university of Connecticut ibraries pge w CD BRITL 589.9.P268 c. 1 PASTEUR # STUDIES ON FERMENTATION 3 T1S3 Q0m37TM fi : r m i. ^ Q m o !&'•■ '.•T-t «1 'H' ,t jt i.*' A?;:. t/ ''^■<'\;-'^ -'^k.^ STUDIES ON FEEMEISTTATION ? STUDIES ON FERMENTATION '^^ THE DISEASES OF BEEE THEIE CAUSES, AND THE MEANS OF PREVENTING THEM L. PASTEUE MLMIiLR ay IXSTITV-TE 01 FRAME, THE KOVAL SOriETI O? I.OXDO.N, ETC. .4 TRANSLATION, MADE WITH THE AUTHOR'S SANCTION, OF " ETUDES SUR LA BIERE," WITH NOTES, INDEX, AND ORIGINAL ILLUSTRATIONS F E A N K F A U L K N E E AUTHOR OF "the ART OF BREVVJ.NG," ETC. AND D. CONSTABLE E 0 B B, B.A. LATE SCHOLAR 01 IVOKCESTER COLL., OXFOr.D \, \ Honbon MACMILLAN & CO 1879 r-^.q-^ c t- l^S^h •Ai;lK)N ANI> SONS, I'UINTEUS, rATEUXOSTEK KOW, U'XDOK. THE MEMORY OF MY r A T H E li, rOEMERLY A SOLDIER UNDER THE FIRST EMPIRE, AND KNIGHT OF THE LEGION OF HONOUR. THE LONGSE I LIVE, THE BETTE-E DO I UNDEESTAXD THE KIXDXESS OF THY HEAET AND THE EXCELLENCE OF THY MIND. TO THY EXAJH'LE AND COUNSELS DO I OWE THE EFFOETS THAT HAVE BEEN DEVOTED TO THESE STUDIES, AS WELL AS TO ALL THE WOEK I HAVE EVEE DONE. AND NOW, HOW CAN I BETTEE HONOUE THESE FILIAL EE3IEMBEANCES THAN BY DEDICATINCt JIY BOOK TO THY ME3M0EY r L. PASTEUR. AUTHOR'S PEEFACE. Our misfortunes inspired me with the idea of these researches, d I undertook them immediately after the war of ISToXaed have since continued them without interruption, ^^with the determina- tion of perfecting them, and tjierebj; benefiting a branch of industry wherein we are undoubtedly surpassed by Germany 71 I am convinced that I have found a precise, practical solution of the arduous problem which I proposed to myself — that of a process of manufacture, independent of season and locality, which should obviate the necessity of having recourse to the costly methods of cooling employed in existing processes, and at the same time secure the preservation of its products for any length of time. These new studies are based on the same principles which guided me in my researches on wine, vinegar, and the silkworm disease — principles, the applications of which are practically unlimited. The etiology of contagious diseases may, perhaps, receive from them an unexpected light. I need not hazard any prediction concerning the advantages likely to accrue to the brewing industry from the adoption of VI 11 AUTHORS PREFACE. such a process of brewing as ni}' stud}' of the subject has enabled me to devise, and from an application of the novel facts upon which this process is founded. Time is the best appraiser of scientific work, and I am not unaware that an industrial discovery rarely produces all its fruit in the hands of its first inventor. I began my researches at Clermont-Ferrand, in the labora- tory, and with the help, of my friend M. Duclaux, professor of chemistry at the Faculty of Sciences of that town. I continued them in Paris, and afterwards at the great brewerj'" of Tourtel Brothers, of Tantonville, which is admitted to be the first in France. I heartily thank these gentlemen for their extreme kindness. I owe also a public tribute of gratitude to M. Kuhn, a clever brewer of Chamalieres, near Clermont-Ferrand, as well as to M. Velten, of Marseilles, and to MM. de Tassigny, of Reims, who have placed at my disposal their establishments and their products, with the most praiseworthy willingness. L. PASTEUR. Paris, June 1, 1876. PREFACE TO ENGLISH EDITION. My first idea of placing before English, brewers a translation of " Etudes sur la Biere " was meagre in the extreme, compared with the final realization of it as it appears in the following pages. Seeing the vast importance of Pasteur's work from a practical point of view, after writing a review of it for the Brctcens' Juurnaly I determined to procure, at any rate for the use of my pupils, a literal translation, illustrated by photo-lithographic copies of the original plates, the thankless task of executing this preliminary translation for so limited a number of readers being most kindly and generously carried out for me by my friend Mr. Frank U. Waite, who, being engaged with me at the time in practical brewing operations, shared my views as to the value of the original work. It was on the completion of this translation that my views and desires expanded. The more I studied the work, the more I was convinced of its immense value to the brewer as affording him an intelligent knowledge of the processes and materials with which he deals, but over and above all this, it was impossible not to feel that the researches of such X rUEFACE TO ENGLISH EDITION. a devoted and accomplished savant as Pasteur, possessed a scientific interest much wider than their mere relation to the art of brewing would imply. As the work of a skilful chemist and a laborious and accurate observer, such a protracted and careful study of the lowest and simplest forms of life, must necessarily be of first imi^ortance to the biologist — to the beginner as an admirable introduction to the study of practical phj'siology in general, as well as to the more advanced student, from the suggestive light which it throws on the nature of analogous phenomena in more complex organisms. I determined accordingly to publuh the work if I could secure the consent of its distinguished author, but at the same time I felt that the publication of M. Pasteur's "Studies" in the form in which Mr. Waite had, at my request, translated it, and illustrated only with inferior copies of the original plates, would not be either advisable or just ; but that I was bound rather to put the book before the English public in as satisfactory and complete a form as lay within my power. Under these cii'cum- stances I was induced to seek the aid of Mr. D. Constable Robb, B.A., of The Oxford Universit}' Museum, who, in taking- Mr. Waite's version as a basis, has so elaborated, annotated, and recast it, that I feel bound to say that much of the value of " Studies on Fermentation," as it now appears, is due to the cure that Mr. Robb has bestowed upon the revision that he so kindly undertook ; a revision the result of which has created a feeling of confidence in the success of the translation as it now .stands, which I could not have had in any mere literal version. To the practical worker the original illustrations alone, which appear in this version, cannot but be of immense value in the microscopical study of the changes in the liquids with which he deals; whilst the many notes and additions, which are a PREFACE TO ENGLISH EDITION. XI feature peculiar to the English edition, more particularly the rendering into the equivalents, with which, unfortunately, practical men on this side of the channel are still most at home, of the metric weights and measures and centigrade tempera- tures, as well as the Index which Mr. Robb has compiled, will, I trust, render the book of still greater service than it other- wise would have been to many of those who may favour it with their attention. The debt which we English brewers owe to M. Pasteur can hardly be over-estimated, and I must be allowed here to express my personal obligations to that distinguished worker for the permission which allows this translation ; and to the French publishers for their help with regard to the interleaved illustrations. The author's preface and dedication are, of course, reproduced, the former making it unnecessary for me to refer more in detail to the contents of the translation. FRANK FAULKNER. The Brewery, St. Helen's, Lancashire, September, 1879. ERE AT A. Page 80, line IS from top, insert * after imsubmerged. ,, 181, ,, 13 ,, ,, ,, " aiter piclliilf/ns. ,, 301, place * before footnote. TABLE OF COIsTTElSTTS, page- Author's Dedicatiox .......... V ,, Preface vii Translator's Preface ix CHAPTEIl I. ON THE IXTIJIATE RELATIOX EXISTING BETWEEN THE DETERIORATION OF BEER, OR THE "WORT FROM WHICH IT IS MADE, AND THE PROCESS Ot BREWING 1 CHAPTER II. ON THE CAUSES OF THE DISEASES WHICH AFFECT BEER AND WORT. § I. Ever}- unliealthy change in the qualitj" of beer coincides -ndth a development of microscopic germs which are alien to the pure fei-meut of beer . . . . . . . . lf> § II. The absence of change in wort and beer coincides with the absence of foreign ors-anisms ...... 25 .CHAPTER III. ON THE ORIGIN OF FERMENTS TROPERLY SO CALLED. § I. On the conditions which cause variations in the nature of the organized prodncts existing in infusions .... 33 XIV TABLE OF COXTEXTS. PAGE § II. Experiments on blood and urine taken in their normal state, and exposed to contact ■with air that has been deprived of the particles of dust which it generally holds in suspension . 40 § in. Experiments on the juice contained in grapes .... 54 § lY. Wort and must exposed to common air 59 § V. New comparative studies on the germs held in suspension by the air of different places which are near each other, but subjected to different conditions affecting the production and diffusion of the particles of dust found in them . . .72 § VI. Yeast may become dry and be reduced to dust without losing its faculty of reproduction 81 CHAPTER IV. THE GEOWXII OF DIFFERENT OllGANISMS IX A STATE OF PUKITX : THEIR AUT0X03IY. § I. Growth of PeniciUium fjlaucum and Asperf/illus (jlauciis in a state of purity. — Proofs that these fimgoid gro-n-ths do not become transformed into the alcoholic ferments of beer or wine. — Preliminary inquirj' into the cause of fermentation . 86 § II. Growth of Mijcoderma vini in a state of purity. — Confirmation of our original conjectures as to the cause of fermentation. — Ilycodenna villi does not change into yeast, although it may give rise to fermentation . . . . . . .108 § III. Growth of Mycinlcnna acdi in a state of purity . . .121 § IV. Growth of Mucor raccmosus in a state of purity. — Example of life more active and lasting when removed from the influence of air 127 CHAPTER V. THE ALCOHOLIC FER:MEXTS. § I. On the origin of ferment § II. On " spontaneous " ferment §111. On " high " and " low " ferments § IV, On the existence and production of other species of ferment § V. On a new race of alcoholic ferments : Aerobian ferments § VI. The purification of commercial yeasts .... 14;} 182 18G 19() 205 211) TABLE OF CONTENTS. XV CHAPTER VI. THE PHYSIOLOGICAL THEORY OF FEE:ME:XT A.TIOX . PAGE § I. On the relations existing between oxygen and yeast . . 235 § II. Fermentation in saccharine fruits immersed in carbonic acid gas 266 § III. Reply to certain critical observations of the German naturalists, Oscar Brefeld And Moritz Traube. ..... 279 § IV. Fermentation of dextro-tartrate of Lime ..... 284 § V. Another example of Life without air. — Fermentation of lactate of lime 292 § VI. Reply to the critical observations of Liebig, piiblished in 1870 . 316 CHAPTER VII. NEW PROCESS FOE THE 3IAXUFACTUKE OF BEEE. § I. Preliminary experiment? 338 § II. Method of estimating the oxygen held in solution in wort . 353 § III. On the qiiantity of oxygen existing in a state of solution in brewers' worts ......... 364 § IV. On the combination of oxygen with wort 371 § V. On the influence of oxygen in combination on the clarification of wort 381 § VI. Application of the principles of the new process of brewing ■nath the use of limited quantities of air .... 387 Appendix 396 Index 403 IIN'DEX TO PLATES. TO FACE PAOE I. Principal disease-ferments met with in wort and beer • . 6 II. Appearance under the microscope of the deposit from "turned " beer 20 III. Toruhe in process of development . . ... .72 IV. Blycodcrma vini functioning as an alcoholic ferment. — Right half, showing appearance of spores just sown ; left half, their appearance after an interval of submerged life V. Iliicor, vegetating submerged, in deficit of air VI. Ferment of jiivcor ........ VII. Yeast-cells — worn out and dissociated (left), after revival in sweet wort (right) VIII. Fertile mould-cells from the outer surface of grapes IX. Various examples of the mode of growth of mould-cells from the outer surface of grapes ...... X. One of the ferments of acid fruits at the commencement of fermentation in its natural medium. .... XI. Sacclinrotnyccs piistoyianus, in course of regular growth . XII. Ferment-cells from a spontaneous fermentation just starting 116 136 138 148 152 154 166 168 184 STUDIES ON FEKMENTATION. CHAPTER I. On the Intimate Relation existing between the Detkrio- RATiON OF Beer, or the Wort from which it is Made, AND the Process of Brewing. At the outset of these " Studies," let us briefly consider the nature of beer and the methods of its manufacture. Beer is a beverage which has been known from the earliest times. It may be described as an infusion of germinated barley and hops, which has been caused to ferment after having been cooled, and which, by means of "settling" and racking, has ultimately been brought to a high state of clarification. It is an alcoholic beverage, vegetable in its origin — a barley wine, as it is sometimes rightly termed.* Beer and wine, however, differ widely in their composition. Beer is less acid and less alcoholic than wine ; it holds more ingredients in solution, and the nature of these ingredients is by no means similar to that of those which are found in wine. These differences in the component parts of wine and beer give rise to corresponding differences in the keeping qualities of the * This expression is found for the first time, it would appear, in Theo- phrastus, B.C. 371. [See, however, Herodotus, Bk. II., chap. 77. Speak- ing of some Egyptians he says, " They drink a kind of wine made from barley (oww 8' eK Kpidtav 7re7ro6r;/xei/a)), for the grape does not grow in that part of the country." Herodotus wrote about 450 B.C. ^schylus (48Q B.C.) has a similar expression, Suppl. 953. — D. C. E.] 2 STUDIES ON FERMENTATION. two liquids. The small amount of aciditj' in beer, its povert}' in alcohol, and the presence of matter that is saccharine, or liable to become so, all operate in imparting to beer a tendency to change, which wine does not possess. That this unequal resist- ance to the aggression of diseases is due to such differences, may be proved by the fact that wine could be made much more liable to change than it actually is, by a diminution of its acidity and its usual proportion of alcohol, or by increasing the proportion of viscid or saccharine matters,* modifications which would tend to assimilate its composition to that of beer. We have remarked elsewhere that the pains devoted to the rearing of vines, and to the ordinary operations of vinification, such as ouiUaffe,f sulphuring, and repeated rackings, as well as the use of cellars and vessels hermetically closed, are entailed by the necessity of counteracting and preventing the diseases to which wine is liable. The same may be said, a fortiori, of beer, inasmucb as it is more liable to change than wine. Manufac- turers and retailers of this beverage have to strive constantly with the difficulty of preserving it, or the wort used in its manufacture. We may readily be convinced of this hj review- ing the usual processes of the art of brewing. When the infusion of malt and hops, which is termed wort, is completed, it is left to cool. It is next put into one or more casks or vats, in which it is made to undergo alcoholic fermentation — the most important of all the processes in brewing. The cooling must be as rapid as possible. This is a condition of success ; otherwise, the wort may deteriorate, which will necessarily lead to deterioration in the quality of the beer. As long as the wort is at a high temperature it will remain sound ; * One of these modifications is a real source of serious danger to the preservation of wine ; for instance, during rainy years, at the time of vintage, the grapes may happen to be covered with earthy matter, consisting principally of carbonate of lime; this will dissolve in wine and partly neutralize its acidity, and the wine will thus become more liable to disease. t Transferring from one cask to another for the purpose of clarifying the wine. STUDIES ON FERMEISTATION. 3 when under 70° C. (158° F.), and particularly when at a tem- perature of from 25° C. to 35° C. (77° F. to '95° F), it wiU be quickly invaded by lactic and butyric ferments. Rapidity in cooling is so essential that to secure it recourse is had to special apparatus.* Even in the preparation of wort, especially when it is effected by successive mashings, in summer, deterioration is imminent : in fact, it is not rare to see the wort becoming acid during the mashes, if these are not accomplished with all possible celerity. After the wort has been cooled, it is mixed with yeast. This is obtained from a previous fermentation, and, after being thoroughly pressed, is added at the rate of from one to two thousandth parts of the weight of the wort, that is, from 100 to 200 grammes per hectolitre (about 4 oz. to 8 oz. average for every 25 gallons). At first sight, this yeast seems free from the possible diseases of the wort and beer ; but this is by no means the case. Now, why do we add yeast to our wort ? This practice is unknown in the art of vinification. The must is alwaj's left to spontaneous fermentation. Why should we not leave the wort to operate in the same manner ? It would be a mistake to suppose that in the brewing of beer yeast is added with the sole object of accelerating fer- mentation, and making it more rapid. Rapidity in fermen- tation is a very questionable advantage, and one which is not desired by brewers, who rather agree in pronouncing it injurious to the quality of beer. It is in the easy deterioration of the wort, or what is tantamount to it, in the facility it afibrds to various spontaneous fermentations, that we find an answer to these questions. The must, through its acidity, due to the presence of bitartrate of potash — which seems to promote alcoholic fermentation — through its proportion of sugar, and perhaps in consequence of some other peculiarity of its compo- sition, always undergoes regular alcoholic fermentation. The * We shall hereafter revert to this rapidity in cooling, to show that it is also of use in the subsequent clarification of beer. B 2 4 STUDIES ON FERMENTATION. diseases of wine, at the commencement of its manufacture, show themselves, so to say, in a latent state only. Therefore a vintage can be left, without inconvenience, to spontaneous fermentation. With wort the case is quite diflferent. Under certain accidental circumstances it is possible that alcoholic fermenta- tion alone may take place in a wort left to ferment spontaneously, and the quality of the beer remain unimpaired, but such an event would be exceptional, and of very rare occurrence. In most cases we should obtain an acid or putrid liquid resulting from the production and multiplication of alien ferments. The addition of yeast is made in consequence of the necessity of exciting through the whole bulk of wort, as soon as it is cold, a single fermentation — viz., the alcoholic, the only one that can produce beer properly so called. The alcoholic ferments concerned in the production of beer will be found represented in several of the engravings in this work. Other ferments we may term " diseased " ; these include all those that may occur spontaneously — that is, whose germs have not been directly and intentionally introduced — amongst the actual alcoholic ferments. The expression, " diseased ferments," is justified by the cir- cumstance that the propagation of these ferments is always accompanied by the production of substances which are acid, putrid, viscous, bitter, or otherwise unpalatable, a consideration of commercial rather than scientific importance. From a physio- logical point of view, all these ferments are of equal interest and importance. The botanist, as a man of science, in contem- plating nature, must give equal attention to all plants, whether useful or noxious, since they are all governed by the same natural laws, among which no order of merit could be estab- lished. The exigencies of industry and health require, however, wide distinctions. The first engraving (Plate I.) represents the difierent diseased ferments, together with some cells of alcoholic yeast, to show the relative size of these organisms. STUDIES ON FERMENTATION. O No. 1 of the engraving represents the ferments of turned beer, as it is called. These are filaments, simple or articulated into chains of different size, and having a diameter of about the thousandth part of a millimetre (about vrxroo" inch). Under a very high power they are seen to be composed of many series of shorter filaments, immovable in their articulations, which are scarcely visible. In No. 2 are given the lactic ferments of wort and beer. These are small, fine and contracted in their middle. They are generally detached, but sometimes occur in chains of two or three. Their diameter is a little greater than that of No. 1. In No. 3 are given the ferments of putrid wort and beer. These are mobile filaments whose movements are more or less rapid, according to the temperature. Their diameter varies, but is for the most part greater than that of the filaments of Nos. 1 and 2. They generally appear at the commencement of fermen- tation, when it is slow, and are almost invariably the result of very defective working. In No. 4 are given the ferments of viscous wort, and those of ropy beer, which the French call filante. They form chaplets of nearly spherical grains. These ferments rarely occur in wort, and still less frequently in beer. No. 5 represents the ferments of pungent, sour beer, which possesses an acetic odour. These ferments occur in the shape of chaplets, and consist of the mycoderma aceti, which bears a close resemblance to lactic ferments (No. 2), especially in the early stages of development. Their physiological functions are widely different, in spite of this similarity. The ferments given in No. 7 characterize beer of a peculiar acidity, which reminds one more or less of unripe, acid fruit, with an odour sui generis. These ferments occur in the form of grains which resemble little spherical points, placed two together, or forming squares. They are generally found with the fila- ments of No. 1, and are more to be feared than the latter, which cause no very great deterioration in the quality of beer, when alone. "When No. 7 is present, by itself or with No. 1, b STUDIES ON FERMENTATION. the beer acquires a sour taste and smell that render it detestable. AVe have met with this ferment existing in beer, unaccompanied by other ferments, and have been convinced of its fatal effects. No. 6 represents one of the deposits belonging to wort. This must not be confounded with the deposits of diseased ferments. The latter are alwa^'s visibly organized, whilst the former is shape- less, although it would not always be easy to decide between the two characters, if several samples of both descriptions were not present. This shapeless deposit interferes with wort during its cooling. It is generally absent from beer, because it remains in the backs, or on the coolers ; or it may get entangled in the yeast during fermentation and disappear with it. Among the shapeless granulations of No. 6 may be discerned little spheres of different sizes and perfect regularity. These are balls of resinous and colouring matter that are frequently found in old beer, at the bottom of bottles or casks ; sometimes they occur in wort preserved after Appert's method. They resemble organized products, but are nothing of the kind. We have remarked before, in " Studies on Wine," that the colouring matter of wine would settle, in course of time, in that form. It is evident that the diflFerent ferments delineated in Plate I. are worthy of thorough study, in consequence of the fermenta- tions to which they may give rise. Care must be taken to isolate the action of each of them in fermentations which we may call pure — a condition of some difficulty, but one that maybe carried out by an adoption of the methods explained in this work. All these diseased ferments have a common origin. Their germs, infinitesimal and hardly perceptible as they are, even with the aid of the microscope, form a part of the dust conveyed through the air. This dust the air is continually taking from or depositing upon all objects in nature, so that the dust that clings to the ingredients from which our beer is manufactured, may teem with the germs of diseased ferments. During the process of fermentation, the occult power of diseased ferments, although it may escape the observation of the brewer, is manifested in a high degree. Principal Disease-ferments met with in Wort and Beer. Imp G€ny-&r0s.PariJ STUDIES ON FERMENTATION. 7 During the lust thirty years, or so, the art of brewing has undergone a radical change, at least in Europe. This change has been effected by a partial abandonment of the process of fermentation formerly used. Thirty years ago only one kind of beer \Vas known ; there are nowadays two distinct kinds — beer fermented at a high temperature, and beer fermented at a low temperature. Each of these is subdivided into many varieties, to which different names are given, according to their strength or colour. This is the case in England, where we find porter, ale, pale ale, stout, bitter beer, and other varieties of beer, although, as a matter of fact, the English have but one kind of beer, all the English beers being fermented at a high tem- perature. Let us briefly examine tlie diflPerences existing between the two kinds of beer. Formerly all beer was fermented at a high temperature. The wort, after having been cooled in the backs, was run into a large vat, at a temperature of about 20° C. (68° F.). Yeast was then added to it, and when the fermentation began to show itself, in the formation of a light, white froth, upon the surface of the liquid, the wort was run into casks, having a capacity of from 50 to 100 litres (11 to 22 gallons) — 75 litres being the commonest size. These casks were placed in cellars, having a temperature of from 18° C. to 20° C. (64° F. to 68° F.). The activity of fermentation soon produced a froth that grew thicker and more and more viscous in proportion to the quantity of yeast it contained. The yeast worked out of the bung-holes and dropped into a vessel placed under the casks ; there it was gathered for subsequent operations. It alwaj^s exceeded the quantity used in the first instance, the ferment increasing greatly during the process of fermentation. The increase in its weight varied with the weight of yeast used and the com- position of the wort. Under the ordinary conditions of brewing, where the weight of the pitching yeast was about one thou- sandth part of the weight of the wort, the increase is said to have been from five to seven times the weight of the yeast ; 8 STUDIES (JN F]:ilMKNTATl()N. but such increase must naturally have been determined by the quality of the wort, the quantity of hops used, the action of oxj^geu, and the proportion of barm employed. The process of fermentation lasted from three to four days. By that time the beer was finished, and had become limpid, the fermentation having been completed. The bungs could then be placed in the casks, and the beer be delivered to the customer.