transl. by Alex. .K. Miller. London. E. & F. N. Spon. 1896. — Biolog. Untersuch. iiber Mist bewohnende Pilze. Bot. Ztg. 1897. - Uber die Variation bei den Bierhefepilzen und bei anderen Sac- charomyceten. Zeitschr. f. d. ges. Brauw. xxi. 1898. Cen- tralbl. f. Bakt. 2. Abt. iv. 1898. - Recherches sur la physiologic et la morphologic des ferments alcooliques. IX. Sur la vitalite des ferments alcooliques et leui variation dans les milieux nutritifs et a 1'etat sec. Compt. rend, du laborat. de Carlsberg. iv. 3. H. 1898. - Neue Untersuchungen iiber die Sporenbildung der Saccharo- myceten. Centralbl. f. Bakt. 2. Abt. v. 1899. — Recherches sur la physiologic et la morphologic des ferments alcooliques. X. La variation des Saccharomyces. Compt. rend, du laborat. de Carlsberg. v. 1. H. 1900. - Recherches sur les bacteries acetifiantes (Troisieme memoire). 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Weinbau in Geisenheim. 1891-92. — Einige Bemerkungen iiber die Vergarung von Mosten mit reinge- ziichteter Hefe. Weinbau u. Weinh. 1892. — Weiteres iiber die Vergarung von Mosten mit reingeziichteten Hefen. Weinbau u. Weinh. 1892. — Untersuchungen iiber reine Hefen. I. Landw. Jahrb. v. Tiel. 1892. — 1. Reinziichtung verschiedener Rassen von Weinhefe ; Versuche iiber die Gartatigkeit derselben und ihre Anwendung in der BIBLIOGRAPHY. . 479 Praxis. 2. Weitere Garversuche mit reingeziichteten Wein- hefen. Ber. d. Kgl. Lehranstalt f. Obst- und Weinbau zu Geisenheim a. Rh. Wiesbaden, 1893. Wortmann, J., Mitteilung uber die Verwendung von konzentriertem Most fur Pilzkulturen. 3otan. Ztg. li. 1893. - Untersuchungen uber reine Helen. II. Landw. Jahrb. 1894. - Anwendung und Wirkung reiner Hefen in der Weinbereitung. Berlin, 1895. - Uber Saureabnahme im Wein. Centralbl. f. Bakt. 2. Abt. iii. 1897. - Uber kiinstlich hervorgerufene Nachgarungen von Weinen in der Flasche und im Fasse. Landw. Jahrb. 1897. - Vorkommen u. Wirkung lebender Organismen in fertigen Weinen. Landw. Jahrb. 1898. - Untersuchungen iiber das Bitterwerden der Rotweine. Landw. Jahrb. 1900. - Uber die Bedeutung der alkoholischen Garung. Weinbau u. Weinh. 1902. - Die wissenschaftl. Grundlagen d. Weinbereitung u. Kellerwirt- schaft. Berlin, 1905. Yabe, K., On the Origin of Sake Yeast (Sacch. Sake). Imp. University Coll. of Agriculture Bull. iii. 1897. Yung, Des poussieres organisees de I'atmosphere. Arch, des sc. phys. et nat. iv. 1880. Zacharias, E., Beitrage zur Kenntnis des Zellkerns und der Sexual- zellen. Botan. Ztg. xlv. 1887. Zalewsky, Uber Sporenbildung in Hefezellen. Verb. d. Krakauer Akad. d. Wiss. Math.-naturw. Sektion. 1. xii. 1885. Zeidler, A., Beitrage zur Kenntnis einiger in Wiirze und Bier vorkom- menden Bakterien. Wochenschr. f. Brau. vii. 1890. — Uber eine Essigsaure bildende Termobakterie. Centralbl. f. Bakt. 2. Abt. ii. 1896. Zimmermann, A., Die Morphologic u. Physiol. d. pflanzl. Zellkerns. 