* A cer- tain amount of yeast still remained in the casks, and caused the beer to become thick, in transit ; but a few days' rest sufficed to restore its brilliancy, and render it fit for drinking or bottling. Here we have an explanation of the term " liigli fermenta- tion," which has been applied to the foregoing process. This process is conducted at a high temperature, which, commencing at 19° C. or 20' C. (66° F. or 68° F.), is raised to 20° C. or 21° C. (68° or 70° F.) by the action of fermentation, which is always accompanied by an increase of heat.f * In some breweries (at Lyons especiallj') fermentation at a high tem- perature is practised in largo vats at about 15' C. (59' F.). The yeast which covers the surface of the liquid is skimmed off and stored in flat tubs. t The initial temperature of the wort must be regulated bj' the quantity of wort subjected to fermentation. In English breweries, where large quantities are brewed at a time, the heat created by the action of fermentation would produce a temperature sufficiently high to aff'ect the quality of the beer, if the yeast were added at 19° C. or 20° C. The following are the temperatures at which the worts are pitched, in the principal London breweries : — For common ale, 60' F. or 15-5° C. ; for pale ale, 58° F. or 14-4" C. ; for porter, G4° F. or 17-8' C. The fer- mentation is commenced in large vats ; from these the beer is run into vessels of a much smaller capacity, in which it completes its fermentation by working off" the yeast and cleansing itself. For white beer of superior qualitj^ the temperature during fennenta- tion must not rise bej'^ond 72^ F. or 22*2° C. ; some brewers never allow it to exceed 18° C. (()5' F.). The temperature is lowered by means of a current of cold water, which circulates through a coil fixed in the vats or other fermenting vessels. 'In the case of poi-ter, the initial heat of which is 64' F. or 17 '8' C, the temperature in the vats sometimes rises to 78° F. or 25"5° C. ; but such an increase in temp(!rature excites considerable ajiprehension. We have seen a tun for pale ale, containing 200 barrels of 36 gallons, STUDIES ON FEKMENTATION. 9 This is not, however, the only reason for the use of the term '^ high fermentation." We have just seen that the fermenting casks were so arranged that most of the yeast produced during the process of fermentation would rise to the upper part of the casks and work out of the bung-holes. In this practical fact we have the actual origin of the expressions '■^ high fermentation^^ and ^^ high beer,'" which are used to distinguish this peculiar fermentation and the quality of beer derived from it. As we have alread)^ observed, all beer was formerly produced by this mode of fermentation, which even at the present time is still practised in the breweries of Great Britain, where beer fermented at a low temperature is absolutely unknown. " Low fermentation " is a slow process, eflfected at a low temperature, during which the yeast sinks to the bottom of the vats or casks. The wort, after cooling, is run into open wooden vats. In cooling, the wort is brought to as low a temperature as 8° 0. (47° F.), or even 6° C. (43° F.), at which point it is maintained by cones or cylinders (styled nageurs, i.e., floats, by the French) floating in the fermenting vats. These floats may be filled with ice if the outside temperature requires it, as is invariably the case in summer. pitched with 600 lbs. of fairly solid yeast. In forty-six hours the attenu- ation was considered sufficient, and the beer, which from an initial heat of o8-r F. or 14-5° C, had risen to 72° F. or 22-2° C, was cleansed to working casks. The large vats in which the fermentation is started may be considered as equivalent to the cuves guilloires of French breweries, the casks in which it is completed and the yeast thrown off representing their 7 5 -litre vessels, improperly called quarts. Notwith- standing the enormous English beer manufacture, and although the fermenting vat, as in making porter, for instance, sometimes attains the capacity of 2,000 to 3,000 litres (400 to 600 gallons), the casks into which it is run are never larger than 15 to 20 hectolitres (300 to 400 gallons); and even at Burton, in the celebrated breweries of Allsopp and Bass, the pale ale is finished in casks of a capacity less than 10 hectolitres, and yet the average turn-out of these immense works reaches 3,000 to 4,000 hectolitres (60,000 to 80,000 gallons) of beer per day. 10 STUDIES ON FERMENTATION. The duration of this fermentation i.s ten, fifteen, or even twenty days. The yeast, which is produced less abundantly than in the case of beer fermented at a high temperature, is gathered after the beer has been drawn off, and is partly used in subsequent fermentations. The term " low fermentation " is derived partly from the lowness of the temperature during the fermenting process, and partly from the fact that the yeast is gathered at the bottom, and not at the upper part of the fermenting vessels.* Beer fermented at a low temperature, of which there are several varieties, differing in colour and quality, is of Bavarian origin.-f- The preference of the public for this kind of beer, and the increased facilities that such a beer affords the trade, are the two reasons why its manufacture has so greatly increased. In Austria, Bavaria, Prussia, and other Continental countries this new method of brewing is almost exclusively adopted. In the Monitmr dc la BraHserie of the 23rd April, 1871, may be found the following significant remarks on the increase in the production of beer fermented at a low temperature on the Continent : " The number of breweries manufacturing high beer is rapidly decreasing, whilst the number of those producing loio beer is still more rapidly increasing. There were in Bohemia, in 1860, 281 breweries in which high fermentation was practised ; in 1865, only 81 of these remained ; in 1870, the number had declined to 18. On the other hand, the number of brew'eries practising low fermentation increased from 135 in 1860 to 459 in 1865, and in 1870 had risen to 831. In 1860 there were 620 breweries in which the two methods were employed ; in 1865 there w'ere 486; in 1870 only 119 remained. The number of breweries at present existing in Bohemia amounts to 968." * [The expressions "high" and "low" fermentation or beer strictly refer to temperature, whereas the other expressions used ("top" or "bottom") refer to the behaviour of the yeast. The French words haute and basse seem to look both ways. — D. C. R.] t It is said that the floating cones or cylinders filled with ice, which enable brewers to manufacture beer at a low temperature, even in summer, were first used in Alsace. STUDIES ON FERMENTATION. 11 In France, we are still in a period of transition ; but year by year the manufacture of " low beer " is increasing, to the evident detriment of its competitor. It is unnecessary to dwell upon certain differences exist- ing between the two kinds of beer, such as may be traced to the preparation and composition of their respective worts. The brewing of " high beer," by hand or machinery, is effected in one operation ; the brewing of " low beer " is accomplished by successive mashings, the temperatures of which are gradually raised. These differences, and others that result from the longer boiling of the wort, in the " high fermentation " process, give rise to diversities in the composition and colour of the worts, from which circumstance " low beers " are sometimes termed ivhite beers, in contradistinction to the others, which have a deeper colour, and are known as dark beers (brims). The name of Strasburg is generally given to " low beer " in France, but sometimes it is called German beer. It is easy to account for the changes introduced into the construction and working of breweries bj"" the new process of " low fermentation." A low temperature is essential not only to the manufacture but also to the preservation of " low beer," and must be secured by the use of ice-cellars in which the temperature may be maintained at 5"" R. or 6° 11.(43° F. or 45°F.), and even at 1°, 2°, or 3° E. (35° to 39 °F.) throughout the year. This necessitates an accumulation of ice and the construction of cellars of enormous extent, for the storage of the beer. " Low beer " is essentially a stock beer, especially if brewed in winter, when due advantage is taken of the low temperature of the season. It is kept in cold cellars until the spring or summer, when beer is consumed in larger quantities. It is calculated that 100 kilos. (1*96 cwt.) of ice is the average quantitj' used per hectolitre (22 gallons) of good beer, between the cooling of the wort and the day of sale.* * 45 million kilos, of ice are annually consumed in the brewery of M. Dreher, in Vienna. The brewery of Sedlmayer, at Municli, uses about 10 million kilos. {Journal cles Brasseurs, 22nd June, 1873.) 12 STUDIES ON FERMENTATION. In the manufacture of " high beer " we find none of these complications, nor have we in that manufacture any similar difficulty of working or expense of construction to contend against. The whole process of brewing, including the delivery of the beer, does not take more than eight days. Why should a mode of brewing so simple, so rapid, and comparatively so inexpensive, have been abandoned by the greater part of Europe in favour of a system disadvantageous to the brewer in so many respects ? It would be a mistake to supj)ose that the sole reason for such a change might be found in the superior quality of "low beer.'^ That such a superiority does exist is admitted as a fact by the majority of beer drinkers ; but taken by itself, this fact is not sufficient to account for the radical transformation that has taken place in the manufacture of beer, as is proved by the example of England, which, we believe, does not possess as yet one single "low beer" brewery, from which circumstance we may fairly suppose that the English liave a decided pre- ference for "high beer." The principal advantage of working at a low temperature lies in the fact that " low beer " is less liable to deterioration, and is less prone to contract diseases than " high beer," especially whilst it remains in the brewery — a circumstance that places the brewer in a position vastly superior to that which he occupied in former times. With the help of ice the brewer can manufacture beer during winter and the early part of spring, for consumption in summer.* "High beer," on the * It should, however, bo borne in mind that these remarks on the relative preservative powers of the two beers hold true on account of three things — differences in the respective modes of brewing, artificial cooling during the process of fermentation, and the storing of the ' ' low beer" in ice-cellars. In itself, perhaps, "low beer" is more liable to change than " high beer ; " that this does not actually take place, is due to the employment of artificial cooling. A brewery which has an average annual production of 10,000 hect. will use cS,000 cwt. of ice. If we add to this the ice used during the retail of the beer, which is best di'unk at 12" C. (54° F.), we shall arrive at the total of 100 kilos, per hectolitre. STUDIES ON FERMENTATION. 13 other hand, must be consumed within a short time of its pro- duction. The brewer is thus compelled to manufacture it as it is wanted, and as orders are sent in, the demand for it being in a great measure dependent upon the state of the weather. Conditions so unfavourable as these must necessarily operate prejudicially against trade. Industry requires more stability and uniformity, both in the production and the sale of its goods. " Low beer '' can be brewed in large quantities at any time to be delivered at any other time, according to requirements ; its manufacture, therefore, is unattended by the inconveniences which we have just noticed.* How is it that the use of ice and yeast operating at a low temperature so greatly facilitates the preservation of our beer * [In connection with the comparison here instituted by M. Pasteur between the drinking and keej^ing qualities of the two kinds of beer, it may be useful to draw the reader's attention to a review by Dr. Charles Graham of the French edition of this work, published in Nature for January 11th, 1877, page 216. At the same time we must remark that Dr. Graham appears to have overlooked M. Pasteur's footnote, page 12, English edition: — "His assertion, that by bottom fermentation store beers can be produced, whereas those produced by top fermentation must be consumed at once and cannot be transported, are certainly strange to an Englishman. So far from these unfavourable comparisons being true in all cases, the exact opposite is generally the case. Bavarian and other bottom fermentation beers are in fact those which can neither be preserved nor transported without the liberal employment of ice ; even that sent from Vienna to London must be kept cold artificially, in order to avoid rapid destruction. As regards flavour, there are many who think a glass of Burton pale ale, or of good old College rent ale, to be superior to any Bavarian beer. The chief cause of the decline in the production of top fermentation beers on the Continent has been the want of attention in the fermentation process ; whereas the English brewer, especially the brewer of high-class ales, has been unremitting in his attention to the temperature in fermentation and to the perfect cleansing of the ale. Now, where such attention is given, it is not difficult to obtain ales which will keep a few years. "While objecting to oui- EngHsh produce being so hastily depreciated by M. Pasteur, our brewers will be the first to avail themselves of his biological researches, in order to render their produce more stable and better flavoured, without having recourse to the general adoption of the vastly more costly system of bottom fermentation."— D. C. E.] 14 STUDIES ON FERMENTATION. and enables us to secure such striking advantages? The expla- nation is simple : the diseased ferments, which we have pointed out, rarely appear at a lower temperature than 10° C. (50° F.), and at that temperature their germs cease to be active. The adoption of low temperatures by brewers is mainly due to this phvsiological fact. On one occasion only have we met with active vibrios (No. 3, Plate I.), at a very low temperature ; these were forming with great difficulty in wort fermenting at 5° C. (41° F.), From this we see that the changes which the manufacture of beer has undergone during the present century have been based mainly on the diseases to which beer is liable, either during or after the process of brewing. The fact that English brewers have not as yet adopted "low fermentation^^ may be accounted for, in a great measure, by the difficulty of enlarging existing breweries, in cities like London, to the extent required for the new method of manufacture. Even in the event of public taste demanding a " low beer," English brewers will hesitate a long time before converting their breweries. Such conversion would impose upon them expenses and difficulties of a very serious nature. If ever such a change should take place, it will probably be inaugurated out of London. It is, however, worthy of remark that English brewers, without adopting "low fermentation," have introduced considerable improvements in brewing, especialh^ in the management of the temperature during fermentation ; this must be preserved within narrow and exact limits, for fear of injury to the product. It might easily be shown that these improvements have resulted from the liability of the beer to contract diseases, although this fact may not have been recog- nized by the brewers who have introduced them. Besides the yeasts which belong to the two principal kinds of fermentation, there exist many varieties of alcoholic ferment that produce, each of them, a special kind of beer. Among these special beers some are deficient in taste, others in aroma, others in brilliancy. Let us suppose that in the manufacture of a beer with one of these yeasts, from which a peculiar flavour STUDIES ON FERMENTATION. 15 is derived, a diflferent and inferior variety is accidentally mixed with that which we intend to use ; in such a case, the inferior variety, the product of which will possess an inferior quality, will exercise such an influence on the brewing as to induce the belief that disease must be present. The microscope, if consulted, will reveal no special organism, nor any of those diseased ferments of which we have given specimens. It is in the study of yeast that we must endeavour to find the cause of the results we observe. This point, which is of the greatest importance to brewers, will become clearer as we proceed. If we examine the practices of the beer trade, in its retail as well as in its export branches, we shall find that many of them aftbrd evidence of the liability of the beer to deteriorate. We may cite some of these. When taken out of the ice-cellars, the beer is kept in casks of small capacity, that it may be the sooner consumed ; when exposed to a high tem- perature, the beer will not keep sound for any length of time, but will speedily effloresce with mycoderma vini or mycoderma aceti. Beer which is intended for bottling should not be kept for more than a month or six weeks. Even in bottling we may perceive the tendency of the liquid to deteriorate.* It is neces- sary that the bottles, immediately after being filled, should be laid on their sides for twenty-four or forty-eight hours ; they ma}'- then be placed upright ; the reason for this is that the air * To preserve bottled beer from deterioration, some bottlers employ, at the moment of filling, a small quantity of bisiilpliite of lime. Others heat the bottles to a temperature of bb° 0. (131° F.) In the north of Ger- many and in Bavaria, this practice has been widely adopted since the publication of the author's " Studies on Wine," and some of M. Velten's writings. The process has been termed pasteurizaiion in recognition of the author's discovery of the causes of deterioration in fermented liquors, and of the means of preserving such liquors by the application of heat. Unfor- tunately this process is less successful in the case of beer than in that of wine, for the delicacy of flavour which distinguishes beer is affected by heat, especially when the beer has been manufactured by the ordinary process. This effect would be less felt in beer manufactured by the process which is advocated in this work. 16 STUDIES ON FERMENTATION. left between the cork and the beer might give rise to the pro- duction of efflorescence. If we lay the bottles down on their sides, the oxygen of this air will be absorbed by the oxidizable substances in the liquid, and there will be little fear of germs developing themselves when the bottles are placed upright. The bottles should not, however, be left on their sides longer than forty-eight hours ; otherwise the supplementary fermentation may force the corks out. Moreover, when the bottles stand upright the products of fermentation collect at the bottom, and not at the sides. Beer which is intended for keeping, if exported or conveyed some distance off, must be surrounded with ice. Without this precaution it will ferment too much or contract some disease. " High beer " cannot stand travelling. This kind of beer should not be exported unless the ordinary proportion of hops has been greatly increased — hop oil acting in some respects as an antiseptic, and preventing the beer from contracting diseases.* The export of English beer to India and the Continent has fallen off of late years, or rather, has not increased to the extent that was anticipated ; in fact, this trade has entailed great losses upon those engaged in it. It is said that an English firm lost as much as £48,000 on one consignment, which on its arrival in India was found to be all turned. There are no breweries in hot countries, where beer would command a very large sale. It is a well-known fact that beer is a remarkably pleasant drink in tropical climates, provided its temperature be a few degrees below that of the atmosphere, but the expenses of its production would be enormous, on account of the immense quantity of ice that would be required in its manufacture and for its preservation. It is in hot countries that beer is most liable to deterioration. Beer is said to be the beverage of northern regions, which * A convincing proof of the influence of hops on the ferment organisms is contained in the fact that beer, even after being raised to ()0° C. or 70" 0. (140" or 160° F.), will, if unhopped, readily take on the butyric fermentation, from which, if hopped, it would remain perfectly free. STUDIES ON FEKMKNTATION. 1^ are deprived of the vine^ b}^ the rigour of their climate. In these regions man has sought in the abundance of grain-fruit a substitute for grapes. To a certain extent this is true ; never- theless, it is an undoubted fact that beer was first brewed in Egypt, a very hot country, whence its manufacture has spread over Europe. It was called Pelusian wine, from Pelusium, a city on the banks of the Nile, which produced a beer that was held in high esteem.* Beyond a doubt, hot countries, even those in which the vine is cultivated, would consume much beer, could it but stand their high temperature. t A considerable quantity of beer is now brewed in British India, but its manufacture entails an enor- mous outlay for ice. The complications which result from the tendency of wort and beer to deteriorate, underlie almost all the details of the process of brewing and the sale of beer, and have been the cause of most of the changes and improvements that have been eifected in brewing, during the present century. * For historical details, see V Encyclopedic, Art. Biere. t As a -wine-producing country France has been highly favoured by nature, but the consumption of beer in France is increasing every year. In 1873 the quantity of beer, paying excise duties, amounted to 7,413,190 hect., which yielded to the Treasury the sum of 20,165,136 fr. These figures are taken from a report published in 1875 by M. Jacqueme, inspector of finances, who remarks that the quantity of beer upon which excise duties are paid represents, probably, not more than one-third of the total production : two-thirds of the quantity brewed evades the duties. 18 CHAPTER II. On the Causes of the Diseases which affect Beek AND Wort. From our preceding observations it will be evident that the manufacture of beer, the arrangement of breweries, and all the processes practised by the brewer immediately depend upon this fact, that beer and wort are fluids essentially liable to change. Thus it becomes a matter of extreme importance that we should have an exact knowledge of the causes and nature of the changes which affect our produce, and it may be that this knowledge will lead us to regard the conditions of the brewing industry from a novel point of view, and bring about important modifications in the practices of the trade. We might vainly search the numerous works which have been written on brewing for information respecting the proposed subject of these studies. At the most we should find the diseases to which beer is liable in the course of its manufacture, or afterwards, vaguely hinted at ; perhaps we might be favoured with certain empirical recipes for disguising the evil effects of those diseases. It will be our endeavour to demonstrate the truth of the proposition we have already laid down, that every change to which wort and beer are liable is brought about solely by the development of organic ferments, whose germs are being per- petually wafted to and fro in the dust floating through the air, or distributed over the surface of tlie dift'erent materials and utensils used in brewing, such as nuiU, yeast, water, coolers, STUDIES ON FEKMEKTATIO^. 19 vats, tubs, casks, shovels, workmen's clotiies, and innumerable other things. It is evident that this proposition bears a marked resembiance to the one which we have demonstrated concerning the diseases of wine.* By the expression diseases of wort and beer, we mean radical changes which so affect the nature of those liquids as to make them unpalatable, especially if they are kept ; such changes produce beer which is sharp, sour, turned, oily, putrid, and otherwise bad. It would be unreasonable to apply the term disease to certain modifications in the quality of beer, which may be produced by practices more or less commendable. Such modifications, too, may result from want of skill in the brewer, from the composition of the wort, from the specific nature of the yeast, or from the inferior quality of ingredients. It is a well-known fact that " low beer," if manufactured according to the ordinary process, has not that same delicacy of flavour which characterizes beer fermented at a lower temperature than 10° C. (50° F.). Fermented at 10^ C. (50°F.),or 12^^ C. (53° F.) or at a higher temperature, it loses the peculiar properties which consumers prize. Nevertheless, in point of soundness it may be as good a beer as one which has been fermented at 6° C. (43° F.), or 8° C. (46° F.). One might say of the former beer that it is inferior to the latter in estimation ; but we could not rightly call it diseased, for we are supposing a case in which disease does not actually exist. § I. — Every Unhealthy Change in the Quality of Beer COINCIDES with A DEVELOPMENT OF MICROSCOPIC GeRMS which are ALIEN TO THE PuRE FeRMENT OF BeER, Our proposition concerning the causes of the diseases of wort and beer might be demonstrated in several ways. The follow- ing is one of the simplest : — Take a few bottles of sound beer, * A statement of this proposition, as far as it concerns beer, appeared first in outline in the author's Etudes sur le vin, published in 1866. C 2 'iO STUDIES ON FEUME STATION. say, for instance, that which is known in Paris as Tourtel's, Griiber's, or Dreher's, from the name of the brewer who manu- factures it. Place some of these bottles in a hot-water bath and raise the temperature to about 60° C. (140° F.). Permit them to coof and then place them by the side of the other bottles that have not been heated. In every case, especially if we conduct this experiment in summer, we shall find that in the course of a few weeks — the length of time varying according to the tempera- ture and the quality of the beer — all the bottles which have not been heated will have become diseased, in some cases even to the extent of being undrinkable. Let us next examine, by way of comparison, the deposits in the heated and non-heated bottles. We shall find associated with the pure alcoholic fer- ment other organisms, filiform and for the most part very slender, and either simple or articulated, as represented in Plate IT., the design of which is taken from actual deposits occurring iu beer that had been kept for some time at the ordinary'' temperature. A number of bottles of beer which had been heated on October 8th, 1871, were compared with those of an equal number of bottles of the same beer which had not been heated. The examinaticm took place on July 27th, 1872. The beer, which had been heated to 55° C. (131° F.), was remarkably sound, well flavoured, and still in a state of fermentation. As a matter of fact, Ave have proved by exact experiments that alcoholic ferments, heated in beer, can endure a temperature of 55° C. (131° F.), without losing the power of germination ; but the action is rendered somewhat more difficult and slower. Diseased ferments, however, existing in the same medium, perish at this temperature, as they do in the case of wine. The beer which had not been heated, had undergone changes which rendered it quite undrinkable. Its acidity, due to volatile acids, was higher than that of the other beer in the proportion of 5 to 1. The beer which had been heated contained A per cent, of alcohol more than the other. The deposits in the heated bottles also showed filaments of disease, but in such rninutc quantity that it was necessary to I'l 11 la ^ ■ \: a' O"^' ^ <@o"/ 9 <f <9 i (§) • '/ / O % <3b u u ^ c^ ©" "^^ ..■ ^ ; -^ </ @ ,^ i ^ A^^l^vf, i (:^ © '^ hfOC, Deyrolle del Appearance under the Microscope of the Deposit from "Turned" Beer. STUDIES ON FERMENTATION, 21 search many fields of the microscope to discover their existence. Those which we found after the heating must have existed in the beer before that operation ; the heat had destroyed them, without sensibly altering their shape or size ; they could neither multiply nor continue to exert any influence upon the compo- nents of the beer.