1896. Zimmermann, 0. E. R., Bakteriologische Diagnostik. Chemnitz. — Die Bakterien unserer Trink- und Nutzwasser, insbesondere der Chemnitzer Wasserleitung. I., II. Berichte der naturwiss. Ges. Chemnitz. 1890, 1894. — Die Peronospora-Krankheit des Weinstocks. Centralbl. f. Bakt. ii. 1887. Zopf, W., Die Konidienfriichte von Fumago. Nova acta Acad. Leop. Carol, xl. 480 BIBLIOGRAPHY. Zopf, W., Entwicklungsgeschichtl. Untersuchungen iiber Crenothnx polyspora. Berlin, 1879. — tJber den genetischen Zusammenhang der Spaltpilzformen. Sit- zungsber. d. Berl. Akad. d. Wiss. 1881. — Zur Morphologic der Spaltpflanzen. (Crenothrix). Leipzig, 1882. — t)ber 'Bacterium merismopedioides. Sitzungsb. des bot. Vereins d. Prov. Brandenburg. Juni, 1882. — Die Spaltpilze. Breslau. 3rd edit. 1885. — Oxalsauregarung (an Stelle von Alkoholgarung) bei einem typischen (endosporen) Saccharomyceten, Sacch. Hansenii n. spec. Ber. d. Deutsch. botan. Gesellsch. vii. 1889. — Die Pilze in morphologischer, physiologischer, biologischer und systematischer Beziehung. (Enzyklopadie der Naturwiss. 1. Abt. 1. Teil. Handb. der Botanik, edited by Schenk. iv. pp. 271-781). Breslau, 1890. Also published as a separate work. — See Liesenberg. 481 INDEX. ACETIC acid, 268. „ bacteria, 92, 23S. „ ,, influence on beer, 104. „ „ influence on wine, 105. „ „ Staining, 7, 95. Acetone, 27. Aeration of wort, 280. „ yeast, 279. Aerobic bacteria, 82. Aeroscope, 60. Agar, 50. Air, Biological examination of, 59. „ Filtration of, 17, 62, 233. ,, Hansen's investigation of, 65. „ Saito's „ 69. „ Sterilisation of, 23. Albuminoids in yeast cells, 9, 228. Alcohol, Amyl, 268. Ethyl, 267. „ Methyl, 268. „ as disinfectant, 28. „ -forming bacteria, 133. Alcoholase, 249. Alcoholic fermentation, Products of, 267. Aldehyde, 268. Ammonium fluoride, 27, 32. Amoebobacter, 80. Amyl alcohol, 268. Amylase in moulds, 256. Amylobacter butylicus, 134, 135. „ ethylicus, 134. Amylomyces (ft), 205. „ Rouxii, 203. Anaerobic bacteria, 54, 82. Anaerobiosis, 240. Analysis of yeast, 311. Anatomy of yeast cells, 290. Antiformin, 27, 33. Antigermin, 33. Antinonnin, 34. Antiseptics, 24, 32. „ Accustoming fungi to, in- creasing doses, 30. „ influence on bacteria, 87. ,, „ on fungi, 178. „ Stimulating action of, 29, 174. Apothecia, 186. Ascococcus Billrothi, 150. Ascogonium, 193. Ascospore formation, 302. Ascus, 169. Aspergillus batatse, 197. flavescens, 182. flavus, 194. fumigatus, 182, 194. glaucus, 182, 191. niger, 174, 194. Oryzae, 195. repens, 174. Wentii, 197. Asporogenesis, 284. Autoclave, 15. Auto-fermentation, 269. Awamori, 395. B BACILLE amylozyme, 134, 135. Bacillus acidi lactici, 107, 109. „ acidificans longissimus, 120. boocopricus, 129. Buchneri, 120, 125. Bulgaricus, 140. butylicus, 134. butyricus, 127. casei, 109. caucasicus, 136. coli communis, 111. cyanogenus, 114. 