* From these experiments we may easily perceive, on the one hand, that beers apparently sound to the taste do not contain these or any other filiform ferments, save in a scarcely appreci- able quantity ; and, on the other hand, that these same ferments appear with the first unfavourable change in the quality of the beers, and that they exist more or less abundantly in propor- tion to the intensity of disease. In certain extremely rare cases it may happen — so, at least, we have been assured, but we have not proved the fact ourselves — that beer may keep sound in bottle, even without the prelimi- nary heating. This exception can only occur in the case of certain beers of a peculiar composition, which are highly hopped, and are made during the favourable months of Novem- ber or December, out of the choicest materials, and fermented with yeast that happens to be pure. In the deposit of such beer, even after a lapse of several months or several years, we should find only the ordinary alcoholic ferment, the slow action of which would merely cause a gradual increase in the quantity of alcohol existing in the beer, and a diminution in the propor- tion of dextrine. This beer might grow old, as wine does, and remain perfectly sound. Very often the whole work of the brewer is jeopardized by the unsuspected presence of diseased ferments, a remedy for * As the deposit in the heated bottles is, as a rule, inconsiderable, it is necessary to exercise some precaution in collecting it. The bottles are taken up ; after some days' rest they are decanted very carefully, -with as little shaking as possible, until not more than one or two cubic centi- metres (about a tea-spoonful) of the liquid remains at the bottom. The bottles are then shaken vigorously, with the object of collecting the whole of the deposit from the bottom and the sides into this small quantity of liquid ; a drop of this is then examined under the microscope. 22 STUDIES ON FERMENTATION. which is only devised after the evil has evoked the complaints of customers. In such a case the brewer avails himself of the kindness of some other brewer to obtain a change of yeast — a custom which is recognized and valued in the trade, since all managers of breweries have an interest in keeping it up. The brewer whose produce is most satisfactory recognizes the fact that unforeseen circumstances may compel him at any moment to change his yeast. We have frequently had occasion to sliow that this necessity for a change of yeast depends, in most cases, on some change brought about by the presence of diseased ferments, the multi- plication of which has resulted fortuitously from some uncon- scious neglect during the process of brewing, or from climatic influences. When we reflect that yeast is a living being, and that the medium which serves as its aliment, and the water in which it lives, are remarkably well adapted for the development of a vast number of other microscopic beings, the comparative purity of yeast should surprise us even more than its deteriora- tion does. Now by means of microscopical observations we might often detect the existence of the evil long before we are warned of it by a defective working, wliich invariably entails great losses.* In proof of this remark we may cite the following facts. In the month of September, 1871, we were permitted to go through a large London brewery, in which the microscopical study of yeast was altogether unknown. We were allowed to make certain experiments in the presence of the managers of the establishment. We first examined porter yeast, which was collected in a channel that received the yeast as it worked out of the fermenting vessels. One of the ferments of disease abounded in this yeast, as may be seen in the accompanying * Since the publication of the author's " Studies on the Diseases of Wine, and the Dangers resulting to Wine and Beer from the Microscopic Parasites found therein," some intelligent brewers havejderived consider- able profit from the application of the theories laid down in that work. STUDIES ON FERMENTATION. 23 sketch, which was taken on the spot (Fig. 1). It was evident that the working of the porter was extremely unsatisfactory, and had, perhaps, been so for a long time ; indeed, we weie told that they had obtained a change of yeast from another London brewery that same day. We made a point of examining this yeast with the microscope. It was beyond comparison purer than the preceding yeast. It is evident that if these brewers had been in the habit of using the microscope they might have detected the unsoundness of their produce before the time when they actuall}'^ made the a & ®^ <© Q QUO Fig. 1. Fig. 2. discovery, which, no doubt, was forced upon them by the com- plaints of their customers, or some other annoying circumstance, that led to their obtaining a change of yeast. We next obtained permission to examine the yeast of the other beers undergoing fermentation, especially those of white beers, such as ale and pale ale. In the sketch which we made of these yeasts one may detect the presence of the filaments peculiar to turned beer (Fig. 2). We examined with much interest the ales which had imme- diately preceded those undergoing fermentation, the yeast of which we had just inspected. We were furnished with two kinds, both in casks, the one fined, the other not fined. The latter was visibly turbid, and, examining a drop of it, we dis- covered three or four filaments present in every field of tlie microscope. The ale which had been fined was nearly clear, but wanting in brilliancy ; it contained about one filament to the field. We asserted in the presence of the head brewer, who 24 STUDIES ON FEKMEXTATTON. had been suiiiinouerl, that these ales were extremely liable to change, that it was liighly necessary to dispose of them without delay, and that they were necessarily already faulty in flavour — a fact which all admitted after some hesitation — attributable, of course, to the natural reluctance which every manufacturer feels to own that his produce is not above reproach. We were shown some of the finings used in the brewery ; the)' were swarming with the same filaments of disease-organisms. We then propounded to the managers certain questions on the subject of the losses which a brewery may sustain from changes in its beer. We had heard from several brewers that the selling price of beer differed so greatly from the cost of its production solely in consequence of the losses which the unavoidable waste of large quantities of beer was constantly causing ; several brewers have in our presence estimated these losses at 20 per cent, of the total production, on the average. At first the English brewers returned somewhat vague answers to our questions ; however, after what had taken place, they doubtless recognized the fact that a mutual understanding between a savant and a practical man may often be of con- siderable benefit to the latter, and in the end they confessed to us that they had stowed away in their brewery a large quantity of beer which had gone bad in cask a fortnight or so after it was brewed. Having avowed thus much they expressed their great anxiety to learn the cause of so serious a change in their beer, which was quite undi'inkable. We examined it under the microscope, without being able to detect immediately any diseased ferments, but being aware that the beer had probably been clarified, by remaining undisturbed for a very long time, and that these ferments might have become inert and precipitated to the bottom of the enormous vats containing the beer, we examined the deposits which had formed at the bottom of these vats. They were composed solely of filaments of disease- organisms, without even the least trace of the globules of alco- holic yeast. The supplementary fermentation of this beer had evidently been nothing but a diseased fermentation. STUDIES ON FERMENTATION. 25 The resemblance between these fihiments and those which, in considerably smaller proportion, accompanied the globules of alcoholic ferment in our preceding observations, the change in the beer, which was almost as bad as beer could possibly be, along with an abundance of filaments, and the change, to a minor extent, in that beer which only jaresented a few filaments in a field of the microscope, impressed those managers of the brewery who were present with an entire belief in the theory which we had been endeavouring to impress on their minds concerninsr the causes of the badness of their beer. Some eight days afterwards we paid another visit to this same brewery, and learnt that the directors had lost no time in acquiring a microscope, and in procuring changes of yeast for all the varieties of beer, which they had put in working since our first visit. There are some periods of the year — early spring, summer, and autumn, for instance — when the working of a brewery is a matter of great difiiculty. The preservation of yeast becomes a subject requiring the most delicate treatment, in consequence of the increase in the temperature. In the early part of autumn the most important ingredients used in brewing are of inferior quality ; the deteriorating influences which have been at work have covered them with a variety of parasites. All these circumstances contribute to facilitate the development of diseased ferments. i:^ II. — The Absence of Change in Wout and Beer Coincides WITH the Absence of Foreign Organisms. The method which we have just pursued in demonstrating the existence of a relation between the diseases of beer and certain microscopic organisms can scarcely leave a doubt, it seems to us, as to the correctness of the principles which we are advocating. In every case where the microscope reveals in a yeast, especiall}^ a yeast which is in a state of activity, products 26 STUDIES ON FERMENTATION. which are foreign to the composition of alcoholic ferment, properly so called, the flavour of the beer is more or less un- satisfactory, according to the abundance or nature of these minute organisms. Moreover, when a finished beer of superior qual'ty loses in the course of time its agreeable flavour, and becomes sour, it may readily be shown that the alcoholic ferment in the deposit existing in bottles or casks, although originally pure, at least in appearance, becomes gradually intermixed with these same filiform ferments or other ones. These facts may be deduced from what precedes ; nevertheless, some prejudiced minds might perhaps urge that these foreign ferments are the consequence of some diseased condition, produced by circumstances of which we know nothing. Although this gratuitous supposition may be difiicult to sustain, we shall endeavour to corroborate our preceding obser- vations by the method of experiment which will be seen to be the more decisive. This method consists in proving that beer never possesses any unpleasant flavour, so long as the alcoholic ferment, properly so called, is not associated with foreign ferments ; that this also holds good in the case of wort, and that wort, liable to change as it is, may be preserved in a state of purity, if it is kept under conditions that protect it from the invasion of microscopic parasites, to which it presents not only favourable nutriment, but also a field for development. By employing this second method we shall, moreover, have the advantage of proving with certainty a proposition that we just advanced, and showing that the germs of these organisms proceed from the particles of dust which the common air wafts about and deposits on every object, or which are spread over the utensils and materials used in a brewer}-, materials that are naturally charged with microscopic germs, which various changes in the store-houses and maltings may multiply to an indefinite extent.* * If we put a handful of germinating barley from a maltster's cistern into a little water, and examine drops of the liquid, after it has become STUDIES ON FERMENTATION. 27 Let us take a glass flask having a long neck (Fig. 3 a), and holding from 250 c.c. to 300 c.c. {i.e., about 9 or 10 fl. oz. ) ; let us put into it some wort, hopped or not, and then draw out the neck of the flask in the flame of a lamp, so as to give to it the shape b (Fig. 3) ; let us next heat the liquid to the boiling point, when the steam will rush with a hissing sound out of the curved end. We may then, without further precaution, permit our flask to cool, or, as an additional safeguard, we may introduce a small quantity of asbestos into the open extremity, at the very moment when the flame is taken away from the flask. Before introducing the asbestos, we may pass it through the flame, and we may repeat this after it has been placed in the end of the tube.* The air which first re-enters the flask must come in contact with the turbid, under a microscope, we shall be amazed at the wonderful number of strange microscopic organisms that swarm on the surface of the grains and on the sides of the cistern. There is no doubt that their presence is injurious to germination, inasmuch as they absorb much oxygen ; more- over, they acidify the grain and cause it to deteriorate. * In these experiments the asbestos is only introduced by way of extra precaution. Originally, in his early experiments in connection with the subject of spontaneous generation, the results of which were published in 1860-62, the author did not use it, and he observed no ill effects resulting from the omission ; now, however, he constantly makes use of it. In studies of this kind novel precautions are never thrown away ; moreover, the presence of this asbestos is a sure bar to the entrance of insects. The author has preserved for a long time a flask, in the slender neck of which an insect is contained; he killed it with a flame just as it was approaching the liquid. Quite recently, M. Calmettes, a young engineer from the Ecole Centrale, when engaged, at 28 STUDIES ON FEllMENTATION. heated glass and tlie hot liquid, and these will destroy the vitality of any germs existing in such particles of dust as this air may introduce. The re-entrance of the air will be effected very gradually — sufficiently so to enable the drop of water which the air, as it enters, forces up the curved tube, to catch all particles of dust. Ultimately, the tube will become dry, but then the passage of the air will proceed so slowly that every foreign particle will get deposited on its interior sides. Experience tends to prove that external particles of dust cannot find their way into flasks of this pattern, having free communication with the air, at all events within ten or twelve years — the longest time that has been devoted to experiments of this kind ; the liquid in the flasks, if originall)'' clear, will not become in the least degree contaminated, either upon its surface or throughout its bulk, although the outside of the flasks may be covered with a thick coat of dust. This is an undeniable proof of the impossibility of particles of dust finding their way inside such flasks. Wort treated thus will preserve its purit}^ for an indefinite time, notwithstanding its extreme liability to rapid change when exposed to the air, under conditions which cause it to come in contact with the particles of dust that air contains. This also holds good in the case of wine, beef-tea, the must of grapes, and, generally speaking, of all liquids which are subject to putrefaction or fermentation, and which possess the faculty, when their temperature is raised to about 100° C. (212° F.), of destroying the vitality of those microscopic germs that are found in dust. A flask such as we have described (Fig. 3) is all that we require when we have to demonstrate the facts that we have Tantonville, in Tourtel's brewery, in carrjnng out certain practical ox- perimonts in connection with the process that will be described in ote of the later chapters of this work, wrote complaining that his flasks had been suddenly invaded by a swarm of aphides, scarcely larger than phylloxeras, and that manj' of them had even penetrated into the inside of the curved tubes. STUDIKS ON FERMENTATION. 29 just reviewed, a more detailed account of which may be found in our Memoir published in the A/iiiaks cie Chimie et de Physique, for 1862, under the title Memoire sur les corpuscules organises en suspension dans Patniospltere. Examen de la doctrine des generations spontanees. The shape of the flask represented in Fig. 4 only differs from that of the preceding one in having a second little tube attached to the globular part of the flask ; this presents great advantages for difi'erent objects of study, and it was adopted in the subsequent investigations detailed in this work. This flask will permit us to study without difficulty every separate kind of microscopic organism in the liquid best adapted to it without fear, if we take reasonable precautions, that the subject of our study may become associated with other organisms, the accidental presence of which cannot fail to seriously affect the results of our observations. Let us use one of those flasks in our experiment on yeast, at the same time expressly assuming that the minute germs of yeast are free from all contamination by foreign germs, an object which we shall learn how to realize by a variety of methods in a subsequent chapter. Let us introduce some wort into our flask (Fig. 4) ; then, Fig. 4. after we have fitted an india-rubber tube to the little supple- mentary tube, let us boil the liquid ; the steam, finding an easier exit through the india-rubber opening than through the drawn-out tube, will rush out through the india-rubber tube, and thus, as it passes, destroy any germs which may be adhering to the sides of the little supplementary tube. If we close tlie india- rubber tube by means of a glass stopper, the steam will imme- 30 STUDIES ON FERMENTATION. diately issue through the bent tube. On permitting the flask to cool, it will then be ready for impregnation. If there were any fear that some foreign germ of an unknown nature might have effected an entrance during cooling, or had not been destroyed by the steam (which is always a little super-heated, in consequence of the resistance which it meets in escaping), we need only place the flasks on a warm stove, and leave them there for a few days or a few weeks to ascertain whether the liquid in them has undergone any change. We should then only use those flasks which contain a sound liquid. At the same time we must warn our readers that this cause of error does not exist once in a thousand times, especiall)^ if we use an asbestos stopper to prevent the entrance of little insects which are attracted by the odour of the liquid, and which instinctively seek to enter at the extremity of the tube, and to pass through it into the flask. In going so far, and incurring such labour in search of their food, they condemn themselves to certain death, for they are sure to be drowned, since it would require an intelligence superior to that which they possess to enable them to get out of the flask ; the liquid, morever, could not fail to undergo some change, in consequence of the particles of dust that the insects would introduce into it. After having passed the flame of a spirit-lamp quickly over the india-rubber tube, the glass stopper, the curved tube, and even over the fingers of the operator, we mav withdraw the glass stopper, and introduce the pure yeast by means of a glass pipette that has been previously heated. This yeast is kept in a vessel also free from the dust floating in the air. However few globules of yeast tlie glass tube may take up, it is sure to introduce a hundred or a thousand times more than is necessary for the impregnation of the liquid. The glass stopper must then be replaced immediately, after having been again quickly passed through the flame. In transferring our yeast from the vessel containing it to the flask, by means of a glass pipette, it is exposed to another cause of impurity, since we cannot avoid bringing it in contact with the common air. If this risk STUDIES ON FEKMENTATJON. 31 frequently troubled us in our experiments, we might banish it or minimize it by some new arrangement ; but this is unneces- sary. We have suffered no inconvenience from this cause, as there does not exist in the atmosphere anything like a con- tinuous supply of that from which the so-called spontaneous generation arises, as was erroneously believed to be the case before the publication of our Memoir in 1862, to which we have already alluded. The following are the results of the experiments conducted in the manner just described. The yeast which we sowed, in ever so small a quantity, seemed to acquire vigour, to bud, and to multiply. Soon, that is to say in the course of twent^^-four or forty-eight hours or longer, according to the temperature, and, more especially, the degree of vitality in the globules, we found that the sides of our flasks were covered with a white yeasty deposit, and noticed on the surface of the liquid a fine froth, which at first appeared like little islets formed by groups of bubbles so minute that they would have been imperceptible had they not been joined to- gether. These patches increased in size, and gradually attached themselves to each other, finally forming a thick froth. In the course of two or three days this froth fell, the fermentation pro- ceeded less rapidly, and then ceased completeh'. The beer was finished. This beer might be preserved for an indefinite period in the flask without undergoing any change. The external air passes freely into and out of the flask, as the pressure of the atmosphere and the temperature vary, and the beer in the course of time becomes flat ; it acquires age in much the same manner as wine does, but it never contracts any taste of disease, it never becomes sour, or sharp, or bitter, or putrid ; it does not even become covered with mycoderma vini as is usually the case with all beer exposed to the common air in the course of trade. After some weeks, or perhaps months, a white ring may show itself on the surface of the liquid on the glass. This is a crown formed by a mass of young yeast globules, which grow there 32 STUDIES ON FERMENTATION. like a moulfl, by absorbing the ox3'geii of the air as it enters the flask. The bulk of the yeast which has been fermenting remains at the bottom of the liquid in the form of an inert deposit. This inertness, however, is apparent rather than real ; the globules may be internally active, without any development of new buds, and the effects of this working may cause them to become more and more languid, and in the course of time may even destroy them. The case is quite different when the yeast with which our wort is impregnated, instead of being pure, is mixed to any extent whatsoever with diseased ferments. Should there be any of these in the yeast with which we impregnate our wort, even though their quantity were so infinitesimal that the most skilful observer could scarcely discover them with the microscope, they would multiply in the flask after the beer had been finished, especially if the beer were left for a short time on a stove. In this manner we may secure an excellent test of the original purity of the yeast which we employ in the impregna- tion of our wort. Thus it may be seen that the absence of microscopic organ- isms that are foreign to the nature of pure yeast may invariably be noticed in the case of a beer which is sound, and which will remain sound for any length of time, when in contact with pure air, at any temperature. We may see, too, that the presence of these organisms may invariably be detected in an unsound beer, the peculiar unsoundness of which depends upon the peculiar species of the organisms contained in it. It would be difficult to adduce clearer proofs than those which we have given as to the intimate relation existing between these organ- isms and the deterioration of beer. The relation between cause and effect, in the succession of physical phenomena in general, is established by proofs tliat are by no means more decisive. 33 CHAPTER III. On the Origin of Ferments properly so called. The new process of brewing-, which it is the principal object of this work to explain, and which will follow as an immediate and inevitable deduction from the novel facts herein demonstrated, cannot be fully understood without a knowledge of all the principles upon which it is founded. One of the most essential of these has reference to the purity of our fermentation. We should gain but little from the use of a yeast uncontaminated by any foreign germs if natural organic substances had the power of organizing themselves by means of spontaneous gene- ration, or by some transformation which took place amongst them, or even by a conversion of certain microscopic beings into certain others. Theories of this kind are still warmly ad- vocated, but, to our thinking, rather from sentimental considera- tions or prejudice than from any basis of serious experimental proofs. Be this as it may, we must free our minds from all suppo- sitions which might qualify the exactness of the principles upon which our new process is founded, or cause any doubt in the minds of our readers as to the possibility of its application and the benefits to be derived from its adoption. § I. — On the Conditions which cause Variations in the Nature of the Organized Products existing in Infusions. We have proved by experiment, in the preceding chapter, 31: STUDIES ON FERMENTATION. that a few minutes' boiling renders liquids, and more especially wort, absolutely free from liability to change when in contact with pure air, that is, air which contains none of the germs of organisms that are continually floating about in the atmo- spheie. What is true in the case of wort is equally so in the case of all organic liquids ; there is not a single one that could not be rendered inaccessible to any subsequent change if it were brought, first of all, to a suitable temperature, which would vary with the nature of the liquids. Amongst them there are some which, like vinegar, lose their tendency to change after having been rapidly raised to a temperature of not more than 50° C. (122° F.) ; others, like wine, require a greater heat. "Wort, to which no hops have been added, should be subjected to a temperature of not more than 90" C. (19-1° F.) ; milk to about 110° C. (230° F.).* It is easy to show that these differences in temperature, which are required to secure organic liquids from ultimate change, depend exclusively upon the state of the liquids, their nature, and, above all, on the conditions that affect their neutrality, whether towards acidity or alkalinity ; for it is not difficult to observe that the least changes in these respects lead to consider- able variations in the temperatures which we must employ. t We could adduce many examples of this. The only diflference between the nature of must and that of wine is caused by fermentation. We may say the same thing of wort and beer, and, better still, of new milk and sour milk. Must, to be secured from change, requires a much higher temperature than wine does ; similarl}^, wort requires a much higher temperature than beer. Milk must be heated to about 110° C. (230° F.), as we have just stated, but sour milk would require 20° C. or 30° C. • We have heard of liquids even less sensitive than these, which re- quired a temperature of 120° C. (248° F.) or more, hut we have had no opportunity of studying them. t See Pasteur, Memoire sur les Generations dites Spontanees {Annalea dc Chiviie et de Physique, t. Ixiv. Z' serie, annee 1862). STUDIES OX FERMENTATION. 