31 108, 482 INDEX. Bacillus cyanofuscus, 116. „ Delbrucki, 108, 120. „ ethaceticus, 134. „ Fitzianus, 133. „ fluorescens liquefaciens, 115, 151. „ fluorescens putidus, 132. ,, foetid us lactis, 115. „ gummosus, 144. „ Hayducki, 120, 125. „ lactis aerogenes, 145. „ „ pituitosi, 145. „ „ saponacei, 113. „ „ viscosus, 114, 145. „ Leichmanni, 125. Lmdneri, 122. „ Listeri, 125. „ lupuliperda, 132. „ luteus, 153. „ Megatherium, 151. „ mesentericus vulgatus, 143. „ nobilis, 112. „ orthobutylicus, 130, 134. „ panis fermentati, 124. „ „ viscosi, 143. „ pneumoniae, 134. prodigiosus, 153. putrificus, 153. rudensis, 116. saccharo-butyricus, 130. Schafferi, 115. subtilis, 152, 175, 182. thermophilus, 86. vasculorum, 151. viscosus, 146. „ bruxellensis, 147. „ sacchari, 142. „ vini, 142. vulgaris, 111, 151. Wortmanni, 125. Zeidleri, 100. Bacteria, 76. „ acetic acid, 92. „ influence on beer, 104. „ „ influence on wine, 105. „ Aerobic, 82. „ Alcohol-forming, 133. „ Anaerobic, 54, 82. „ Butyric acid, 125. „ cell contents, 78. „ wall, 79. „ chemical composition, 81. „ colouring matter, 80. „ fermenting cellulose, 132. „ Forms of, 76. „ influence of antiseptics, 87. Bacteria, influence of light, 86. „ pressure, 87. „ temperature, 85. „ vibration, 87. involution forms, 78. iron, 162. lactic acid, 106. Migula's system of, 89. Nitrifying, 164. nutrition, 81. organs of motion, 6, 83. Phosphorescent, 80. Propagation of, 83. Slime-forming, 142. Spore-forming, 84. Sulphur, 164. with diastatic enzymes, 153. „ inverting enzymes, 151. „ proteolytic enzymes, 153. Bacteriocysts, 80. Bacterium aceti, 82, 95. aceticum rosaceum, 104. acetigenum, 101. acetosum, 101. ascendens, 102. curvum, 102. gelatinosum betae, 150. gummosum, 143. industrium, 101. Kiitzingianum, 96. lactis acidi, 107, 109, 110, 112, 124. „ erythrogenes, 115. „ longi, 114. mannitopaeum, 123. orleanense, 103. oxydans, 100, Pastorianum, 96. prodigiosum, 111, 115. Sacchari, 151. Schiitzenbachi, 102. soya, 196. synxanthum, 115. vermiforme, 141. vini acetati, 1 03. xylinoides, 103. xylinum, 100. Bacteroids, 80. Beer, Belgian, 121. Cloudiness of, 9. Diseases in, 256. Filtration of, 16. Lactic acid in, 121. Sarcinae in, 155. Slime-forming bacteria in, 145. Sterilisation of, 23. " Weissbier," 121, 341. -wort, Action of yeasts on, 261. INDEX. 483 Beer wort, Aeration of, 280. „ „ Production of acid in, 263. „ ,, „ alcohol in, 261. „ „ „ glycerine in, 261. Beggiatoa alba, 164. Biological examination of air, 59. „ „ ice, 71. ,, „ water, 69. „ relationships of yeasts, 270. Blastoderma salmonicolor, 398. Botrytis cinerea, 176, 183. Bottcher's moist chamber, 12, 44, 51. Bottom fermentation yeasts, 283, 285, 311, 318, 320, 323. Bouquet in wine, 265. Brewery yeasts, 317. Broth, Nutritive, 41. Butter, Abnormal, 115. „ Torula yeasts in, 400. Butyric acid bacteria, 125. CALCIUM, 172. „ bisulphite, 34. Carbohydrates in fungi, 