35 (36° F. or 54° F.) less. Wine, when fresh, ceases to be liable to change after it has been brought to a temperature below 100° C. (212° F.) ; it requires a temperature of more than 100° C. in the presence of carbonate of lime. As regards the explanation of the influence which acidity or alkalinity exerts in diminishing or increasing the tempera- ture required to protect infusions and organic matters from ultimate change, although this is a subject which claims special study, we are inclined to believe that acidity permits, and alkalinit}' prevents, the penetration of moisture into the interior of the cells of the germs belonging to infusions, so that in heating the outer cases of these cells in an alkaline medium we heat the germs in a dry state ; and in heating the outer cases in an acid medium we heat the germs when they are moist. We all know that these conditions make a great difference in the resistance which bodies offer to the action of heat. A particle of mould which, in a moist state, cannot survive a temperature of from 60° C. to 100°. C. (140° to 212° F.), will preserve its fecundity even if heated to 120° C. (248° F.), if it has been previously well dried.* The nature of the spontaneous productions which we see appear in organic liquids is affected to a remarkable extent by the smallest change in the compositions of those liquids. Generally speaking, as we have often proved in our former works, a feeble acidity is unfavourable to the development of bacteria and infusoria, and, on the other hand, favourable to the growth of mould. A liquid which is neutral or of feeble alka- linity behaves in an exactly inverse manner. Those who support the theory of spontaneous generation seek to find in the natural differences between the organic pro- ductions of various liquids which are simultaneously exposed to the same atmosphere, an argument in favour of their doctrine. These differences, however, are only an effect of the greater or less fitness of a peculiar liquid for a certain kind of growth. When an acid organic liquid, such as the must of grapes for * See mv Meraoire sur les Generations dites Spontanees, already cited. D 2 .'36 STUDIES ON FERMENTATION. example, is exposed freely to the air, it is besieged simul- taneously by spores of miicecUnes, as well as germs of bacteria, leptofhnx, vibrios, dc, but the latter germs are checked in their development, if, indeed, they are not actually destroyed, by the acidicy of the liquid ; and we should never find them there in their adult state. Most moulds, on the other hand, thrive in acid liquids, and therefore they are generally found alone. If we began by saturating must with carbonate of lime, previously dried or otherwise, we should observe opposite phenomena ; bacteria, lactic ferments and butyric vibrios would invade our fields long before the spores of mould had time to grow, since the germina- tion of these proceeds very languidly in neutral or alkaline liquids, and an infusion once occupied by a living organism has much trouble in nourishing others ; the early developments consume the alimentary substances, especially the oxygen. Differences as marked as these in the adaptability of certain liquids to certain growths give rise to innumerable illusions, and are one of the chief sources of error in the study of this subject. If we sow in aa acid liquid, such as must, an alcoholic ferment, the development of which is not arrested by the acid character of our medium, it will multiply there, and we shall have no difficulty in growing it over and over again in the same acid medium. This being the case, let us suppose that our alcoholic ferment is impure — let us say, mixed with filaments of turned wine, which are due to a ferment checked in its development by the peculiar qualities of the must. In repeating the growth of this alcoholic ferment in the must the filaments which were, by supposition, present in our first sowing, and which cannot reproduce themselves in the must, or do so with great difficulty, will become very scarce in the fields of our microscope ; they will not, however, cease to exist, for the repetition of our attempts to grow them only serves to spread the original germs over a larger surface ; and, although the eye fails to detect them, they will only have become more difficult to discover. At this point the experimentalist is in danger of falling into error, for when STUDIES ON FERMENTATION. 37 he no longer perceives the foreign ferment he will be inclined to believe that it has vanished, and that he may regard the alcoholic ferment as being free from all impurity, without testing for the purity by a direct experiment. An example of this mistake is to be found in a recent work by M. Jules Duval. This writer has published a theory accord- ing to which yeast becomes transformed into lactic ferment, and likewise into other ferments — that of urea, for example — the only condition of change, according to M. Jules Duval, being that we should cultivate it in suitable mediums. The proofs by which he supports his conclusion are altogether inadmissible, and a simple glance at his experiments enables us to detect innumerable causes of error. M. Duval believes that yeast becomes transformed into lactic ferment from the fact that he obtained a fermentation which furnished lactate of lime and lactic ferment from some sour milk to which he had added some glucose, chalk, and phosphate of ammonia before impregnating it with yeast ; but he took no steps to secure himself against introducing into his medium — which was, as a matter of fact, well adapted to lactic fermen- tation, inasmuch as it was a little alkaline — an alcoholic ferment containing impurities. This was the crucial point in his experiments. M. Duval recognizes this, but he deceives himself when he says, without proof: — "My alcoholic ferment is pure, for I have grown it over and over again in must, pre- served in flasks prepared after the manner of those which M. Pasteur uses in his experiments." Here we have merely a simple assertion ; a direct experimental test might have proved that the yeast was impure. Yeast cannot transform itself into lactic ferment. No matter what the medium may be in which it is sown, if it is actually pure it will never present the least trace of lactic ferment or of any other ferments. By certain changes in the nature of the medium, the temperature, and other conditions, the cells of the ferment may become oval, elongated, spherical, and larger or smaller in size, but they will never produce the most minute quantity of 3'S STUDIES ON FERMENTATION. lactic ferment or lactic acid. The whole theory of the transmu- tation of ferments, which M. Duval has published, is imaginary.* We have asserted that the source from which we obtain our supply of the substances that we expose to contact with the air may likewise furnish a reason for the development of vegetable or animal organisms which make their ap- pearance in our infusions. This may be easily under- stood. If we expose the infusions to temperatures more or less high, with the object of destroying the vitality of the germs which they may contain, we completely suppress all germs originating from two different sources — those which the infusions maj^ have acquired directly from the atmospheric air, or from the dust upon our utensils, and those which may have been introduced by the materials used in manufacturing our infusions or decoctions, which materials must have been brought from some distance. An infusion, after having been heated to a sufficient degree, can harbour and nourish only such germs as are conveyed to it by the air after the heating. These floating germs are far from being as varied in their nature as it pleases those to believe who are tied down by their arbitrary and faulty interpretation of the knowledge that we have acquired, and the discussions in which we have taken part, during the last fifteen years. Air, unless in violent agitation, can hold only the most minute particles in suspen- sion. The observations which have been recorded concerning organisms of spontaneous growth found in infusions, have always been made in sheltered places — in rooms or laboratories, the atmosphere of which is, relatively speaking, verj'- still. For this reason, in liquids that have been subjected to heat, the flora and fauna — if we may use such an expression — are very poor, and all the more so because, as we have recently • Jules Duval (of Versailles), Nouveaux fails concernant la mutahiUte des germes microscopiques. Role passif des etres classes sous le nom de ferments. (See the Journal d'Anatomie et de Physiologie, edited by C. Robin, Sept. and Oct. 1874, and Compte-rendus de V Acadrmie des Sciences, Nov. 1874). M. Bechamps had previously fallen into similar errors. STUDIES ON FERMENTATION. 39 had occasion to remark, a great number of the organisms which would spring into existence of their own accord, if they were allowed sufficient time for germination, are kept back by others of a more rapid development. The truth of this is evident from the fact that we may observe greater variations in the nature and number of species of living organisms, if we divide one infusion amongst several vessels which are immediately closed up again, than if we leave our infusion in contact with an unlimited volume of surrounding air. By this means we expose each portion of the liquid to no other germs than those existing in a state of suspension in the volume of air introduced into the vessels ; and it so frequently happens that we obtain a variety of germs which, coming into contact with a liquid adapted to their nutrition, without having mixed with others, finally multiply there, in consequence of no other organisms of greater activity occurring to impede their slow and laborious propagation. The nature of the products resulting from raw infusions — that is, those obtained, without heating, from the maceration of organic substances, such as leaves, fruits, grains, or the organs of plants or animals — is much more varied. The reason of this is that such substances generally carry with thera not only the particles of dust existing in air that is in motion, but also micro- scopic parasites, which find a congenial resting-place on their surface or its vicinity. We may cite a few accurate observations on this point, for the subject is one of great interest. If we boil an infusion of hay, and then expose it to contact with the air in a room, all its productions will be derived from such germs as a comparatively still air can carry about ; thus, we shall very rarely find any colpoda in our infusion, for the germs of these infusoria, consisting of rather large cells, can scarcely exist in a state of suspension in motionless air, in spite of their extraordinary diffusion in nature. On the other hand, we almost invariably find colpoda in macerations of raw hay. This difference is easily accounted for ; the particles of dust adhering to the surface of hay, especially that which comes from marshy districts, contain the germ-cells of colpoda in 40 STUDIES ON FERMENTAIIOX. abundance. The reason is obvious ; rain falls on a meadow and forms little pools of water about the roots of the herbs that grow there ; this water remains for some time, and very soon swarms with a multitude of infusoria, especially colpoda. Dry- ness follows ; the colpoda becomes encysted, and forms a dust that is wafted by the winds on to the blades of grass, so that the mower will carry away not only his hay, but also myriads of colpoda, as well as spores of mucedines and other organisms,* The maceration of pepper will give us some infusoria hardly ever found in other infusions, the reason of this being that such infusoria exist where the pepper grows — in other words, their germs are exotic. That the infusion of a special plant should give us special infusoria, is scarcely more surprising than the discovery of a particular parasite or insect existing on a particular plant, and not on others of a different species that grow near it. Thus it happens that the ferment-germs of must exist on the surface of grapes, whether detached or in clusters. It is only natural that we should find the organ or the plant that is destined later on to become the food of a parasite serving as a habitation for the germs of that parasite. ^ II. — Experiments on Blood and XJkine taken in their Normal State, and Exposed to Contact with Air THAT HAS BEEN DEPRIVED OF THE PaRTICLES OF DuST "WHICH IT Generally holds in Suspension. Recourse to the application of heat, in the first place, is an excellent means, as we have just seen, of procuring organic liquids free from all disturbing germs ; but there is a still more remarkable and instructive, wc may even say more unlocked for, method of securing this result, which may be described as in some measure borrowed from the nature of things. It consists in seeking purity in the natural liquids of animals and plants. It is difficult to understand how the liquids circidating in the organs of animal bodies, such as blood, urine, milk, amniotic • On this subject see the observations of M. Coste {Compte-rendus de ' Academie, t. lix. pp. 149 and 308, 18G4). STUDIES ON FERMENTATION. 41 fluid, and so on, can possibly secrete the germs of microscopic organisms. There would be excellent opportunities for these germs to propagate themselves if they actually did exist in the liquids appertaining to the animal economy. Life in all pro- bability would become impossible in the presence of such guests. A proof of this is to be found in the multitude of diseases which many of the greatest minds of modern times attribute to parasitic developments of this nature. Medical men of high authority agree in thinking that the questions of contagion and infection will find solutions from the obscurity in which the}^ are now involved in a careful study of ferments, and that hygieuists and physicians should labour to secure by every possible means the destruction of the germs of ferments, and should strive to check their development, and prevent the evils which they cause when developed. Great progress has already been made in this direction, and we deem it a signal honour that our researches on the subject have been considered, even by those by whom this progress has been accomplished, as the source from which they derived their first inspirations. We shall, doubtless, be excused by our readers if we recall this fact in relating certain historical details which are especially necessary in order that they may comprehend the principles that we are endeavouring to explain in these " studies." It is a matter of regret to us, however, that the facts which we have established should have been accredited with any importance beyond that which is their due. The exaggeration of novel ideas invariably leads to a reaction, which, again, overshooting the mark, brings into disrepute even those points in which such ideas are perfectly j ust, or, at all events, worthy of serious consideration. There are certain symptoms of such a reaction in the case of our theories : they are evident in the tendency of unreflecting minds to give a total denial to the fact that certain diseases may be derived from certain ferments — organized and living ferments — of the nature of those which have been discovered in the course of the last twenty years. We should be guided by facts, whichever side of the question 42 STUDIES ON FERMKNTATION. we espouse, and by facts alone we should test the truth of doubtful discoveries. We are but on the threshold of the exploration of our subject, and we should strive to discover new facts in connection with it, and should deduce from these, what- ever they may be, only such conclusions as they may strictly warrant. Unfortunately, there is amongst physicians a tendency to generalize by anticipation. Many of them are men of rare natural or acquired talent, endowed with keen powers of intellect, and the art of expressing themselves fluently and persuasively ; but the more eminent they are, the more they are occupied by the duties of their profession, and the less leisure they have for the work of investigation. Urged on by that thirst for know- ledge which belongs to superior minds, and perhaps, in some measure, through associating with the upper classes of society, which are becoming more and more interested in science, they eagerly seize upon easy and plausible theories, readily adapted for statement which is general and vague just in proportion to the unsoundness of the facts on which they are based. When we see beer and wine undergo radical changes, in consequence of the harbour which those liquids afford to microscopic organisms that introduce themselves invisibly and unsought into it, and swarm subsequently therein, how can we help imagining that similar changes may and do take place in the case of man and animals ? Should we, however, be disposed to think that such a thing must hold true, because it seems both probable and possible, we must, before asserting our belief, recall to mind the epigraph of this work : the greatest aberration of the mind is to believe a tJtinrj to be, because we desire it. One of the most distinguished members of the Academy of Medicine, M. Davainne, who was the first to give his attention to rigorous experiments on the influence that organic ferments exercise on the production and propagation of infectious diseases, declares that the idea of his researches on splenic fever and malignant pustule was suggested to him by his perusal of our work on butyric fermentation, published in 1861. In 1850 this gentleman and M. Rayer discovered in the blood STUDIES ON FERMENTATION. 43 of animals attacked by these diseases minute filiform bodies, to wbich they paid little attention, and which M. Davainne recollected, when he came across our Memoir. He had the foi'e- sight to conjecture — a conjecture that was soon confirmed most decisively by his researches — that the former disease, known under the name of sang cle rate, might be the production of a fermentation analogous to the butyric, in which the minute fili- form bodies observed by Rayer and himself, in 1850, played the part which vibrios fill in butyric fermentation. Within two years of this the first works of Messrs. Coze and Feltz appeared. These clever and courageous experimentalists avowed that their beau- tiful researches had been suggested to them by the perusal of my work on putrefaction, published in 1863. We might also quote the striking and admirably conceived experiments of Dr. Chauveau, on castration.* We cannot, however, refrain from reproducing here a letter addressed to us in 1874 by the cele- brated Edinburgh surgeon, Mr. Lister: — " Edinburgh, Feb. 10, 1874. " Dear Sir, — Will you permit me to beg your acceptance of a pamphlet which I forward to you by this post, and which describes certain inquiries into a subject upon which you have thrown so much light — the theory of germs and fermentation ? It gives me pleasure to think that you will peruse with some interest what I have written about an organism that you * Chauveau' 8 experiments were directed to show that the operation listournage, employed by veterinary surgeons for castrating animals by twisting and subcutaneous rupture of the spermatic cord, an operation which, though leading to the mortification and subsequent absorption of the testicles, is commonly attended with no other mischief to the animal, does, nevertheless, lead to septic eflFects of a serious character, provided that septic germs — decomposing serum containing vibrios, for example — be introduced into the blood current. From the fact that the operation is ordinarily harmless, M. Chauveau concludes that septic organisms are not produced by the action of the constituent gases of the atmosphere — always present in the blood — upon albuminous matter when outside vital influences ; whilst, from the success of the direct experiment of intro- ducing septic germs, he concludes that the phenomena always arise from the actual presence of such genns. — D. C. B. 44 STUDIES ON FERMENTATION. were the first to study in your Memoir on lactic fermentation. I do not know if you ever see the records of British surgery. If you have perused them you may have observed from time to time notices of the antiseptic system which I have been endeavouring for the last nine years to bring to perfection. Permit me to take this opportunity of offering you my most hearty thanks for having demonstrated by your brilliant researches the truth of the theory of putrefactive germs, and for having afforded me in this manner the sole means of per- fecting the antiseptic system, " Should you ever come to Edinburgh I am sure that you will be truly gratified to see in our hospital the extent to which the human race has profited by your work. I need hardly add, that I should have great satisfaction in showing you how greatly surgery is indebted to you. " Excuse the freedom which I have taken in addressing you, on the grounds of our common love for science, and believe in the profound esteem of yours, very sincerely, Joseph Lister." The wad dressing of Dr. Alphonse Guerin, surgeon at the Hotel-Dieu, Paris, which has already rendered great assistance to surgery, and has been the subject of a very favourable report at the Academy of Sciences in Paris, was invented by its author in consequence of certain reflections suggested to him by the perusal of our researches. The commission which framed the report made, through M. Gosselin, some wise reservations in the case of certain theoretical ideas which M. Guerin had not sufficiently proved by experiment. We have no doubt, how- ever, that when the matter is thoroughly examined, facts will confirm the truth of the wide views entertained by the surgeon of the Hotel-Dieu. Dr. Declat has founded quite a new system for the treatment of infectious diseases, which is based upon the use of one of the best known antiseptics, phenic acid. His theory, which he affirms was suggested to him by our studies on fermentations, is, that the diseases which transmit themselves are, each of STUDIES ON FERMENTATION. 45 them, the product of a special ferment, and that both medicid and surgical professors of therapeutics should make it their study to prevent exterior ferments from penetrating into the liquids of our economy, or in the event of these ferments having found their way into the system, to discover antiferments for their destruction, without effecting any change in the vitality of the histological elements of the liquid or tissues. There is no doubt that extreme caution must be exercised in dealing with questions of this kind, as M. Sedillot has autho- ritatively remarked ; but at the same time it cannot be denied that the more such questions are discussed with exactness, the more those celebrated practitioners who originated them are confirmed in the ideas which first guided them. We may give another example of this. In 1874, in consequence of a communication addressed to the Academy of Sciences by Messrs Gosselin and A. Robin, on the subject of ammoniacal urine, we made the observation that we should endeavour to ascertain if, in all such cases, the urinary fluids were not rendered ammoniacal by the presence of the little ferment of the urea, which we have previously noticed,* and which has since then been discussed with remarkable in- telligence by M. Van Tieghem, in the thesis which he main- tained for his doctor's degree. The suggestion and the considerations that justified it led to a discussion before the Academy of Medicine, in which contrary opinions were main- tained. We lost no time in submitting these to the test of facts. We could not find a single person suffering from ammoniacal urine, in whose case the little ferment which we mentioned was not to be detected. Our predictions were thus completely justified. As early as 1864 the Gazette Hehdomadaire de Medecine et de Chirurgie published an account of urine made ammoniacal in the bladder, the author of which, Dr. Traube, makes the follow- ing observations : — " It was believed that, in consequence of the * See Pasteur, Me moire sur Its Generations elites Spontanees, pp. 51 and 52, 1862. 46 STUDIES ON FEIIMENIATION. retention of the liquid, and the resulting distension of the bladder, that organ became irritated and produced a larger quantity of mucus, and that this mucus was the ferment which caused the decomposition of the urea, by virtue of a chdmical action peculiar to itself. This belief can no longer be held in presence of M. Pasteur's discoveries. This observer has demonstrated, in the most conclusive manner, that ammo- niacal fermentation, like alcoholic and acetic, is produced by living beings, whose pre-existence in the fermentable liquid is a necessary condition of the process. The preceding fact offers a remarkable proof of M. Pasteur's theory. In spite of the long duration of the retention, alkaline fermentation was not produced by an excessive secretion of the vesical mucus or of pus : it only began from the moment when the germs of vibrios passed from the outside into the bladder."* Finally, we may conclude with perfect truth that the liquids of our economy, blood and urine, for instance, may aiFord a * Davainne, Compte-rendus de VAcademie des Sciences, t. Ivii. p. 220, 1863. Coze and Feltz, Recherches cliniqnes et experimentaJes siir les maladies infect ie uses, Paris, J. B. Bailliere, 1872. Summary of all their works published before 18fi5. Dr. Lister, Medical and surgical journals, particularly the Lawet, 1865-67. Dr. GuEaiN, Compte-rendus de VAcademie dfs Sciences, March 23, 1874, and May 28, 1874, also the Report of M. Gosseliu, December, 1854. Dr. Sedillot, Compte-rendus de VAcademie des Sciences, November, 1874, t. Ixxix. p. 1108. Pasteur, Memoir e sur la fermentation appelee lactique. {Annales de Chimie et de Physique, t. lii. S" serie, 1875.) — Animalcules infusoires, vivant sans gaz oxygene litre et determinant des fermentations. {Compte- rendus de VAcademie des Sciences, t. lii. 1861.) — Recherches sur la putre- faction. {Compte-rendths de VAcademie des Sciences, t, Ivi. 1863.) GossELiN, Robin, and Pasteur, Compte-rendus de VAcademie des Sciences, January 5, 1874. Urines ammoniacnles. Traube, Gazette hebdomadaire de medecine et de chirurgie. Sur la fermentation alcaline de Vurine, April 8, 1804. Chauveau, Putrefaction dans Vanimal vivant. {Compte-rendus de VAcademie des Sciences, April 28th, 1873.) STUDIES ON FERMENTATION. 47 harbour to different ferments, even in the inmost parts of the organs, when external causes enable such ferments to find their way into those liquids, and that diseases of greater or less gravity result from this cause. On the other hand, it must be admitted that the bodies of animals in a state of health afford Fig. o. no means of entrance to these external germs. At the same time, direct experiment alone can convince the mind as to the truth of this latter assertion. Let us take some of the sub- stances that are to be found inside living animals in perfect health, and expose them, in the same condition in which life has formed them, to contact with pure air. For this purpose we must provide ourselves with a glass flask, joined to a copper tap by means of an india-rubber tube, as shown in Fig. 5. The two branches of the tap should be about Fig. 6. 48 STUDIES OX FERMEXTATIOX. twelve centimetres long (about i-in.), the one which is free tapering off like the end of a pipe. In order to destroy all germs which may exist in the flask, we must join the free end of the copper tube to a platinum tube kept at a very high temperature, after having carefully introduced into the flask a small quantity of water, and expelled all the air by converting the water into steam. Then as we allow the flask to cool, the air which re- enters it will necessarily pass through the hot tube (Fig, 6). We may cause the water in the flask to boil at a temperature Fig. 7. of more than 100° C. (212° F.) by fitting to the free end of the platinum tube a glass tube bent at riglit angles, which we plunge to any depth in a deep vessel filled with mercury (Fig. 7). Whilst the water is boiling under pressure, we must separate the tube which is plunged in the mercury ; the water in the flask will STUDIKS ON FERMENTATION. 