182. „ Reaction of yeasts with, 258. Carbolic acid, 28. Carbon, 173. Carlsberg bottom yeast I., 261, 281, 320, 323. II., 261, 284, 320, 323. flask, 39. Carragheen moss, 42. , Cell nucleus, 5, 300. „ wall, 300. Cellulose, Bacteria-fermenting, 132. Chalara mycoderma, 217. Chamberland flask, 37. Cheese, Abnormal, 115. „ Ripening of, 1 10. „ Sarcinae in, 154. „ Torula yeasts in, 400. Chemical constituents of fungi, 178. Chlamydomucor Oryzsc, 205. Chlamydospore, 201. Chloride of lime, 32. Chlorine, 27. Chloroform, 27. Cilia, 6, 83. Citric acid from citromyces, 190. Citromyces, 190. „ glaber, 190. „ Pfefferianus, 190. Cladosporium butyri, 115. Cladosporium herbarum, 221. Cladothrix dichotoma, 162. Clostridium butyricum, 127, 129. „ gelatinosum, 150. „ Pastorianum, 131. Cloudiness in beer, 9. Cohn's nutritive fluid, 41. Colonies, Giant, 43. Conidia, 169. „ in moulds, 169. Cream, Souring of, 116. Crenothrix Kiihniana, 162. Crystalloids in moulds, 167. Cultures, Drop, 53. „ Indian ink point, 53. „ Liquid, 40. Plate-, 42. Stab-, 43. Streak-, 42. „ on gypsum blocks, 303, 311. „ on solid substrata, 297. DEMATIUM pullulans, 218. „ „ on grapes, 271. Dextrose, Yeast fermenting, 259, 267. Diseases in beer, 245, 256. Disinfectants, 24. „ Testing power of, 25. Disinfection in practice, 31. Dispora caucasica, 136. Distillery mash, 118. „ yeasts, 342. Drop cultures, 53. Drying methods, 5. ELECTRICITY, Influence on fungi, 177. Endomyces decipiens, 306. Endotryptase in yeast, 250, 256. Enzymes, 180. „ in Penicillium, 190. „ of acetic acid fermentation, 94. „ of bacteria, 151. „ ' of lactic acid fermentation, 108. „ of yeast, 255. Proteolytic, 153, 250, 256. .. Reducing, 256. Erysiphe Tuckeri, 222. Ether, 27. „ as disinfectant, 28. Ethyl alcohol, 267. 4-84 INDEX. Eurotium aspergillus glaucus, 193. Exosporium, 189. FATTY oils in moulds, 167. Fermentation, Products of alcoholic, 267. Theories of, 229. Fermentative power of mucor, 208. „ „ yeast, 270. ,, „ „ juice, 251. Film-formation, yeasts, 292. Filter, Cotton wool, 233. „ Nordtmeyer, 15. „ Pasteur- Chamberland, 15. Filtration of air, 17, 63. „ beer, 16. milk, 17. „ water, 18. Fischer's nutritive fluid, 41. Flagella, 6, 83. Flasks, 36. Carlsberg, 39. Chamberland, 37. Freudenreich, 37. Hansen, 38, 419. Jorgensen, 38, 419. Pasteur, 36. Foodstuffs, Inorganic, for yeasts, 226. „ Organic, for yeasts, 227. Formaldehyde, 27, 32. Formalin, 32. Formic acid, 268. Freudenreich flask, 37. " Frohberg " yeast, 325. Fructose, Yeast fermenting, 267. Fumago, 222. Fungi, Carbohydrates in, 182. Chemical constituents of, 178. influence of antiseptics, 178. electricity, 177. light, 176. pressure, 177. temperature, 175. vibration, 177. nutritive, physiology of, 171. Fusarium, 217. G GELATINE, 42, 50. Gemmae, 201. Giant colonies, 43. Ginger-beer plant, 140. Glucose, Yeast-fermenting, 259, 267. Glycase, 247. Glycerine produced during fermentation, 253, 264, 267. Glycogen in yeast cells, 8, 227, 302. Gonidia in Crenothrix, 85. Granules in yeast cell, 9, 301. Granulobacter, 131, 135. „ saccharo-butyiicum, 126, 131. Grapes, attacked by Botrytis, 186. „ „ Oidium Tuckeri, 222. Penicillium, 189. ,, „ Peronospora viti- cola, 223. „ Dematium pullulans on, 271. Gypsum blocks, Cultures on, 303, 311. H H-SIMATIMETER, 55. Hansen flask, 38, 419. Hops, Spontaneous heating of, 132. Hydrofluoric acid and fluorides, 27, 32, 46. Hydrogen fermentation, 133. „ peroxide, 27, 35. ICE, Biological examination of, 71. Invertase in bacteria, 151. „ Monilia, 213. „ yeast, 246, 255. Involution forms, Bacteria, 78. Iron, 172. „ bacteria, 162. Isomaltose, 260. JOHANNISBERG, I., 344. II., 284, 286, 344. „ II., Spores of, 306. Jorgensen's flask, 38, 419. „ moist chamber, 51. K KEPHIR, 135. Koji, 195. Koumiss, 138. INDEX. 4S.-> LACTASE in yeast, 248, 255. Lactic acid, 268. „ bacteria, 106. ,, in the brewery, 121. distillery, 118. „ „ leaven, 124. „ „ preserved foods, 125. „ „ wine, 123. Lactobacillus caucasicus, 136. „ fermentum, 121. Lactomyces inflans casei grana, 400. Lactose, Torula yeasts fermenting, 399. Laevulose, Yeast fermenting, 267. Leaven, Lactic acid in, 124. Leben, 139, 413. Leptothrix ochracea, 162. Leuconostoc mesenterioides, 79, 148. Levure caseeuss, 283. „ de sel (a), 365. •„ „ (0), 394. „ „ (y), 394. Light, influence on bacteria, 86. „ „ fungi, 176. Lime, 27. „ Milk of, 35. M MAGNESIUM, 172. Maltase, 247. „ in yeast, 255. Maltose, Yeast fermenting, 260, 267. Mash, Distillery, 118. „ Yeast, 119. " Maya," 140. Mazun, 139. „ Torula yeasts in, 401. Melibiase in yeast, 255. Melibiose, 247. Membrane of moulds, 167. Mercuric chloride, 26. Methane fermentation, 133. Methyl alcohol, 268. Micro-biological research, 10. Micro-chemical examination, 8. Micrococcus acidi laevolactici, 1 10. .. amarificans, 156. Billrothi, 150. „ candidus, 156. „ carneus, 156. „ casei amari, 116, 156. „ „ liquefaciens, 111. ,, cinnabarium, 156. concentricus, 156. Micrococcus flavus, 156. Freudenreichii, 114. gummosus, 144. lacticus, 156. luteus, 156. malolacticus, 124. ureae, 156. Microscope, 1. Microscopical preparations, 1. Mikrosol, 34. Milk, Abnormal, 113. Filtration of, 17. Peptonised, 41. Sarcinse in, 155. Slime-forming bacteria in, 145. Sterilisation of, 22. Torula yeasts in, 399. Moist chamber, Bottcher's, 12, 51. „ Jorgensen's, 51. „ Ranvier's, 11. Molasses, Slime - forming bacteria in, 148. Monilia, 210. „ Candida, 210, 213, 259. „ Invertase in, 213. ,, sitophila, 214. Montanin, 33. Morphology of yeast cells, 290. Moulds, 165. Conidia in, 169. Crystalloids in, 167. Fatty oils in, 167. Membrane of, 167. Nucleus of, 167. Protoplasm in, 167. Spherical yeast, 168. Sporangium, 169. Spores of, 169, 170. Zygospores, 169. Mucor, 197. alpinus, 202, 207. alternans, 202. circinelloides, 202, 209. erectus, 202, 209. Fermentative power of, 209. javanicus, 204, 209. Mucedo, 199, 209. neglectus, 207. Praini, 204. pyriformis, 210. racemosus, 201, 202, 207, 209. Rouxii, 203, 209. spinosus, 202, 209. stolonifer, 204. Must, see Wine must. Mycoderma cerevisiae, 258, 407- „ humuli, 398. „ rubrum, 398. 