49 continue to boil at the ordinary pressure. "We must then leave the flask to cool. It will gradually become filled with air that has been heated to a high. temperature, more than sufficient to burn up all the organic particles of dust which that air could have contained. When the flask is cold we must close the tap and detach it, and proceed to prepare other similar flasks. It will be advisable to close the tap when the temperature of the flask is still a few degrees above that of the surrounding atmo- sphere : this precaution will cause the air in the cooled flask to have a lower pressure than that of the external atmosphere. During the interval which must elapse between the prepara- tion and the use of a flask, it is a good thing to keep the free branch of the tap inclined towards the ground, to secure the inside of its tube from the deposit of external particles of dust. Whether this precaution be adopted or not, we must take care to heat this branch in the flame of a spirit lamp just before we bring our flask into requisition. If we have to study blood, we must take it from a living animal — a dog, for example. We must expose a vein or an artery of the animal, and make an incision into which the end of the free tap-branch, which has previously been heated and allowed to cool, must be introduced and fixed by a ligature in the vein or artery. On opening the tap, the blood will rush into the flask ; it must then be closed, and the flask placed in an oven at a certain temperature. We have successfully accomplished these manipulations, thanks to the kind help of our illustrious colleague and friend, M. Claude Bernard. The operation is nearly the same in the case of urine. The end of the free branch of the tap is introduced into the passage of the urethra ; the tap is turned at the moment when the urine is emitted, which is then allowed to pass into the flask, until it is a third or half filled. The following were the results of our experiments : — Blood underwent no putrefactive change even at the highest tempera- tures of the atmosphere, but retained the odour of fresh blood, or acquired the smoll of lye. Contrary to what we might have 50 STUDIES ()> FEllMENTATIOX. expected, the direct oxidation of the constituents of blood by slow combustion was rather sluggish. After subjecting our flasks to a temperature of 25^ C. or 30° C. (77° F. to 80° F.) in an oven for several weeks, we observed an absorption of not mora than 2 or 3 per cent, of oxygen, which was replaced by a volume of carbonic acid gas of about an equal bulk.* Nearly the same results were obtained in the case of urine ; it underwent no radical change ; its colour merely assumed a reddish brown tint ; it formed some small deposirs of crystals, but without becoming at all turbid or putrefying in any way. The direct oxidation of the urinary substances was likewise very sluggish. An analysis of the air in one of the flasks, made , forty days after the commencement of the experiment, gave the following results : — Oxygen . . . . . . . . 19-2 Carbonic acid.. .. ., .. 0-8 Nitrogen 80-0 100-0 * We must mention one curious result, which relates to what have been called the crystals of the blood. We could hardly have recourse to a better method of preparing these crystals, at least in the case of dog's blood, which seems to yield them with the greatest facility in any quan- tity we might desire to procure. Under the circumstances just recounted, in which dog's blood exposed to contact with pure air underwent no putrefactive change whatever, the crystals of that blood formed with a remarkable rapidity. From the first day that it was placed in the oven and exposed to an ordinary temperature, the serum began gradually to assume a dark brown hue. In proportion as this effect was produced, the globules of blood disappeared, and the serum and the coagulum became filled with very distinct crj^stals, of a brown or red colour. In the course of a few weeks, not a single globule of blood remained, either in the serum or coagulum ; every drop of serum contained thousands of these crystals, and the smallest particle of coagulum, when bruised under a piece of glass, presented to view coloux-less and very elastic fibrine, associated with masses of crj^stals, without the slightest trace of blood- globules. Where our observations were protracted, it sometimes hap- pened that all the fibrine collected into one hyaline mass, which gradually expelled every crystal from its interior. STUDIES ON FERMENTATION. 51 These experiments on blood and urine which we have just mentioned date from 1863.* Ten years afterwards, in 1873, they were confirmed in an important and striking manner by the results of a very able series of experiments, which were carried out in our laboratory, by M. Gay on, who was formerly a pupil in the Ecole Normale Super ienre. M. Gayon proved that what held good in the case of blood and urine, also held good in the case of the substances contained in eggs. He found that the whites of eggs might be exposed for any length of time to contact with air, as also might the yolks, or the white and yolks mixed, without any putrefactive change or fermentation result- ing, and without the smallest microscopic germ showing itself, the sole condition being that the air must be freed from all organic particles of dust, germs of mould, bacteria, vibrios, and other organisms which it holds in suspension. This was only a part of the important facts brought to light by M. Gayon. Amongst other things, he proved that spontaneous putrefaction in eggs is invariably caused by the development of organized ferments, thereby correcting the opposite statements an- nounced by M. Donne and M. Bechamp, who were led by their observations to believe that the change in eggs took place quite independently of the action of vibrios and niucedines.f It is almost superfluous to remark how greatly the results of these experiments on blood, urine, and the components of the egg are opposed to the doctrine of spontaneous generation, as also to most modern theories on the generation of ferments. As long as experiments relating to the question of so-called spontaneous generation were made on heated substances, the advocates of heterogenesis had some grounds for asserting that such materials could not satisfy the conditions of spontaneous life, and that we should obtain different results by using natural organic liquids, which, if exposed to contact of pure air would * Pastetje, Comptes rendus de V Acndemie des Sciences, t. Ivi. p. 738, 1863. f Gayon, Comptes rendus de V Academic des Sciences, and AnnaJes Scientifiques de I'Ecule Nornmle Suverieure, 1874-7o. E 2 52 STUDIES ON FERMENTATION. doubtless serve for the production of new beings which did not issue from parents which resembled them. This novel enuncia- tion of the hypothesis of spontaneous generation, the only one, we think, that could be defended after the publication of our Memoir, in 1802, is condemned by the preceding facts. The same facts completely upset the hypothesis recently maintained by Messrs. Fremy and Trecul on the subject of the causes of fermentation. " Side by side with the immediate, definite principles which may be formed by synthesis," saj-^s M. Fremy, " such as glucose, oxalic acid, and urea, other substances of greatly in- ferior stability exist, the constitution of which is considerably more complicated, containing all the elements of living organs, such as carbon, hydrogen, oxygen, nitrogen, and even phos- phorus and sulphur ; and often salts of lime and of the alkalies besides. These bodies are albumen, fibrin, casein, the congeners of vitellin and others. Chemical synthesis cannot reproduce them. It is impossible, in my opinion, to regard them as im- mediate, definite principles. I designate them by the general name of semi- organized bodies, because they hold an intermediate place between the immediate principle and the organized tissue. " These semi-organized bodies, which contain all the elements of organs, have the power, like a dr}^ seed-grain, of existing in a state of organic immobility, and of becoming active under circumstances which favour organic development. By reason of the vital energy that they possess they undergo a succession of decompositions, giving origin to new derivatives, and to the advent of ferments, not by any process oi spontaneous r/eneration, but by a vital energy/, which pre-exists in the semi-organized bodies, and is simpl}'- carried on, when this energy manifests itself, in these most varied organic changes." After having expressed these hypothetical and confused opinions, M. Fremy continues : — " I do not consider, then, that these semi-organized bodies serve merel}' as nourishment for certain animal and vegetable organisms, which may be the sole agents of fermentations, but I give them a direct role and STUDIES ON FERMENTATION. Oo admit that, under the influences which I have already cited* they may assume a real and complete organization, and produce ferments which are not derived, as we have seen, from a germ or an ovum but from a semi-organized body, the vital energy of which has become active. f It will be seen that these opinions are quite different from those which M. Pasteur has maintained in his works, since they attribute the origin of alcoholic and lactic ferments to an albuminous substance. Taking the case of alcoholic fermentation alone, I assert that, in the production of wine, it is the juice of the grape itself which, in contact with air, produces grains of yeast, by the transformation of the albu- minous substances. M. Pasteur, on the contrary, maintains that the grains of yeast are produced by certain germs. "| We have combated these propositions, so extraordinary and unsupported by any rigorous experiments, before the Academy of Sciences, where they were first enunciated. On that occasion we related the facts in connection with blood and urine, which we have just discussed. Could there be any more forcible argument against the theory of our honourable colleague than those facts ? Here we had natviral albuminous substances, forming part of matter eminently liable to putrefactive change and fermentation, which produced no ferments of any sort whatso- ever when brought into contact with air deprived of its organic particles of dust. Under no known circumstances is albuminous matter trans- formed into grains of yeast or any other organized ferment, and, to our thinking, nothing can be more chimerical than the gratuitous hypothesis of hemi-organism. We shall proceed to new proofs of this, dealing this time with a liquid formed by the life of a vegetable. * Amongst tliese influences one of the most important, according to M. Fremy, is " organic impulse" — another gratuitous assumption. t Fremy, Comptes rendus de V Academic des Sciences, t. Iviii. p. 1167, 1864. X Fremy, Gomptes rendus de V Academie des Sciences, t. Ixxiii. p. 1425, 1871. M. Trecul shares M. Fremy's opinions, and extends them to the development of diffei'ent fungoid growths. 54 STUDIES ON FERMENTATION. ^ III, — Experiments on the Juice contained in Grapes. In the course of the discussion which took place, at the Academy, on the subject of the generation of ferments, properly so called, much was said about the oldest known fermentation, that of wine. We at once resolved to demolish M. Fremy's theory, by a decisive experiment on the juice of grapes. We prepared forty flasks, capable of holding from 250 c.c. to 300 c.c. (from 9 to 11 fl. oz.) and shaped as represented in Fio-. 8. These we filled with filtered must, which was perfectly J Fig. 8. bright, and which, like all acid liquids, would remain sound, after having been boiled for a few seconds, although the ends of the long curved necks of the flasks containing the must might remain constantly open for months or years. We washed, in a few cubic centimetres of water, part of a bunch of grapes, washing the grapes separately, or the grapes and the wood together, or even the wood of the bunch alone. This washing was easily accomplished by means of a perfectly clean badger's-hair brush, the water receiving all the particles of dust adhering to the surface of the graj^es and the wood of the bunch. By means of a microscope we easily proved that this water held in suspension an infinite number of organized corpuscles, some of them bearing a very close resemblance to the spores of fungoid growths, others to alcoholic ferment, others to mycoderma vini, and so on.* • This observation had already been made by Anthon and II. Hoffmann. "If we scrape the surface of a gooseberry with a blunt knife," saysH. Hoffmann, "and put under the microscope the scrapings, which ai'e of a whitish colour, we shall recognize amongst many varieties of shapeless dirt, earthj^ particles and other things, the same fungoid spores that we find in the expressed juice, but we shall see them there studies! on fermentation. We next pi'oceeded to put on one side ten of our forty flasks, to serve for subsequent corroboration ; in ten others, by means of the tube which is represented on the right hand side of the flask (Fig. 8), we put a few drops of the water in which the bunch of grapes had been washed ; in a third series of ten flasks we put a few drops of the same liquid, after having previously boiled it. Lastly, we introduced into the ten remaining flasks a drop of grape-juice, taken from the inside of an uninjured grape. To do this we had to bend the right-hand tube of each of our last ten flasks, drawing it out to a fine point and closing it in the flame, as represented in Fig. 9 A. This ^•>\ Fig. 9. fine closed point was filed round near its extremity and then thrust, as represented in Fig. 9 B, into a grape placed on a hard substance ; when the point b was felt to touch the substance supporting the grape it was broken ofi" by a slight pressure sideways at the point a, where the file marks had been made. We had taken care to secure a slight vacuum in the flask in infinitely larger quantities. Some of them will be of a dusky colour {Stemphyliwn, Glados^jorium), and others will be colourless ; the shape of these latter will be round or oval, and cylindrical. Most of them will bear resemblance to beads of the chaplets of Oidium, Monilia, Torula (that is to say, to spores of certain Hyphomycetes), which have been detached and carried off by the wind, and have attached themselves to the fruit. Some of these spores will be already provided with short germinating filaments. (Annales des Sciences Naturelles, Bataniqtie, t. xiii. p. 21, 1860). 56 STUDIES ON FKRMENTATION. beforelumd ; this now caused a drop of the juice to be drawn into the flask. We then drew out the fine point, and closed it immediately in the flame of a spirit lamp. The vacuum was produced by heating the flasks in our hands, or over the flame of a lamp, thus causing a little air to be forced out through the end of the bent tube, which we then closed up with the lamp. When the flask was cool, the slight difference of pressure sufficed to force into it some of the juice contained in the grape, as we have just described. The drop of juice that is sucked into the flask generally remains in the curved part of the fine tube ; to mix it with the must we must incline the flask so as to bring the must in contact with the drop ; after that we may replace the flask in its natural position. The following are the results presented by our four series of comparative experiments in the different cases. The first ten flasks — our standard flasks, containing must boiled in contact with pure air — showed no signs of organized pro- ducts ; the must might have remained in them for any number of years without change. Our second series of flasks, which contained the water in which the grapes, separately and in bunch, had been washed, had undergone alcoholic fermenta- tion in every instance ; this had manifested itself in all the flasks in the course of about forty-eight hours, the tempera- ture being at about summer heat. At the same time that the yeast made its appearance in the form of little white lines, which gradually joining together formed a deposit on the sides of the flask, we perceived minute flakes of mycelium forming ; sometimes as a single fungoid growth, sometimes combined with another, or with many together — these growths being quite independent otherwise of the yeast or alcoholic ferment. In several cases, too, mycoderma vini showed itself on the surface of the liquid in the course of a few days, , Vibrios and lactic ferments, properly so called, could not make their appearance, on account of the nature of the liquid. The flasks of our third series, containing the water in which the bunch of grapes had been washed, and which we boiled STUDIES ON FERMENTATION. 57 before our experiment, remained as free from change as the flasks of our first series had done. Lastly, our fourth series of flasks, containing the drops of juice taken from inside the grapes, remained equally free from change, although we could not be certain of having removed, in every case, without exception, all causes of error which must inevitably occur sometimes in so delicate an experiment. These experiments cannot leave the least doubt on our minds : That must, if boiled, will never ferment when in contact with air that has been freed from the germs which exist in it in a state of suspension. That must may be fermented, after boiling, by introducing into it a very small quantity of water, in which a bunch of grapes has been washed. That must will not ferment if we introduce into it some of this same water which has been boiled and afterwards cooled. That must will not ferment if we introduce into it a small quantity of the juice contained in a grape.* It follows, then, that the ferment which causes grapes to ferment in the vintage tub must come from the exterior, and not the interior of the grapes. Thus, the hypothesis of MM. Trecul and Fremy, according to which albuminous substances transform themselves into grains of yeast by the action of a peculiar vital force, is annihilated ; a fortiori, there can no longer be a question concerning Liebig's theory, on the * The experiments that we have described give rise to a useful remark. All the organic liquids, boiled or not, in the course of time must take up oxygen from the air. At the same time, and certainly under this influ- ence, they assume an amber or brownish colour, but this eflfect is only produced when the liquids are placed under conditions of unalterability. Should fermentation or the development of fungoid growths be possible, scai'cely any change of colour will take jDlace. Doubtless this non- coloration may be attributed to the fact that these organisms consume the oxygen necessary for coloration. In these experiments on must, all the unchanged flasks assumed a pale yellowish brown colour ; those which fermented or contained fungoid growths remained colourless, or nearly' so. 58 STUDIES ON FERMKNTATIOX. transformation of albuminous substances into ferments, by a process of oxidation. Our readers ma}^ be curious to know what M. Freray has been able to oppose to such crucial experiments ; they could scarcely have imagined the following : — " In my experiments, which I have varied in every possible manner," says that gentleman, " I have found that it is almost impossible to discover alcoholic fermentation, appreciable by its results, in a single drop of grape juice, and I may add that this fermentation must be still more difficult to discover when this drop has been drowned in a large quantity of juice that has been previously boiled/'* It will be admitted that we were justified in saying, at the commencement of this paragraph, that we should demolish the theory which was opposed to ours, and which its advocates have been constrained to defend by hypotheses manifestly false. At the meeting following the one in which M. Fremy declared that minute quantities do not ferment, we had the malicious satisfaction of showing a great many very small closed flasks, into each of which we had caused a single drop of the must of crushed grapes to be introduced by suction. We broke the thin points of many of them, in the presence of the Academy, and every one of them showed by a sharp hissing, which was audible at a distance, that fermentation was proceeding in the drop of liquid they contained. M. Fremy was there, but he made no remark. We may cite some very curious facts on the subject of the period at which the germs that develop yeast are in a con- dition to be able to cause fermentation. On July 25th, 1875, in the neighbourhood of Arbois (Jura), the grapes were still green and of the size of peas. We went to a vine that was far from paths and roads, and there, with a pair of small scissors, cut some grapes from off a bunch and let them fall with their short stalks into tubes half filled with * Comptes rnuhts de V Academic, sennce da 28 Ocfohre, 1872. STUDIES ON FERMENTATTOX, 59 gooseberry must, previously rendered unalterable by boiling. These tubes we closed again with all possible precautions, using corks which had been passed through the flame of a spirit-lamp and carried them to our laboratory, where we left them to themselves. Some days afterwards we saw diverse fungoid growths appear in most of the tubes, but not one of them then, or subsequently^, presented the least appearance of fermentation. The germs of yeast at that period of the year did not exist either upon the woody part of the bunch, or upon the grapes. In Chapter V. we shall return to observations on this subject. § IV. — WoKT AND Must Exposed to Common Aik. If the principles which we have laid down possess all the value that we attribute to them, if the cause of change in natural or artificial organic liquids does not exist in those liquids themselves, if change considered in itself depends upon the nature and number of the particles of dust in various places, if it is, besides, radically affected by the composition of the liquids, it must necessarily follow that wort or must, whilst, under certain circumstances of exposure to air, it remains absolutely free from life and its results, will, under other circumstances, give rise to a variety of organisms and their corresponding fermentations. This is, in fact, the lesson which direct proofs will teach us. Before entering upon these new observations in detail, we must call the reader's attention to the difficulty, as experience has shown, of interpreting correctly the facts connected with the spontaneous impregnations of organic liquids. Gay-Lussac crushed some grapes under a bell-jar filled with mercury, after having washed them in hydrogen, to expel the air adhering to the grapes and the sides of the jar. Having waited for several weeks without detecting any signs of fermen- tation, he introduced some bubbles of oxygen, and fermentation showed itself the following day. Gay-Lussac concluded that 60 STUDIES ON FERMENTATION. the fermentation of must could not commence without the help of oxygen.* Under the conditions of his experiment nothing could be truer, and we must admire the diffidence with which this great natural philosopher interpreted the fact that he had observed. Another French natural philosopher, however, M. Cagniard- Latour, observed that the ferment of alcoholic fermentation was a little cellular plant. What was its origin in Gay-Lussac's experiment? The advocates of the doctrine of spontaneous generation were ready with their explanation, and we have seen how MM. Trecul and Fremy, following many others, did not hesitate to maintain that the little plant with all its particles was produced by the action of oxygen on the albuminous substances contained in the juice of the grapes. The experiments, which we have given in the preceding paragraph, show us positively that germs of the ferment of must exist on the surface of the grape, and that, consequently, Gay-Lussac's experiment has a more simple and natural explanation. The germs of the ferment existing on the surface of the grape become mixed with the juice of the grape when the latter is crushed ; these germs remain inactive in the presence of hydrogen ; they vegetate as soon as oxygen is introduced to them. Moreover, the results of our labours in connection with spontaneous generation, in 1862, teach us that in Gay-Lussac's experiment the germ of the ferment might also have had its origin either in certain particles of dust adhering to the sides of the glass bell, or upon the mercury ; and, in a laboratory where alcoholic fermentation is studied, dust invariably contains dry cells of ferment. The necessity of oxygen for the success of the experiment is surprising, when we reflect that * Gay-Lussac, Annales de Cliimie, t. Ixxvi. p. 245 ; read at the Institute, December 3rd, 1810. Tjong before Gay-Lussac, it had been remarked that atmospheric air had a great iutluenco on fermentation. See M. Chevroul's articles on the historj' of chemistry in the Journal des Savants. STUDIES ON FERMENTATIOX. 61 alcoliolic fermentation often takes place in liquids that are not exposed to contact with air ; but we sliall prove by experiment that, notwithstanding what may happen duinng fermentation, oxygen has the greatest influence on the readiness with which ferment develops itself, and that this gas is indispensable to the revival of withered cells, and still more so to the germination of special cells, which we may consider to be the true germs of the little plant. The advocates of the doctrine of spo7iianeous generation have based most of the objections which they vainly urge against their opponents upon erroneous interpretations of certain facts relating to tlie spontaneous impregnation of organic infusions. Taking a very wrong view of the essential condi- tions of the phenomena, they require that the assertors of the diffusion of the germs of microscopic organisms should be com- pelled to place at any one point of space, so to say, all the germs of the products of infusions ; a demand which really borders on absurdity. They believe, or feign to believe, that we are bound to admit the existence of germs of must in all places and at all times, on the banks of rivers and on the loftiest moun- tains, and so on. " Fermentations," said one of these gentle- men one day, before the Academy, " cannot depend upon chance particles of atmospheric dust. How is it possible that germs of yeast can be present everywhere throughout the universe, ever ready to fall upon must ? " It is an established fact that grapes crushed in any part of the globe whatsoever, even on a glacier or at the highest elevations, can set up a fermentation. The expla- nation of this pretended impossibility is most simple, for we know, from the facts related in the preceding paragraph, that grapes carry on their skins the germs of their own ferments. In experiments relating to the kind of organisms which we are discussing we must never fail to take into account the action of the particles of dust spread over the articles that are used. Very often an effect that should be attributed to gferms adhei'lng: to the vessels and utensils used in experiments, the origin of which may be altogether special, is erroneously imputed to the dust- 62 STUDIES ON FKRMENTATION. forming germs — that is to say, to those germs which exist in a state of suspension in the air. In our Memoir of 1862, which we have quoted several times, we have explained that it is almost impossible to draw any serious conclusions from experiments made in a basin of mercury, because of the organic particles of dust which always exifjt in that metal, and which, without the knowledge of the operator, pass into the interior of the vessel, where they produce certain changes which one is tempted to impute to heterogenesis. In all classical works an experiment of Appert's, reproduced by Gay-Lussac, is given. This, through a faulty interpretation, led to the hypothesis of the continuity of the causes of fer- mentation, if we may use such a term, in the atmospheric air.