486 INDEX. ilycoderma vini, 407. Myxo-bacteria, 79. N NITRIFYING bacteria, 164. Nitrogen, 172. Nucleus of moulds, 167. Nutrition of yeasts, 225. Nutritive broth, 41. fluid, Cohn's, 41. „ Fischer's, 41. „ Pasteur's, 40. „ Raulin's, 42. „ Voges and Proskauer's, 41. substrata, 40. OIDITTM lactis, 106, 113, 115, 214. Tuckeri, 222. Oil globules in yeast cells, 9. Ozone, 27, 35. PAPIN'S digester, 15. Paraplectrum foetidum, 112, 131. Parasites, 81. Pasteur flasks, 36. Pasteur's nutritive fluid, 40. Pasteurisation, 21. Pediococcus acedulefaciens, 158. acidi lactici, 119. cerevisise, 155. damnosus, 157. odoris mellisimilis, 157. perniciosus, 157. sarcinaeformis, 156. viscosus, 145. Penicillium album, 113, 190. „ Camembert, 189. candidum, 113, 190. „ Enzymes in, 190. glaucum, 113, 175, 187, 190. „ italicum, 190. luteum, 190. „ olivaceum, 190. Roquefort, 189. Peronospora viticola, 223. Petri dishes, 50. Phenol, 28. Phosphorus, 172. Photographs, Micro-, 7. Phycomyces nitens, 207. Pichia californica, 367. farinosa, 368. membranaefaciens I., 366. II., 367. III., 367. Radaisii, 368. Tamarindorum, 367. taurica, 367. Plate-cultures, 42, 50, 53. " Podkvassa," 140. Point cultures, Indian ink, 53. Poisonous substances in fungi, 182. Poisons, influence of minute doses, 29, 174. Potassium, 172. Preserved foods, Lactic acid in, 125. Pressed yeast, 342. Pressure, influence on bacteria, 87. » » fungi, 177. Products of alcoholic fermentation, 267. Propagating apparatus, Bergh and Jorgensen, 417. ,, „ Hansen and Kiihle, 415. Proteolytic enzymes, 153, 250, 256. Proteus vulgaris, 151. Protoplasm in moulds, 167. Pure culture, Despatch of, 419. „ dilution methods, 47. „ physiological methods, 44. ,, Preparation of, 43. Pure culture of yeast on large scale, 414. Pyricit, 34. R RANVIER'S moist chamber, 11. Raulin's nutritive fluid, 42. Reducing enzymes, 256. Rennet, 111. Resting cells, Yeast, 295. Rhizopus japonicus, 205, 208, 210. „ nigricans, 204. Oryzffi, 205, 210. Tamari, 208. „ tonkinensis, 206, 210. " SAAZ " yeast, 325. Saccharobacillus Pastorianus, 121. Saccharomyces acidi lactici, 370. „ ' anomalus, 278, 368. „ „ in beer, 257. „ „ Spores of, 306. „ in soil, 276. INDEX. 487 Saccharomyces apiculatus, 258, 402. „ ' „ Habitat of, 271, 275. ,, aquifolii, 358. Bailii, 363. Batatae, 197, 359. „ Bayanus, 356. „ brassicse, I., II., III., 395. „ Carlsbergensis, 323. „ cartilaginosus, 359. „ cerevisise I., 258, 277, 285, 290, 293, 326. ., „ Spores of, 304, 308. „ Classification of, 314. „ Comesii, 377. , ellipsoideus I., 258, 263, 290, 294, 351. „ „ Spores of, 308. „ II., 258, 263, 277, 286, 290, 294, 354. „ „ in beer, 257, 312. „ „ Spores of, 308. exiguus, 258, 262, 361. (lava lactis, 365. foetidus I. in beer, 258. fragilis, 371. guttulatus, 383. Hansenii, 366. hyalosporus, 363- ilicis, 358. intermedium, 347. Johannisberg I., 344. „ II., 284, 286, 344. „ II., Spores cf, 306. Jorgensenii, 362. Kephyr, 400. Kreuznach, 344. levure de sel («), 365. Logos, 356. Ludwigii, 258, 278, 281. 298, 301, 306, 374. „ Spores of, 306. raali Duclaux, 365. „ Risler, 360. Marxianus, 258, 278, 298, 360. mellacei, 381. membranaefaciens, 258, 278, 297, 366. Saccharomyces membranaefaciens in soil, 276. „ minor, 366. „ Monacensis, 323. „ Miilheim, 344. „ multisporus, 360. „ octosporus, 282, 301, 377. „ Pastorianus L, 258, 263, 277, 282, 284, 290,294,345. ,, ,, in beer, 257, 312. „ „ Spores of, 308. „ Pastorianus II., 258, 263, 277, 290, 294, 347. „ Spores of, 308. „ Pastorianus III., 258, 263, 277, 290, 294, 349. „ „ in beer, 257, 312. „ „ Spores of, 308. Piesport, 344. „ pinophtorus enervans, 392. „ melodus, 392. Pombe, 380. „ pyriformis, 141, 358. „ Rouxii, 364. Sake, 359. „ Saturnus, 370. Soya, 196, 364. „ thermantitonum, 357. „ turbidans, 354. Tyrocola, 400. „ unisporus, 365. validus, 349. „ Vordermanni, 358. „ Walporzheim I., 344. „ Willianus, 355. „ Zopfii, 362. Saccharomycodes Ludwigii, 374. Saccharomycopsis capsularis, 384. „ guttulatus, 383. Saccharose, Yeast fermenting, 259, 267. Sachsia suaveolens, 2 1 4 Sake, 195. Salicylic acid, 35. Salt yeasts, 394. Saprophytes, 81. Sarcinae, 153. „ acidificans, 154. .. alutacea, 154. „ aurantiaca, 154. „ butyrica, 154. 488 INDEX. Sarcinae, Candida, 154. casei, 154. flava, 154. fusca, 155. Hamaguchiae, 196. lutea, 154. maxima, 155. mobilis, 155. ,, rosacea, 155. „ rosea, 115. „ rubra, 155. Sarcinae in beer, 155. „ cheese, 154. „ milk, 155. Schizosaccharomyces comesii, 377. mellacei, 286, 381. „ octosporus, 377. „ Pombe, 286, 380. Sclerotia, 168. Sclerotinia Fuckeliana, 1 83. Sclerotium of botrytis, 185. Semiclostridium commune, 150. Slime-forming bacteria, 142. „ „ in beer, 145. „ „ in milk, 145. „ „ in molasses, 148. ,, „ in wine, 142. Soda, 27, 35. Sodium hypochlorite, 27. „ sulphite, 27. Soja, 196. Solid substrata, Cultures on, 297. Sphaerotilus dichotoma, 162. Spherical yeast, moulds, 168. Spontaneous generation, 232. Sporangia, 169. Spore-formation, 281, 302. Spores, Bacteria, 84. moulds, 169, 170. ,, of Saccharomyces anomalus,306 „ „ cerevisiae I., 304, 308. ellipsoideus I., 308. II., 308. Johannisberg II., 306. Ludwigii, 306. Pastorianus I., 308. II., 308. „ III., 308. Sporodinia grandis, 207. Stab-cultures, 43. Staining methods, 5. Steam, Disinfection by, 14, 35. Sterigma, 187. Sterigmatocystis niger, 194. Sterilisation, 13. „ Discontinuous, 20. Sterilisation, Fractional, 20. of air, 23. „ of beer, 23. „ of glass and metal objects, 14. „ of liquid and solid