* When we decant bottles of must, which has been preserved by Appert's method, into other bottles, all the latter soon set up a fermentation : this constitutes the experiment. If it were proved that the must, whilst being decanted, came in contact with atmospheric air alone, as Gay-Lussac believed, we should be compelled to admit, according to the theory of germs, that the must had come in contact with some particles of ferment in the air during decanting. And again, if it were shown that the experiment could succeed in any place whatsoever, we must come to the conclusion that germs of ferment exist everywhere in a state of suspension in the air. " I have taken,"" writes Gay-Lussac, " a bottle of must that had been preserved for a year and was perfectly transparent, and have decanted the must into another bottle, which I then carefully corked and exposed to a temperature of 15° C. to 30° C. (59° F. to 86° F.). Eight days afterwards the must has lost its transparency ; fermentation has taken place in it, and soon my must has become transformed into a vinous liquor, sparkling like the best champagne. A second bottle of must that had been preserved for a year, like the preceding one, * Gay-Lussac, Annaks deChimic, t. Ixxvi. p. 247, Mcmoire citt', 1810. STUDIES ON FERMENTATION. but which had not been brought into contact with the air, has presented no signs of fermentation, although placed under conditions most favourable to its development." The result of this experiment, when roughly made, is cor- rectly described by Gay-Lussac ; in other words, it may be proved that if, at the time of vintage, we prepare some bottles of must, after Appert's process, and, in the course of time, open them and decant their contents into other bottles, we shall soon see the must ferment and deposit yeast. It is, neverthe- less, equally certain that the inferences which have been drawn from this celebrated experiment have been founded on error, and that the germs of yeast are very rarely derived from the particles of dust floating in the air with which the must comes in contact. The germs in question are, in our opinion, generally derived, not from the air, but from the sides of the bottles, from corks, from the string employed in corking, from corkscrews, and from a variety of other things. The reason for this is that any room, vault, cellar, or labora- tory where the grapes, or must, or vintage, are handled — unless special precautions, of which Appert and Gay-Lussac certainly never thought, are taken — all the utensils, as well as all articles of clothing, and all the sides of the bottles which the hands have touched, are contaminated by cells of ferment derived from must that has fermented, or by germs from the surface of grapes and clusters. Thus, at the moment of decanting the must, a thousand accidental circumstances may lead to the introduction of those germs, the origin of which, as we have seen, may be actually traced to the very grapes which served for the manufacture of the must. In other words, we believe the inference that the germs of yeast which cause the experiment to succeed, are derived from particles of dust floating in the air of the place where we decant the contents of our bottles, to be altogether an erroneous one. Since the preceding remarks were written, we have endea- voured to repeat this experiment of Gay-Lussac's in such a manner as we believed would confirm our views, by varying the 64 STUDIES OX FERMENTATION. conditions in such a way as would cause it to succeed or fail, according to the circumstances of the manipulation emplo3'ed. On December 7th, 1874, we took two bottles of must which we had preserved, after Appert's process, in our laboratory from the beginning of October, 1873. Both of these were covered with dust — the dust that floated about in our labora- tory. We decanted them as follows : — One bottle, which we handled without special precautions, we uncorked by means of an ordinary corkscrew, and decanted into another bottle that had been well waslied, as bottles are washed when they are to be used subsequently. This bottle was taken from a number that had been standing upside down on a drainer for a fortnight. We took no precaution to remove the dust which covered the exterior of the bottle of must, or to purify the washed bottle. The second bottle of must, on the other hand, was decanted after we had removed the dust that covered it ; its cork was cut off close to the string, and the flame of a spirit lamp was passed over the string and the surface of the cork ; and, as a final precaution, the corkscrew was passed through the flame. As for the bottle into which we subsequently decanted the must, we first plunged it in a hot- water bath kept at 100° C. (212° F.), then took it into a garden to cool upside down in the open air. After these precautions we removed it, and imme- diately decanted into it the must from the second bottle. The first bottle showed signs of growths, both on the surface and at the bottom of the must, the day after the operation, and manifested the first symptoms of alcoholic fermentation on December 16th. The contents of the second bottle remained perfectly unchanged after being exposed to the warmth of a stove for several months. Can anything be more conclusive than these facts ? They are in perfect keeping with the views that wo have recently expressed, and with the principles that we have maintained for nearly twenty 3^cars, on the subject of the causes of change in organic liquids. It is by no means our intention to assert that in the atmo- STUDIES ON FERMENTATIOX. 65 Spheric air there exist no germs of ferment in a state of suspen- sion, as fine dust. Beyond all doubt they do so exist in that state ; but, as a rule, in comparatively small number, their abundance or scarcity being dependent upon circumstances which control their multiplication, favouring or restricting it, as we are about to prove. On May 2nd, 1873, we uncorked two ordinary bottles filled with wort, prepared in December, 1872, after Appert's process. To avoid the causes of error which we have mentioned, we uncorked the bottles in the following manner : — The cork was cut oif to the level of the neck ; the cork and string were next passed through flame, regardless of burning and charring them ; we then gently extracted the cork by means of a corkscrew which also had been passed through the flame. The bottles thus prepared were placed on the table of an underground room, in which we were continually making ex- periments on alcoholic fermentation. Is^ bottle. — On May 7th we observed little particles of fungoid growth on the surface of the liquid, and at the bottom were large flakes of mycelium. On May 11th a veil of mycoderma vini had formed: there were no signs of fermentation. On May 13th vigorous fermentation commenced; it lasted until May 23rd. The microscope revealed yeast in globules of two sizes, the larger of which were considerably less numerous than the others. There were no signs of lactic or butyric ferment. 2ncl bottle. — May 7th, particles of fungoid growth on the surface of the liquid, and also a veil oi mycoderma vini. On May 11th, 13th, and up to the 23rd, no signs of fermentation were visible. On May 30th fermentation was active. The microscope showed us yeast mixed with butyric mbrios. In this case, alcoholic ferments had come into existence, anr^ from the precautions taken at the moment when the liquid was brought into contact with the external air, it is certain that the advent of the germs of those ferments, as also those of the p 66 STUDIES ON FERMENTATION. other organisms which made their appearance — the fungoid growths, mycodertna vini and vibrios — could only be accounted for by the fall of particles of dust floating about in the room. It follows, then, that under certain circumstances germs of alcoho- lic ferment may be found floating in the air ; but we can readily show that the peculiar conditions of the place had a large share in bringing about the results obtained by the foregoing experi- ment. The same day. May 2nd, 1873, we uncorked, with the pre- cautions that we have already described, four other bottles of the same must. These were placed in a room which was used less frequently than the preceding one, and in which experi- ments relating to fermentation were seldom conducted. l.s'^ bottle. — On May 8th we observed on the surface of the liquid large, frothy pieces of mycelium [mucor mucedo or mucor racemoms) . The liquid was perfectly bright.* May 30th. — No signs of actual fermentation yet visible. 2nd bottle. — On May 8th we noticed a thin, greasy-looking scum on our liquid, which had become turbid and acquired a sour smell. The microscope showed that this scum was formed of mycoderma aceti. On May 30th the scum had assumed a whitish appearance, and seemed to be dead ; there was a green spot of penicitlium glaucitm upon it. No signs of fe7'mentation. 3rrf bottle. — May 8th, patches of fungoid growth on the sur- face of the liquid. May 30th, thick and abundant fungoid growth, but no fermentation. Ath bottle. — May 8th, little patches of fungoid growth, and a scum of mycoderma vini. May 30th, still no fermentation. Up to the month of August, 1873, not one of these bottles gave the least sign of alcoholic or other fermentation. On December 16th, 1872, we uncorked four bottles of wort, which also had been preserved by Appert's process ; these we * It is well to notice that under the influence of fungoid growths, jiroperly so called, the wort of beer speedily becomes bright. We may say that fungoid growths, by their rapid development, clarify the must, which serves to nourish them. STUDIES ON FERMENTATION. 67 placed on an oven, where there were always vessels fermenting, at about 25° C. (77° F.), but where none of the manipulations required for the starting or final study of fermentations were practised. The next day a fungoid growth, but unaccompanied by any signs of fermentation, made its appearance, and this state of things lasted for five months, after which we ceased to keep these bottles under observation. On May 26th, 1873, we uncorked, with all the necessary pre- cautions, ten bottles of wort, which had been preserved from April 9th, and then left them undisturbed in a room where we were constantly engaged in the study of fermentation. On the following day, some patches of fungoid growth ap- peared on the surface of the liquids. May 30th. — Fermentation commenced in one of the bottles. May 31st. — A second bottle likewise began to ferment. June 9th. — Four bottles, including the two preceding ones, were now in a state of fermentation. The six bottles that had not fermented were thereupon covered with caps of paper, taken from the centre of a ream of paper and passed through a flame. After this, and up to August 1, when our observations were discontinued, these six bottles underwent no fermentation. From these examples, which are confirmed by many others that we shall have occasion to mention in the course of this work, it will be seen that the germs of alcoholic ferment are not present in every little point of space, constantly ready to fall upon any object, not even in those places M^here one is per- petually dealing with that kind of growth.* If we conduct our experiments with exactness^ we very soon learn that all that has been written on the facility with which saccharine * It has already been observed in our Memoir on spontaneous genera- tion, that alcoholic fermentation is not always to be obtained by sowing wads of cotton or asbestos, charged with the particles of dust which float through the air, in saccharine musts that are in contact with much air. The air which furnished the particles of dust, in the experiments to which we are alluding, was taken outside the laboratory, in a neighbouring street. F 2 68 STUDIES ON FERMENTATION. musts may be made to ferment, by being rapidly brought into contact with the surrounding air, is greatly exagge- rated. The germs of ferments, especially of alcoholic ferments — the yeast of beer and the yeast of wine — are not nearly as abundant in atmospheric air, or in the particles of dust spread over the surface of things, as are the spores of fungoid growths. It is easy to understand this, for spores are generally borne by aerial organs in a state of dryness, so that the least breath of wind catches them up and carries them away, whilst ferments are composed of moist cellules that do not readily become dry. The vacuous flasks, partly filled witli organic liquids, which are opened and closed again immediately, frequently give us fungoid growths, but very rarely alcoholic fermentation, although in the latter respect they may not be absolutely sterile. "We may cite some proofs of this. On June 19th, 1872, we prepared seven flasks of saccharine liquid, impregnated with yeast — our flasks were of 300 c.c. capacity (about 10 fl. oz.) — and contained 100 c.c. of the liquid ; we then drew out their necks to a small opening, which was sealed during boiling, after the steam had expelled all the air. On June 29th we opened them in the principal room of our laborator3\ On July 9th, two of the seven flasks gave no sign of organized products ; the others were swarming with mycelia, submerged or fruiting on the surface of the liquid, and either with or without bacteria entangled in their flakes. In two of the flasks there were visible at the bottom of the liquid some white streaks, which is an indication sometimes of the presence of alcoholic ferment, but much more frequently of a little cellular plant resembling it in appearance, but purely aerial in its growth, that is to say, taking no part in fermentation. Some days afterward, we saw bubbles of gas rising from the bottom of one of these flasks, and then fermentation proceeded so rapidly STUDIES ON FERMENTATION. 69 that we were obliged to open the neck to avoid an explosion. We append a sketch of its ferment (Fig. 10). o ^ Fig. 10. The other flask with the white streaks showed no signs of any fermentation. In this kind of observation we rarely succeed in obtaining active ferments, the reason being that we deal with volumes of air that are too limited for the few germs of ferment that exist in a state of suspension in it. We are more sure of success if we expose a tolerably large surface of saccharine liquid to the open air, because, under such circumstances, even if the exposure is of short duration, a con- siderable volume of air will pass over the surface of the liquid. On May 29th, 1873, at five o'clock in the afternoon, we placed in the underground room previously mentioned, at a height of about two feet, ten porcelain dishes having surfaces of from thirty-five to forty square inches. We had just taken them from boiling water, and after allowing them to cool we placed in each quantities of wort to about one-third of an inch deep, which we poured from bottles uncorked with every precaution against the chance of the wort coming into contact with anything besides the floating particles of dust. On May 30th, at five o'clock, that is after twenty-four hours of exposure to the air of the room, we emptied the contents of the basins separately into glass flasks with long necks, which had been treated with boiling 70 STUDIES ON FERMENTATION. Avater and then cooled, necks downwards. The beak of the basin, b}' means of which the liquid vras poured, and the funnels — for we used a separate funnel for each flask — had been passed through the flame. The whole ten flasks were then placed in an oven at a temperature of 25° C. (77° F.).* On June 1st six of the flasks gave signs of fermentation, and next day all the flasks were fermenting. The following are some of the numerous microscopic observa- tions which we made on the liquids and their deposits. On June ] st the liquid in one of the six flasks which had begun to ferment was covered with a continuous scum of nvjco- dcrma vim, below which appeared a filamentous network belong- ing to a mycelium, or other fungoid growth. But neither in the liquid itself nor in the deposit could we perceive any cells of the ferment of beer; the field, however, was swarming with active butyric vibrios, rather thick and short, their length being about twice their diameter. This fermentation was exclusively butyric. On June 2nd another of the flasks showed no vibrios, but alcoholic ferment in small quantity, much lactic ferment, con- sisting of little particles contracted at the middle and non- mobile, and, finally, some slender filaments, resembling those represented in Plate I., Nos. 1 and 2. On June 3rd we examined the liquid in the flask which showed the most marked fermentation. In addition to the flakes of fungoid growths, checked in their development through ■want of air, we found at least five distinct productions, which are represented in the accompanying sketch (Fig. 1]). aaa. — Thick cells of alcoholic ferment, the size of which is given in our sketch : thus, -^^-f-^ indicates that the correspond- ing figure is ^^'-V millimetre in length (rather more than -oinr inch). hbb. —Small alcoholic ferment, such as we generally see in the * The decanting into the flasks is necessary, because of the possibility of the fermentation in the basins being masked . See further on the note on p. 75. STUDIES ON FERMENTATION. 71 must of acid and sugared fruits — especially in filtered grape raust. Its dimensions varied from 1 to li, or 2, 450tlis of a millimetre.* Fig. 11. ccc. — " Low " yeast, of a type resembling that existing in other preparations fermenting in tlie room. ddd. — Enlarged, distended spores of mucor racemosns. These are scarce, and have an old appearance. We shall come to them again in a subsequent chapter, where we shall explain their real significance. eee. — Short vibrios, occurring either contracted or not near the middle. Some were motionless, others vibrating to and fro and executing other movements. These forms belong to the butyric and lactic ferments shown on Plate I. * This small ferment is very curious, although it scarcely affects industrial fermentation. It was first described in 1862. (Pasteue, Bulletin de la SocietS chimique, 1862, page 67, and following : Quelques faits nouveaux au sujet des levures alcooliques.) It has since been described by Dr. Eees under the name Saccharomyces apiculatus. (Dr. Eees, Leipzig, 1870 : Sur les champignons de fermentation alcooliques. See also Dr. Engel, These pour le Doctorat, Paris, 1872.) If we carefully filter some grape must at the time of vintage, we may be sure that we shall see it appear in the clear liquid at the bottom of our vessel, without intermixture with any other ferment. Should we not filter the must this ferment will appear all the same, but it wiU soon become associated with another, thicker in appearance and more elongated, which also is one of the ferments peculiar to the fer- mentation of grape must. 72 STUDIES ON FERMENTATION. The preceding series of experiments shows us that, in the case of wort exposed to the air, germs of divers organisms, amongst which various ferments — butyric, lactic, and alcoholic — are to be found, fall simultaneously from the particles of dust floating in the air. We must observe, however, that we were dealing with the air of a laboratory in which we were constantly studying analogous fermentations, and that a different atmo- sphere would, most likely, give us different results. We shall see a proof of this in the following paragraph, where we shall also find some new facts tending to prove that the germs of alcoholic ferment do not exist amongst the particles of dust floating in the air, in anything like the quantity usually supposed. § V. — New Comparative Studies on the Germs held in Suspension by the Air of Different Places which are near each other, but Subjected to Different Conditions affecting the Production and Diffusion OF THE Particles of Dust found in them. We may compare the character and the greater or less abun- dance of similar germs existing in neighbouring localities, by studying the changes which take place in similar liquids exposed simultaneously to the action of the air in those localities. To do this, we must prepare a large number of flasks of the same size, free from air, and containing about equal quantities of a particular liquid^ — the same being used for all. We must open the same number of these flasks in each of the localities we have selected, and permit the air with all its particles of dust . to rush into them ; then we must close our flasks again, and observe, day by day, the appearances they present. The results obtained by these means will not furnish us with con- clusions applicable to every kind of germ that the air contains at any given moment, but only with conclusions which apply to those germs which can develop in the particular liquid em- ployed. Thus, for example, we could draw no inference as to the nature and relative number of bacteria or vibrios, in the i? ^«> <x^ <^ <0 °q .%. ^ fei3 ^ <«5 -"C" g» ^ ® 60 § "0^. 4. UJ u ■<JP^ e# 0. caqg* § ©- J o CO ©. ^ ©D ct> /j.OO Deyrolle del Ficart sc ToRiM,.K iM Process ok Devei.oi'mknt. /f7^ l'<iny Crai . /'or STUDIES ON FERMENTATION. /d case where we employ an acid liquid ; for organisms of that kind we must have recourse to neutral or slightly alkaline in- fusions. On the other hand, liquids having a feeble acid reaction would favour the growth of tnucedines, mycoderms, and certain ferments, as for example, the alcoholic. On November 26th, 1872, we opened and reclosed thirty flasks containing must kept from the last vintage. Ten flasks were opened at the bottom of the garden of the Ecole Normale. Ten on the landing of the second floor. Ten in the principal room of our laboratory, which had been swept out shortly before, by which operation the dust of the floor had been raised and put in motion. Difierent objects made their appearance in a certain number of the thirty flasks on the following days ; but from December 17th, things remained stationary. The following observations were made at that date : — Of the ten flasks from the bottom of the garden, only one had undergone any change. Of the ten flasks from the interior of the building, four had undergone change. Of the ten flasks from our laboratory, all had undergone change. The difierence in the number of germs held in suspension in the three difierent places whence we had taken our air was, therefore, considerable. The difierence in the nature of the germs was equally marked. Those flasks of our first two series which had undergone change presented no trace of tomlce, or anything besides fungoid growths, whilst three of the last ten contained toruke associated with fungoid growths.* * We may remind the reader that in 1862, in our Memoire sur les Generations dites spontanees, we applied the expression of torula to all the little cellular plants of spontaneous growth, excepting mycelium, pro- pagated by budding, after the manner of the ferment of beer. At the same time, stress was laid upon the frequent occurrence of their germs, especially in our laboratory, where studies on fermentation were, even then, carried on. Plate III. represents two of these ferments. 74 stui)ip:s on fermentation. On May 29th, 1873, eighteen flasks, free from air and with necks drawn out to a fine point, containing must, were taken into one of our rooms at the Ecole Normale. A jet of gas from an ordinary burner was passed over the surface of the glass down to the surface of the liquid, with the object of burning any particles of dust that might have been deposited from the atmosphere of the laboratory ; the points of the flasks were then broken with a pair of ordinary scissors that had been passed through the flame of a spirit lamp ; and, lastl}', the tops of the flasks were taken off", just above the surface of the liquid, and the eighteen flasks thus became transformed into eighteen basins, each con- taining about 100 c,c. (about 3| fluid ounces) of grape must. These eighteen basins were placed on a table in the room, and left there for five daj'S, precautions being taken to prevent any one from entering the room. The basins were examined on June 2nd : they all contained little flakes of floating mycelium, but none of them had any white streaks on their sides — a proof that they were destitute of torulce — the liquid had remained very bright. AYith the contents of nine of the basins we filled two long-necked flasks that we had prepared for our purpose — that is to say, had heated to a certain point, just before using them, with the object of re- moving from their sides any foreign germs which they might have picked up in the laboratory. Up to July 10th, when we deemed it useless to carry our observations further, these flasks presented no signs of fermentation whatever. With the con- tents of the remaining nine basins, we filled two other long- necked flasks, but before doing so, we kept the basins for twenty- four hours (June 2nd to 3rd) in the basement of our laboratory. These two flasks soon set up an active fermentation, and de- posited an abundance of yeast — an additional proof of the great difference in character of the germs floating about the room and those floating about the laboratory. On June 3rd we exposed, simultaneously, in the aforemen- tioned room and also in our laboratory, seven basins prepared as just described. On June 8th all the basins in the room showed STUDIES ON FERMEIs'TATION. 75 signs of fungoid growths, without any trace oi mycoderma rini, or lines indicative of the presence of tonilce, whilst six of the seven in our laboratory had their sides covered with a white precipitate, and on the surface of the liquids a layer of isolated patches of fungoid growth. The liquid in these latter basins was poured into a long-necked flask, which it nearly filled, and in the course of forty-eight hours it began to show signs of alcoholic fermentation.* This is another striking proof of the difference between the number of germs of ferment and iorulacece in the air of our laboratory and that of an ordinary room. We append drawings of the torulce found in the six labora- tory basins (see Sketches I., II., III., IV., V., VI. of Fig. 12). The abundance of the germs of these organisms in our laboratory is very striking, and is doubtless due to the nature of the work * It is to be remarked that in this case, as in the case recorded § IV. p. 70, in order to detect with certainty any alcoholic ferment, the contents of the basins were transferred to a long-necked flask ; since where, as in the basins, a liquid has a large surface exposed to spon- taneous impregnation, the strictly alcoholic fermentation may escape observation. The reason of this is that, when a liquid of large surface but small depth is exposed to the air it affords a suitable medium for the active development of moulds, which, by absorbing the oxygen which would dissolve in the liquid, checks the growth of the ferment, or even prevents its germination altogether. As a matter of fact we shall see that for their growth and multiplication ferment cells, and still more ferment germs (the difference between the two will appear in Chap. V.), require a larger supply of oxygen just in proportion to their age, state of desiccation, and distance from the budding condition. Now, if the spores of moulds be present and effect a settlement in the liquid, the increase of the ferment, or even its actual germination, is prevented. But by collect- ing the liquid in a deep and narrow vessel, such as a long-necked flask, after it has been exposed to spontaneous impregnation, we deprive the moulds almost completely of oxygen, and so allow the feiTaent to exert its peculiar energies. The mere act of transferring enables the liquid to take up a sufficiency of oxygen, and a liberation of gas very speedily shows that fermentative action is going on. We must add further that sometimes in a liquid of large surface and shallow depth, in which but little ferment is formed, the evolution of carbonic acid gas may fail to be detected, by reason of its diffusing itself into the air slowly as it is formed. '6 STUDIES ON FERMENTATION. carried on there, as well as to the power of endurance peculiar to the germs, or the minute vegetative cells of these micro- scopic plants — a tenacity of life which prevents them from losing their reproductive powers, even after being dried up into dust. But varied as are the formations represented in Fig. 12 — and it will be observed that in IV. and VI. we have shown four 6-3^ Y n ^„ 4^ vr ] \o cP ^ VI Fig. 12. distinct forms, marked a, a; b, b ; m, m ; n, n respectively — it must not be supposed that they correspond necessarily to dis- tinct species. From the ends of a compound organism like those in No. Ill, a little spherical cell may detach itself and then, by a process of budding, give rise to a series of other STUDIES ON FERMENTATION. 