sub> strata, 15. of milk, 22. Stimulating action of antiseptics, 29, 174. Streak-cultures, 42. Streptococcus hollandicus, 114. „ lacticus, 109, 112, 116. „ mesenterioides, 148. Structure of yeast cells, 298. Substrata, Nutritive, 40. Succinic acid, 268. Sugars, Fermentation of, 267. „ Synthesis of, 246. „ Synthetically prepared, 247. „ Yeasts fermenting certain, 259. Sulphur, 172. „ bacteria, 164. Sulphuric acid, 35. Sulphurous acid and sulphites, 27, 34. TAO-TJIUKG, 197. Tane-Koji, 195. Temperature, influence on bacteria, 85. „ „ fungi, 175. „ „ yeast cells, 277. Thamnidium elegans, 207. Theories of fermentation, 229. Thermobacterium Zeidleri, 100. Thymol, 27. Tobacco, Fermentation of, 160. Toluol, 27. Top-fermentation yeasts, 311, 319, 329. „ Pure cultures of, 341. Torula a = Torula Holmii, 388. b = „ mucilaginosa, 395. c = „ cinnabarina, 396. amara, 401. colliculosa, 393. epizoa, 394.- novae carlsbergiae, 260, 388. in beer, 257. yeasts fermenting lactose, 399. Torulas, 259, 385. Turbidity in beer, 9. Tyrothrix, 112. U ULTRA-MICROSCOPE, 8. INDEX. 489 VACTTOLES in yeast cells, 301. Variations in the Saccharomycetes, 280. „ in yeasts occurring in prac- tice, 288. Vibration, influence on bacteria, 87. „ „ fungi, 177. Vinegar process, Orleans, 93. Pasteur's, 93. „ „ " Quick," 93. Vitalistic theory of fermentation, 235. Voges and Proskauer's nutritive fluid, 41. W WATER, Biological examination of, 69. „ „ examination of, by Hansen, 71. „ „ Holm, 69. „ „ Jorgensen, 70. „ „ Lindner, 74. „ „ Wichmann, 73. Filtration of, 18. " Weissbier," 121, 341. Willia anomala, 368. „ Saturnus, 370. Wine, Lactic acid in, 123. Production of acid in, 265. „ alcohol in, 265. „ bouquet in, 265. „ glycerine in, 264. Pure cultures of yeasts in, 266. Slime-forming bacteria in, 142. must, Action of yeasts on, 263. yeast in soil, 274. yeasts, 343. Wort, Aeration of, 280. see Beer -wort. YAM brandy, 197. Yeast, Analysis of, 311. „ Biological relationships of, 270. „ Fermenting power of, 270. ,, Pure culture of, on large scale 414. Yeast cells, Counting, 55. „ effect of nutritive fluid, 279. „ temperature, 277. film-formation, 292. Pvesting, 295. spore-formation, 302. Structure of, 298. Yeast deposits, 290. Yeast juice, Fermentation of, 252. „ Preparation of, 248. Yeast mash, 119. Yeast propagating apparatus, Bergh and Jorgensen, 417. „ „ Hansen and Kiihle, 415. Yeasts, 225. Brewery, 317. Culture, 317. Distillery, 342. habitat in soil, 274. Nutrition of, 225. Pressed, 342. Top-fermentation species, 311, 319, 329. „ Wine, 343. Yoghourt, 140. ZOOGIXEA, 79. Zygosaccharomyces Barkeri, 372. Zygospores, 169. Zymase, 181, 249. „ Properties of, 254. BELL AND BAIN, UlillTKD, PRINTERS, GLASGOW. 32 r CO CO CD CD CD it UNIVERSITY OF TORONTO LIBRARY f remove the card 1 ^ V 1 from this Pocket. Acme Library Card Pocket Under Pat. " Ref. Index File." Made by LIBRARY BUREAU