77 rairiute spherical cells reproducing the form shown in Nos. I. and II. The forms figured in No. III. represent one of the types of mycoderma vini, which is often found in this branching arbor- escent state ; but it frequently also occurs in short forms, and it is in this shape that it is generally met with on the surface of wines. It is true that the nature of the substratum has a great influence on the changes of aspect in the organisms which we are describing, but this is not the sole cause of their morpho- logical modifications. We are strongly inclined to believe that each of the cells, or vegetating forms so represented, difiering so greatly as they do in aspect, and begotten, all of them, spon- taneously in certain appropriate liquids, in a laboratory where researches on fermentation are pursued, is capable of furnishing a distinct variety. In fact, there is not a single one of the cells in the six varieties in Fig. 12, which, taken alone, has not its own peculiar characteristics, which, by hereditary trans- mission, it can impart, in a greater or less degree, to all the individuals of the generations that succeed it. We may remark, on the other hand, that nothing can be more favourable to the isolation of different varieties of toruJa or mycoderma vini than the spontaneous impregnations to which we submit our liquids. For when suitable liquids contained in flasks exhausted of air are impregnated with the particles of atmospheric dust, by opening the flasks for a moment and then immediately re-sealing them, it must generally happen that we admit only one species of reproductive organism, so that we shall have a vegetation exclusively of one kind, as being derived from the same mother-cell. If we could take from a crowd composed of men and women separate couples, and forthwith transport each couple to a separate isolated and unpeopled island, they would, in the course of time, beyond doubt form so many distinct tribes. It is very remarkable that some of the torulce in Fig. 12 are not ferments ; they do not cause sugar to decompose into alcohol and carbonic acid, any more than the mycoderma viioi 78 STUDIES ON FERMENTATION. does ; but, nevertheless, there may be an absolute similarity in aspect, development, shape, and size between the alcoholic ferment, properly so called, and these toru/acece. We must here cite a case in proof. On May 28th, 1872, in one of the rooms of our laboratory, we broke the fine points of a series of flasks, similar to those used in our previous experi- ments, containing must of grapes and deprived of air. We then closed up the ends immediatel}' after the sudden entrance of the exterior air. One of these flasks developed only one kind of organism, which belonged to the torulacece. On June 7th this was sufliciently abundant to cover all the sides with a white deposit, and the surface of the liquid appeared quite free of any mycodenna vini. To assure ourselves that we were actually dealing with one kind of tnruhe, unattended by fungoid growths, we waited until June 14th, but the aspect of things remained unchanged. On that day we opened the flask ; there was no escape of gas to indicate that the interior pressure was greater than the exterior. AYe then subjected the plant to microscopic examination. It was quite homogeneous, and formed of a mass of cells, absolutely identical in aspect and bize with old cells of ordinary yeast (Fig. 13). & ^ © Fig. 13. We distilled all the liquid, of which there was about 100 c.c, (3 1 fl. oz.) without obtaining any trace of alcohol in our distillate; we collected 33 c.c. (about an ounce) of the distillate, which we distilled again, and we submitted the distillate a third time to distillation ; but even then there were no more signs of the presence of alcohol than there had been in our first distillate.* * Here we had to seek for a most minute quantity of alcohol, that no alcoholometer could have indicated. A certain sign of the presence of alcohol is contained in the first few drops distilled ; these alwaj's assume the form of little drops or strice, or, better still, oily tears, when alcohol is present in the distillate. The distillation should bo eirecteJ with a STUDIES ON FERMENTATION. 79 "We may safely conclude that our torula, in the course of its development in must, with a weight that would have been very appreciable, did not produce, by its action of multiplica- tion io-{«o of 1 c.c. of alcohol. Under the following conditions we obtained a slightly different result, which, nevertheless, confirmed the preceding one. On July 5th, 1872, we opened and closed twelve flasks similar to those we had used before, the sole difference being that they contained yeast water * sweetened with ten per cent, of sugar. One of these flasks furnished us similarly with one kind of torula, which bore the greatest resemblance to the ferment of beer. When this torida was beginning to spread all over the bottom of our flask, we shook up the liquid, and turned the flask upside down, with the object of submerging the torula and depriving it of air, at least at the bottom of the neck. For some days, and even months, there were no signs of the libera- tion of gas. On July 22nd, 1873, after the interval of a year, we opened the flask (which gave no indication of the existence of an interior pressure) and endeavoured to discover the pres- ence of alcohol, by means of successive distillations, as just described. In the two first distillates there seemed to be no alcohol, but in the third we detected its presence in ver}^ small quantity. We shall see, later on, that the formation of such a small quantity of alcohol may be attributed to the fact of the plant having been submerged when in full growth, and to its having continued to live for some time after its submersion quite independently of the oxygen contained in the air of the flask. small long-necked retort and a Liebig's condenser. "We must carefully watch tlie neck of the retort at the moment of boiling ; should the liquid contain y^joo P^^^ ^^ ^^^ volume of alcohol, we shall observe the indica- tions given above for a short, but appreciable time, -^j]^ of alcohol is difficult to judge, but with care and practice we may do it without fail- ing. Collecting a third of each distillate, and supposing the limit of appreciation to stop at thousandths, in three distillations we may easily detect the presence of lo^ooo of 1 c.c. of alcohol in a total volume of 100 c.c. * Yeast when macerated in water imparts to it certain soluble nitro- genous materials. The solution so obtained, filtered from yeast globules, is known as yeast ivater. — D. C. R. 80 STUDIES ON FERMENTATION. From the previous facts it is obvious that there exist certain productions of various aspects, the germs of which are parti- cularly abundant in the dust of a laboratory where the pheno- mena of fermentation are studied, productions essentially aerial, and incapable of giving rise to fermentation, although it may be impossible for the microscope to distinguish their forms from those of true alcoholic ferments. The idea of some physiological bond between these plants and the ferments which resemble them in so remarkable a manner, is one that impresses itself forcibly and, so to say, instinctively upon the mind. This remark holds good also in the case of mycoderma vim, properly so called, when compared with alcoholic ferments. There appears to be no other difference between the mycoderma vini and the torulce of which we are speaking, than that afforded by peculiarities of physical structure and a certain greasiness in the cells of the former which permits it to exist, in the form of a scum, upon the surface of liquids, that is to say unsubmerged. We have frequently, but without success, endeavoured to bring about the conversion of these unsubmerged torn Ice and mycoderma vini into alcoholic ferments ; in other words, we have never succeeded in imparting to these torulm or to myco- derma vini, which bear so striking a resemblance to alcoholic ferments, the permanent fermentative character peculiar to the latter. At one period of our researches, in 1862, and more recently, in 1872, we thought that we had discovered the con- ditions under which such conversion might be possible, but, as we shall explain in a subsequent chapter, our experiments were affected by certain errors that had escaped our notice. * It is possible that this greasiuess in the cells of the common mycoderma vini arises simply from the composition of the liquid in which it vegetates. It is in saccharine liquids that the submerged toruJoi are found; fer- mented liquids more readily give birth to the forms of toruJcc and myco- derma vini which exist as a scum. In all probability, however, there is no radical diflference between these two kinds of little cellular plants of aerial growth, the floating torulw and mycoderma vini. STUDIES ON FEKMENTATIOX. " 81 ^ VI, — Yeast may become Dry and be Reduced to Dust WITHOUT LOSING ITS FACULTY OF ReI'RODUCTION. In the preceding paragraphs we have given examples of liquids becoming impregnated with self-sown alcoliolic ferments. We shall proceed to show that this little cellular plant may actually exist in the form of dust, floating in the air, after the manner of spores of fungoid growth and the encysted forms of certain infusoria, without losing its powers of reproduction. On December 16th, 1872, we collected and pressed all the yeast resulting from a brewing of about one hectolitre (about 22 gallons). From the centre of the cake we took a few grammes (50 or 60 grains) of yeast, which we mixed in a porcelain mortar with five times its weight of plaster — both mortar and plaster having been heated, just before, in an oil bath, to a temperature of about 200° 0. (392° F.), and then cooled rapidly. The powder thus prepared was immediately done up in a twist of paper, which had been passed through the flame of a spirit lamp, and the twist and its contents were then placed in an oven at a temperature of from 20° C. to 25° 0. (about 75° F.). The object of these several precautions was to free the powder composed of the yeast and plaster, if not from the germs contained in floating particles of dust, at all events from those contained in the dust existing on the surface of the articles we used — the mortar, plaster, and paper. On December 18th, we took up with a platinum spatula. Fig. 14. previously passed through the flame, a pinch of the j^cast-and- plaster powder, and sowed it in a two- necked flask (Fig. 14) 82 STUDIES ON FEUMENTATION. containing some pure wort. We then placed the flask in an oven, at 20° C. On December 21st, three days after we had sown the powder, fermentation began to manifest itself by the appearance of patches of froth on the surface of the wort. On December 10th and 20th the yeaat was sensibly developing, although there was no liberation of gas to denote the presence of actual fermentation. The yeast, examined under the microscope, appeared very pure. On March 5th, 1873, we took another pinch of the yeast- and-plaster powder from the twist of paper, and placed it in a flask of pure wort, as in the foregoing experiment. On March 9th, that is, after having been subjected to a heat of 20° C. (68° F.) in the oven for four days, fermentation began to manifest itself by the appearance of patches of froth on the surface of the wort. From this it was evident that the yeast had not been destroyed, but only retarded in its revival. On July 25th, 1873 — that is, after a lapse of seven months and a half — we resumed our experiments, and sowed some more of the yeast-and-plaster powder in another flask of wort. On August 2nd, eight days from the time of our sowing, the little islets of froth appeared on the surface of the liquid. Observed under the microscope, the yeast still seemed pure, and resembled the original yeast ; we append a sketch, which will give an idea of its shape (Fig. 15). :? k Fig. 15. On November 7th, 1873, we once more sowed some of the powder. This time the yeast was dead ; we observed the flask whicli contained it, day by day, until February 1st, 1874, without detecting the sHglitest sign of fermentation or de- velopment of the yeast that we had sown. On Februarj^ 1st, we made a microscopical examination of the yeast, and found STUDIES ON FERMEMATIOX. 83 it mixed with the plaster and absolutely inert at the bottom of the saccharine liquid ; its cells were isolated, very old-looking and granulated, without any appearance that might denote the possibility of their ever budding again. Thus we determined that alcoholic ferment may be dried at the ordinary temperature of the atmosphere, and preserved, in the form of dust, for a period of seven months or longer, without losing its faculty of reproduction. This faculty evi- dently diminishes in the course of time, for our dried yeast, after having been kept for seven months and a half — all the other conditions of the two experiments having been precisely the same — required about eight days to develop sufficiently to reveal fermentation, whilst, immediately after the drying, it only required three or four days to accomplish the same thing. Side by side with these experiments on alcoholic ferment, we carried on exactly similar ones with yeast obtained from "high fermentation " breweries. On December 16th, 1872, we prepared a powder of this yeast and plaster as before. Our last sowing took place on July 25th, 1873, in a flask of pure wort, which showed signs of fermentation on July 27th. We append a sketch (Fig. 16) which gives the general aspect of 6 Pig. 1(J. this " high ferment," when revived after such a lapse of time ; it had preserved the distinctive features of the cells of "high ferment." These facts can leave no doubt whatever as to the possi- bility of cells of yeast existing, in a state of suspension in the air, in the form of fine dust, particularly in a laboratory where researches on alcoholic fermentation are pursued. 84 CHAPTER IV. The Growth of Different Organis.ms in a State of Purity : Their Autonomy* Our observations in the preceding chapter will have shown that organic liquids, natural or artificial — the wort of beer amongst others — if exposed to contact with the air, rapidly develop various forms of life. This is a natural consequence of the mode of impregnation. The fertility of the liquid depends on the various microscopic germs which are deposited * In the course of this work we shall combat, by means of experi- mental proofs which appear to us irrefragable, the opinions which many writers entertain on the subject of certain transformations of organisms — that of peniciUium gJnuciim into ferment, or mycoderma ; of bacteria into lactic ferment ; of ferment into vibrios ; of mycoderma aceti into fer- ment, and so on. Nevertheless, we shall pronounce no a priori opinion on the question whether the inferior organisms, which will be the subject of this chapter, and which include yeast and the fei-ments properly so called, are perfect beings in their habitual form, or whether they are susceptible of polymorphism. It is with this reservation that we employ the word autonomy. If we claim polymorphism for any species, we shall not do so without furnishing proofs. Some organs detached from higher organisms, and some beings in a certain phase of their existence, may reproduce themselves iinder a special form, with special j^roperties, when brought into media and under conditions that are unfit for the production of the plant or animal under its other shape or ordinary mode of repro- duction. Modern Science affords many examples of this, and certain alcoholic ferments present us with analogous facts ; but to wish to stretch these facts beyond their due significance, and to admit a polymorphism that cannot bo proved, in consequence of a belief that it is possible, or on the faith of confused observations, is to indulge in gratuitous assertion from a mere spirit of system. STUDIES ON FER^IENTATION". 85 in it by the common air, and these, again, as regards their nature and number, are dependent upon the situation of the vessel containing them, its height above the ground, the time of year, the disturbance of the atmosphere, and other causes. The fortuitous association of other forms, in growths which we believe to be uniform and independent, constitutes one of the principal difficulties that occur in the study of the lower organisms, particularly that of fungoid growths. The fact that the germs of many of these little beings exist in the atmo- sphere in the form of dust, invisible to the naked eye, or, as such, spread over the surface of the diiferent matei"ials and objects used in experiments, exposes the student to constant risk of wrongly interpreting the results which, come under his notice. He has sown a plant, and is observing the course of its development. Without his knowledge, spores of another plant have got mixed with his growth, and germinated. In his ignorance, he will attribute all that he sees, all the changes Avhich he describes and which he sketches, and all the con- clusions which he draws, to the one plant which engages his attention. If he is dealing with bacteria, vibrios, and, generally speaking, the infinite variety of mobile microscopic organisms, his embarrasment will be greater still. Again, inasmuch as the medium which serves as substratum for growths has a considerable influence on the fertility of the germs in contact with it, as well as on their ulterior development, it often happens that germs deposited fortuitously by the particles of dust which fall from the atmosphere or collect on objects are fertile and multijaly with rapidity, whilst those which have been directly sown, no matter in what number, remain sterile, or multiply very slowly. If we place in a young wine some mycoderma aceti, we shall obtain mycoderma vini; by placing some mycoderma mni in an old wine, especially if it is a little acid, we shall obtain mycoderma aceti* It is from facts of * See, on this subject, the author's Etudes sur le Vinaigre, Paris, 1868, p. 76, note ; and esj)ecially Etudes sur le Vin, 2nd Edition, 1873, p. 19. 8G STUDIES ON FKRMENTATTOX. this kind, wrongly interpreted, that many errors have crept into our knowledge of the lower organisms, and that we are con- stantly seeing old discussions crop up, both on the subject of so-called spontaneous generation and on that of the theory of fermentation. At every step in the course of this work we shall see the trace of these complications, as well as the influence they have had on the progress of our knowledge. In opposition to these results, we will study the case of wort sown directly with germs distinctly of one kind, unmixed witli any other. § I. — Growth of Penicillium Glaucum and Aspergillus Glaucus in a State of Purity — Proofs that these Fungoid Growths do not become Transformed into THE Alcoholic Ferments of Beer or Wine, — Pre- liminary Enquiry into the Cause of Fermentation. Let us again take one of the flasks furnished with two necks such as we have already described, and let it be supposed that this flask contains a quantity of saccharine wort, brewed some considerable time ago, which has undergone no change what- ever, except in colour, the slow process of oxidation having gradually darkened the original colour of the liquid. What we have to do is to drop into this unchanged and fertile liquid some grains or spores of penicilliiim which are free from the slightest trace of the spores or germs of other microscopic organisms. One means of effecting this consists in taking up with a pair of metallic forceps, previously heated, a piece of platinum wire, one or two centimetres (about J in.) in length, which we also pass through the flame of a spirit lamp, and with which, as soon as it is cold, we touch a mass of sporanges of a growth of penicUliam, No matter how few spores ma}- be taken up on the end of the platinum wire, we shall liave far more than we require for the impregnation of the liquid. At the moment of charging the STUniKS ON FRiaiENTATION, 87 point of the \\'iro we withdraw the glass stopper which closes the india-rubber tube on the right-hand neck of the flask (Fig. 14) and drop the wire through that tube ; we then I'eplace the glass stopper, after having, by way of additional precaution, passed it rapidly through the flame of the lamp. There is no doubt that we expose ourselves to error in consequence of having to convey the wire through the surrounding air, and also, in consequence of having previously to open the flask ; but, as we have already remarked, this double cause of error has never, we may say, interfered with the exactness of our experi- ments, the volume of air. with which we are concerned being exceedingly limited. Moreover, our flask being in free com- munication with the exterior air, by means of the opening in the curved, slender tube, there is no inrush of air when we withdraw the stopper. The chance of encountering a spore or fecund germ and introducing it into the flask on the wire that is charged with the others, is so remote that we have considered it unnecessary to adopt a more perfect apparatus, which might easily have been devised had we felt that it was necessary. A more serious cause of error may occur in the preceding method ; resulting from the possible impurity of the spores taken from a field of peniciiliiim which has developed in contact with common air. This field receives, every instant, and has received throughout its growth, particles of dust which have fallen from the atmosphere ; thus, it may not be, and, as a matter of fact, is not, free from the germs of other fungoid growths.* * Some observations in the preceding chapter enable us to account for the vast number of germs which are constantly falling on the surface of everything. We may here allude to the use we have made of flasks, shaped as in Fig. 17, and holding from 250 c.c. to 300 c.c, which are a third part filled with an organic liquid, and are closed up when boiling. They contain no air when cool, and are opened in series of 10, 20, &c., out of doors, and closed up again immediately. The air rushes violently into the vacuum, and thus we introduce about 200 c.c. of air, with all the particles of dust contained in that air, into each flask. It has been proved that a certain number of these flasks undergo change in the 88 STUDIES ON FERMENTATION. The operator, without knowing it, may frequently sow, besides the penicillium, which is all he can see, spores of mucor Diucedo and mycoclerma vini, in short, of all the most common fungoid growths. This process of impregnation, therefore, does not afford us sufficient safeguards, but b3aneans of the following device we shall Fig render it more satisfactor3^ Let us take a series of flasks shaped as in Fig. 17, containing an organic liquid suitable to the develop- ment of fungoid growths, that is to say, slightly acid — yeast- water, plain or sugared, the wort of beer, or Raulin's fluid* course of time, the number of those changing and the nature of their changes being in close proportion to the probable number and nature of the floating germs able to develop in the particular nutritive liquid used. If we work at great elevations, far from houses and the dirt of towns and inhabited plains, as we did at Montanvert, near the 3fer de 0/ace, change will seldom occur. The opposite will be the case if we work in a place like the living-room of the little, dirty, • ill-kept inn at Montanvert. In a laboratory where fermentation is studied we obtain certain kinds of germs which often differ from those found in the air of the open country. If we desii'e to have organisms in all our flasks, we have only to stir up the dust on the ground or on sur- rounding objects at the moment when we open the flasks. This simple and easy experiment clearly shows us that it is impossible for a field of sporanges of fungoid growth, existing in an uncovered vessel or on the surface of a fruit, to escape becoming mixed with germs that are foreign to the little plant ; in other words, the student who sows spores of penicilUam, which ho has collected from one place or another on a brush, exposes himself to serious causes of error. * M. Jules Rauliu has published a well-known and remarkable work on the discovery of the mineral modium best adapted bj' its composition STUDIES ON FERMENTATION. OV will answer our purpose ; let us boil the liquid, and having pre- viously drawn out the necks, let us close the ends in the flame of a lamp whilst the steam is escaping, as soon as we judge that the air has been nearh^ all expelled. Having prepared ten or twenty of these flasks in this manner, when tliey are cold we may break their points in any place we may be in. The air will rush into the flasks, and we must then seal them up again in the flame of the lamp, and put them aside for future obser- vations. In a certain number of these flasks, as we have already explained in our experiments carried on after this fashion, we shall see some fungoid growths appear, first in the shape of flakes of mycelium floating in the liquid, and after- wards coming to the surface to fructify. Now, it often happens that penicillium glaucum appears alone, so numerous are the spores of this fungus floating in the air. Under such con- ditions, M^e shall evidently obtain a field of sporanges quite free from the presence of other organisms. If we now take ofi" the neck of one of the flasks containing the pure penicillium, and take out some of the germs with our platinum wire, * we to tlie life of certain ordinary fungoid growths ; lie has given a formula for the composition of such a medium. It is this that we call here *' Raulin's fluid " for abbreviation. Water 1,500 ] Carbonate of Magnesia . . 0-4 Sugar Candy 70 Sulphate of Ammonia. . . . 0'25 Tartaric Acid 4 Sulphate of Zinc 0*07 Nitrate of Ammonia 4 Sulphate of Iron 0'07 Phosphate of Ammonia . . 0*6 Silicate of Potassium .... 0-07 Carbonate of Potassium . . 0'6 | J. Eaulin. Paris, Victor Masson, 1870. These pour le dodorat. * If we do not wish to take the chance of pi'ocuring the pure penicillium by means of these spontaneous sowings, effected by opening and then closing in the flame a certain number of flasks with drawn-out points, we may utilize one of the flasks, which, having been opened and closed again, has notwithstanding developed no organized forms, as follows : — We impi'egnate the contained liquid directly, by dropping into it from a metallic wire spores taken from any growth of penicillium exposed to the common air ; and then from the new field of sporanges formed by this sowing in the flask that has been re-closed, we must, later on, take the pure spores that we require. This method is quicker and almost as safe. 90 STI Dir.S ox FERMENTATION. shall thus obtain with most certainty spores of penicillium free from impurities. Our readers will excuse the length of these details and the minutiae of our precautions, but we shall again and again see that to neglect them, or any part of them, is to expose ourselves to hazard in drawing sure conclusions from facts which come under our observation. Fig. 18. On June 17th, 1872, we placed some pure spores of pruicil/ium in a series of three flasks containing wort (Fig. 18), observing all the precautions that we have indicated. We shall designate We should add that, if we wish to use for our purpose spores oi penicillium from a closed flask, in which the plant has fructified, we must be careful not to leave the plant too long closed up. A few daj-s after the sowing the growth of the fungus is arrested, in consequence of all the oxygen being absorbed, and its place being sujiplied by a mixture of carbonic acid and nitrogen ; and the spores, if ki'pt too long in this atmosphere, will all perish. STUDIES ON FERMRNTATTO.V. 91 these flasks by the letters A, B, C. On the following day the spores germinated, and the liquid became full of flakes of mycelium, some of which came to the surface to fructify. The temperature varied between 25° C. and 30° C. (77° to 86° F.). On June 22nd, small patches, with whitish borders and green centres, developed on the liquids. "We then shook up the flask A, in order to submerge the plant and the spores.* We also shook the flask B, after observing the precaution of sealing up the slender bent tube.f The flask C was attached on one side to an aspirator, on the other to a tube filled with cotton, and every day we renewed the air in it. During the weeks and months over which our observations extended, there was not the least formation of yeast in these flasks ; moreover, we have frequently repeated this and other experiments of a similar kind, without ever detecting the appearance of either ordinary yeast or any other true alcoholic ferment. The experiments may be made with saccharine juices that are highly favourable to the development of bacteria and lactic ferment. These latter appear equally incapable of trans- formation into yeast, which has never been seen to develop in experiments where they were used, if proper precautions have been taken to secure a pure growth. Should we neglect any of the precautions that are necessary to secure the purity of our spores, we may of course obtain difierent results. If, for instance, we sow spores oi pmicillium grown in free contact with * To shake tlie liquid witliout danger of introducing exterior particles of dust, we apply the flame of the spirit lamp to the drawn-out neck of the flask, and close up the open end ; we may then shake our flask with- out risk. We must afterwards reopen the end of the drawn-out neck for the purpose of re-establishing communication with the exterior air. t The flask B was closed with the lamp in consequence of one of the objects of these experiments being to test M. Trecul's experiments on the transformation oi penidlliwn into ferment. Strangely enough, according to M. Trecul, as we shall see later on, the spores of penicillium refuse to change into ferment, if the vessels in which they are sown are not " perfectly air-tight." 92 STUDIES ON FEltMENTATIOX. the atmospliere, and consequently exposed to tlie i)articles of dust floating therein, we shall frequently observe, mixed with the fruiting hyphse of the fungoid growth, 5^east and mycoderma vini and toruke, or even bacteria and lactic ferment. Thus we shall he led to believe, in all good faith, that we have under our eyes examples of the changes of spores of fungoid growth into cells of ferment, or proofs of the conversion of bacteria or lactic ferment into the same cells. Causes of error of this nature have induced some German naturalists to believe that they have succeeded in proving, beyond the possibility of doubt, that a number of fungoid growths may produce alcoholic ferment, and that they have clearly demonstrated that spores of these fungi maj'' become transformed into yeast. In 1856 M. Bail, and, about the same time, Berkeley, and, later on, H. Hoffmann and Hallier, have successively entertained these views, which were introduced into science by M. Turpin. We have combated them since the year 1861.* Since that period they have lost rather than gained ground abroad, in spite of the growing favour bestowed on the Darwinian system. One of the mycologists who enjoy the most legitimate authority beyond the Rhine, M. de Bary, has arrived, as we have, at absolutely negative results. A simple perusal of what has been written in favour of the transformations which we are discussing causes us to entertain the gravest doubts as to the correctness of results which are quoted as decisive. We need only give one example, which we extract from a paper by M. 11. Hoffmann. *' In some cases," this author writes, " and under favourable circumstances, I have been able to see the ferment produce fila- ments, both small specimens that could be examined immediately under the microscope, and also large specimens, and I have recognized, amongst other varieties, pcniciUium glaucum, asco- pliora mucedo, ascophora elegans, and periconia hyalina, sometimes isolated, sometimes intermixed. This result is most easily to be * BuUdin dc la Societc PliilomatJrique. STUDIES ON FERMENTATION. 93 obtained by the following method ; — Pour a small quantity of water into a test-tube, which should then be placed slant- ingly : introduce some fresh yeast into the middle part of the tube, and close the tube with a wad of cotton, to prevent the entrance of exterior particles of dust. In this vapour-filled receptacle we shall sometimes see flakes develop. It seems that Messrs. Berkeley and G. II. Hoffmann have also obtained penicillium from ferment, by a similar process."* Why, however, should it not be admitted that penicillium found under such conditions may be derived from spores of that growth, adhering to the sides of the tube before it is closed with the wad, or mixed with the yeast that is put into the tube ? The facts alleged during the last few years by M. Bail, who persists in his views, in spite of their apparently greater exact- ness, are also far from being satisfactory.! M. Trecul, who is almost the only one in France, besides Messrs. Ch. Robin and Fremy, to participate in these errors, does not confine himself to afiirming the change of the spores oi p)^nicilliiim into yeast, and vice versa; his system is a far more extensive one. " According to my observations," he says, "there would be the following series of changes : albuminous matter changed into bacteria, or directly into alcoholic ferment, or into tnycoderma ; bacteria into lactic ferment, there becoming im- moveable ; lactic ferment into alcoholic ferment ; alcoholic ferment into mycoclerma cerevisiw ; finally, mycoclenna cerevisice into penicillium. J M. Trecul does not stop here. He goes on to explain the * Hermann Hoffmann, Etudes Mycologiqnes sur la Fermentation. Botanische Zeitung and AnnaJes des Sciences Naturelles, ■4'' serie, t. xiii. p. 24, 1860. t Communication sur VOrigine et le Developpement de queJques Cham- pignons, Dantzig, 1867. X Trecul, Comptes rendus de V Academic, t. Ixxiii. p. l-lo4 ; Docember 28, 1871. 94 S'lUDIES O.N FERMENTA'IIOX. principal of these changes, as tliough his testimony were quite beyond refutation : "If we use a perfectly filtered wort, containing no granula- tions, and prepared at a temperature between 60° C. and 70° C. (140° F. and 1 60° F.), there will first of all appear a multitude of fine granules that will develop into active bacteria, which, losing the faculty of motion, will constitute lactic ferment, as I have repeatedly pointed out. A few days after the appear- ance of the first granules, we shall perceive others rather larger in size and isolated. These will increase in size, and, in the course of time, assume the form of little globuloid, or elliptic cells ; they will not commence to bud before they have attained a comparatively large size, approaching that of ordinary j-east, consequently, there will be a considerable interval of time, during which the young cells will present no buds, especially if we work at a low temperature, as from 20° C. to 35° C. (68° F. to 95° F.) "As for the transformation of the spores oi penicillium into alcoholic ferment, the possibility of which M. Pasteur also denies, I have very often obtained it by using liquids, such as boiled wort and sugared barley water, which had stood for a month or six weeks without setting up an alcoholic fermentation. These liquids, sown with spores of different forms of pciiicilliuni, chosen when young and in full growth, fermented after a vary- ing number of days, even at a temperature of 12° C. (54° F.), the condition of fermentation being that the flasks were closed with very elastic corks, which had been boiled for a quarter or half an hour ; these corks, as I have already pointed out, it is best to keep for a month after the boiling, to make sure, bj' dry- ing them thoroughly again, of destroying any mycelia adhering to them. It is necessary to keep the flasks stoppered that the corks may be always moist, and it is also advisable to shake the flasks once or twice a day, to secure the submersion of the spores. If these conditions are carried out, we shall soon see the spores increase in size, gradually lose their green colour and then bud, and often a very active fermentation will manifest itself. All STUDIES ON FERMENTATION. 95 the spores will be transformed, if the flasks are perfectly air-tight."* Such is the manner in which M. Trecul regards these changes. His is entirely a system of spontaneous generation, worked out into minutest detail, from the transformation of the albuminous substances to the formation of cells of the higher organisms, passing from the disintegration of the original substances to the formation of very fine granules, from these to the creation of active bacteria, which last, in their turn, become lactic ferment through the simple cessation of their faculty of moving and so on. We regard all this as purely imaginary. As a matter of fact, M. Trecul's argument is based on the successive phenomena which manifest themselves in filtered wort " containing no granulations." As M. Trecul reasons, this condition is a neces- sity, for he starts with the assertion that the albuminous sub- stances in the wort become changed into granulations " that will develop into active bacteria." This is another of M. Trecul's illusions. No doubt we may filter hopped wort to almost per- fect clearness, but we can only do this when it is cold. If we filter it warm, it will be bright as long as it remains warm, but as soon as cold it will appear turbid, in consequence of the great number of minute granules floating in it. Again, cold wort, however little it may be or has been in contact with air, under- goes a process of oxidation, and this oxidation, which acts principally on the colouring or resinous matter, causes a deposit of fine granules, the number of which is constantly increasing as oxidation goes on. These granules form an absolutely inert precipitate which, under no possible circumstances, can become * Teecul, Comptes rendus deVAcademie, t. Ixxv. p. 1169, November 11, 1872. A proof of M. Trecul's carelessness in experiments of this kind is the fact that in studying the fertility of an impregnated wort, he often obtains different productions. Our experiments give opposite results. If we sow nothing, we obtain nothing. If we sow a plant, we obtain a similar plant ; or, should there be any difference, the change may be traced, bej'ond question, to its origin in the plant sown, and is the con- sequence of some alteration in the conditions of our experiment. 06 STUDIES ON FERMENTATION. transformed into active bacteria. Nothinj^ can be easier than to prove this fact, by taking some of our two-necked flasks (Fig. 4) and preparing pure wort in them, by boiling, and then leaving it to cool and undergo the process of oxidation. In wort thus exposed to the air — the air being pure and free from germs — there will be formed a granular deposit, which will never become active or transform itself into any kind of organism whatever. "We may also remark that whilst M. Trecul used wort in his experiments, he does not tell us if it was hopped or not. Had M. Trecul informed us that the facts which he described applied to unhopped wort, we should reply that a temperature of 60° C. or 70° C. (140° F. or 160° F.) is quite insufficient to kill the germs of bacteria existing in such a wort. Hopped wort should be heated to 70° 0. or 75° C. (160° F. to 170° F.), that it may remain inert after having cooled down in contact with pure air ; unhopped wort must be heated to about 90° C. (194° F.). In short, whatever may be the case, it must be evident to our readers that the active bacteria observed by M. Trecul existed in the form of germs in the wort that he used, and that what he observed was nothing more than the development of these germs when brought into contact with the air held in solution in the liquid. As for the success of M. Trecul's experiments on the penicillimn, we have no doubt that that gentleman has sown germs of yeast or tonilce, which bear so striking a resemblance to yeast, at the same time that he sowed spores, since he took his spores from sporangia of penicilUum that had been exposed to contact with ordinary air. The conditions under which M. Trecul conducted his experiment rendered it difficult for the spores — although, relatively, much more numerous than the contaminating germs of ferment with which they happened to be associated — to make way against these latter, since the spores were unable to continue their development in a medium that was deprived of oxygen. On the other hand, the cells of S'JUDIES ON FliRMENTATlON. 97 ferment might easily have multiplied to such an extent as to make the discovery of the spores that had been sown a matter of some difficulty, since those spores would have been lost amongst the great number of cells of ferment. This was, pro- bably, one of the causes which led to the mistaken notion that such spores underwent conversion into cells of ferment. Now although botanists describe several varieties of i^eniciUiam glaucum, we do not suppose that the cause of the difference between our results and those obtained by M. Trecul can be attributed to our having operated upon a different variety of penkilUuin from that which he used. Supposing that there had been a great difference between our two varieties, still M. Trecul declares that he has realized the phenomenon with numerous varieties of this fungus. M. Trecul expresses himself as follows : "I have used spores oi peniciUium of several varieties in these experiments : Firstly, thick, green, elliptic spores of a variety of penicilUum that grows on lemons ; secondly, elliptic spores of a bluish colour and smaller than the preceding, of another variety oipenicillium found on lemons ; thirdly, spherical spores of the variety ievrnQdi penicil- Uum crustaceum ; fourthly, spores of the 2ienicillium that develops on the yeast of beer.* This criticism of M. Trecul's opinions was written on the occasion of a discussion at the Academy, after we had been induced to read over again the remarks which he had published on the subject. We were so impressed by the positive manner in which his conclusions were stated, that we asked ourselves, once more, which of us could be mistaken, and once more, also, we applied ourselves to fresh experiments, which we conducted with every possible precaution, following, as far as we could without falling into the errors of which we accuse the learned botanist, the mode of procedure that he adopted. As his descrip- tions struck us as being at times insufficient, we resolved to ask him for certain explanations vim voce (November 3rd, 1873), which he gave us with the greatest willingness. * Comptes rendus des Seances de VAcadeinie des Sciences, t. Ixxv • p. 1220; Nov. 18, 1872. H 98 1STUJ)IKS ON FERMENTATION. " Every variety of pcnicillium" said M. Trecul, " especially when young and vigorous, is amenable to transformation into ferment. This is the way in which I operated : I had some little flasks of 30 c.c. to 40 c.c. (about I5 fluid ounces) in capacity, filled quite full with wort, or, at least, con- taining very little air, closed perfectly air-tight with corks which I had kept for a quarter of an hour in boiling water. These flasks when corked were heated to 60° C. or 70° C. (140° to 158° F.). After they had cooled I uncorked them, and intro- duced into them the spores which had been prepared as follows : I placed on a piece of glass some spores of the variety of peni- cillium that I wished to study, taken with a pair of forceps from a mouldy lemon, and I mixed these spores with a drop of wort and observed them under the microscope to assure myself that they contained nothing of a foreign nature ; then I poured my drop of wort from the piece of glass into one of the flasks, which I recorked and laid down. The transforma- tion into ferment took place next day." Provided with these new data we set to work again and pre- pared a series of little flasks which were filled quite full with hopped wort, or contained but very little air, as M. Trecul had recommended. These we heated in a hot water bath to 70° C. (158° F.) at least ; we then impregnated them, observing the necessary precautions, which we described at the commencement of this paragraph — not working in the evidently defective manner in which M. Trecul had done. Taking his spore-seeds from a field of sporanges exposed to the air, and afterwards manipulating them, in contact with the air, in water on a piece of glass, before he made his microscopical examination, his experiments were conducted under circumstances in every way conducive to the introduction of causes of error. One of the most serious of those causes is that which results from the sub- stratum of the spores as taken from a mouldy lemon. If M. Trecul will examine under the microscope the water in which any lemon has been washed — even a sound lemon, unattacked by any fungoid growth — he will immediately see the cause of error to which his method of working exposes him. Germs of STUDIES ON FERMENTATION. 99 microscopic organisms exist abundantly on the surface of all fruits. Having impregnated the liquids in our flasks with spores from a quantity of pure sporangia grown in a closed vessel, gathered on the point of a platinum wire, which had first been heated and then allowed to cool, we found that in each ease, without exception, germination took place, then a mycelium was developed, which soon, however, ceased to grow from want of a proper supply of air ; but not in any single case was there the faintest trace of fermentation, formation of yeast, or appearance of bacteria or lactic ferment. We repeated these experiments, using unhopped wort instead and obtained similar negative results. We had previousljr determined that it was necessary to heat flasks of hopped wort to 70° C (158° F.), at least, and those of unhopped wort to 90° C, (194° F.) to secure them from further change. In short, contrary to the assertions of M. Trecul, M. H. Hoflfmann, and other naturalists, it is not true that the spores of 2)eniciUium can change into alcoholic ferment. Regarded from another point of view, growths of pure penicillium will give us some remarkable results, the interpreta- tion of which seems to us to be intimately connected with the physiological theory of fermentation that we shall discuss in a subsequent chapter. It is a question as to the production of alcohol whilst the life of the plant is carried on under certain conditions of growth. If we distil saccharine liquids on the surface or in the body of which we have grown penicillium, and repeat the distillation in the manner that we have already described for the detection of the minutest quantities of alcohol, we shall readily find that those liquids frequently do contain a little ordinary alcohol. Moreover, if we regard the quantities of alcohol produced, which are always very minute, seldom exceeding 1 or 1'5 thousandth of the total volume of the liquids, we shall find that there is no fixed proportion between this alcohol and the weights of the plants formed. It is possible, for instance, that H 2 100 STU])IKS ON FERMENTATION. we niny obtain more alcohol from one plant than from another weighing a hundred times as much. Often, however, when the vegetation is abundant we cannot make out the occurrence of alcohol in spite of the sensitiveness of the process described (p. 78). What can be the cause of these varying results relating to the production or non -production of alcohol in the vegetation of the little plant ? The numerous experiments that we have made seem to demonstrate positively that they are dependent upon variations in the amount of air or oxygen that is supplied to the fungoid growths, whether, that is, the vegetating my- celium alone be submerged, or the whole plant with its organs of fructification. When the plant has at its disposal an excess of oxygen, as much as its vitality can dispose of, there is no alcohol, or very little, formed. If, on the other hand, the plant vegetates with difiiculty, in presence of an insufficiency of oxj'gen, the proportion of alcohol increases ; in other words, the plant shows a certain tendency to behave after the manner of ferments. Some time ago, wishing to assure ourselves that the spores of penicillium could not become transformed into ferment, we sowed some pure spores in small flasks, holding from 50 c.c. to 100 c.c. (from 2 to 4 fl. oz.), which contained very little air, and which were sealed hermetically after the sowing. Under these conditions, the germination and growth of the spores proceeded with great difficulty, and soon ceased through want of air. The total weight of the little plant was too small to be determined. In cases of this kind, if we distil the whole of the liquid we shall often see the alcohol appear in the second distillation, even though the weight of the plant may have been scarcely appreciable. If, on the other hand, side by side with experiments of this kind, we grow ^xxxq penicillium in flasks containing air and having quantities of saccharine liquids equal to the quantities in the small flasks of which we have been speaking, the plant, in consequence of the large volume of air at its disposal, will develop vigorously, and in the course of even STUDIES ON FERMEiNTATION. 101 a few da5^s will have become perceptibly heavier. In dis- tilling the subjacent liquid, however, we shall generally find that it contains no alcohol at all, even though the weight of the plant be half a gramme, or more (6 or 7 grains). These results apply to all the fungoid growths that we have studied, but they vary considerably with the nature of the organisms. Aspergillus glaucus is, in this respect, one of the most curious. On June 15th, 1873, we impregnated three flasks of wort, A, B, C, with pure spores of aspergillm glaucus. The development was rapid and the fructification abundant. On June 20th, we shook up the liquid and the supernatant fungoid growths in the three flasks ; the flasks A and B were then treated as follows : — "We distilled the liquid in A to discover the presence of alcoholj but could find none. HE: 'IIIIHlhllllilllll'lllillllllHliiihll.l'i Fig. 19. The flask B was connected with a test flask (Fig. 19), into which the liquid, together with its fungoid growth, was trans- ferred from B. The next day, June 21st, the mycelium which 102 STUDIES ON FERMENTATION. was on the surface of the liquid, in the neck of the flask, was studded with bubbles of gas ; these we dispersed by shaking. On June 22nd, many others had formed again, and a large flake of mycelium that had risen from the bottom of the test flask had been stopped at the bottom of the neck, quite distended by gas bubbles. We liberated the gas b}^ shaking, but the bubbles formed again by the next day, and this effect continued for several days ; nevertheless the liberation of gas was not con- tinuous, as is the case in an ordinary fermentation. On July 20th we drew off the liquid and distilled it ; it was still very sweet, but though it contained a sensible quantity of alcohol, the microscope failed to detect a single cell of ordinary alcoholic ferment. These results show that the aspergilliis when in full growth, with plenty of air at its disposal, does not yield alcohol, and that if we submerge it, so as to prevent the oxygen of the air from readily coming into contact with its various parts, it decomposes sugar, after the manner of yeast, forming carbonic acid gas and alcohol. These effects were still more marked in the case of the flask C, the liquid in which, after having been shaken up, was not decanted to any great depth in our test flask, as had been the case with B. From June 21st, there was mycelium on the surface of the liquid, studded with large bubbles of gas, which formed again after having been liberated by shaking. This last flask was examined on November 1st, 1873. Its aspect was un- changed ; the liquid was covered with mycelium loaded with sporanges and borne up by large old bubbles that had not disappeared. The following was the analj'sis of the liquid : — Alcohol 1-2 Glucose . . .. .. •• 84"0 Dextrine (?) 320 The liquid was very bright, and contained an amorphous granular deposit, formed by the wort after it had been boiled, at the time when we prepared our flasks. We crushed a small STUDIES ON FERMENTATION. 103 quantity of mycelium that had risen to the surface of the liquid, and obtained a field such as is represented iu Fig. 20. Amongst Fig. 20. the ordinary filaments of mycelium belonging to the plant, which are not represented in our engraving, and which were not moro 104 STUDIES ON FERMENTATION. than -jjyyj of a millimetre (nearly y-VTr i^O i^ diameter, we per- ceived much larger ones, swollen and contorted in the most singular manner, and measuring as much as -y'-^ of a millimetre across their broadest parts. There was also a multitude of the ordinary spores of aspergilhis mixed with others of larger size, and big, inflated cells, with irregular or spherical protuberances, full of granular matter. As there are all the stages between the normal spores of the plant and the big cells, and between these latter and the filaments, it must be admitted that the whole of this strange vegetation results from spores which change their structure under the influence of special conditions to which they are exposed.* Beyond all doubt these cells and irregularly shaped segments, in vegetating with difiiculty, gave rise to the fermentation, which, although insignificant, was sufficiently marked to produce more than a gramme (15 grains) of alcohol. The oxygen of the air failing, or existing in insufficient quantity for the regular develop- ment of the filaments of mycelium belonging to the plant, and for the germination of its submerged spores, filaments and spores vegetated as the yeast of beer might have done if deprived of oxygen. If we study the vegetation of as^ycrgillus glaucus with this preconceived idea, we shall soon recognize the fact that these spherical forms of mycelium are the result of a greater or less deprivation of air. The filaments of this mycelium which develop freely in the aerated liquid are young and transparent, small in diameter, and exhibit the ordinary ramifications. Those which are situated about the centre, in the denser or * Since -writing the above we have experienced some doubt as to whether the forms of development represented in Fig. 20 are actually those of the aspergilhis glaucus, which we supposed our fungoid growth to be. In some of the later sketches of our observations we find similar forms, which belong to a bluish kind of peniciJHum, with rather large spores. Fortunately, this doubt affects our argument in no essential particular. It matters very little what variety of fungoid growth it is that gives rise to alcoholic fermentation attended by peculiarities of shape that only occur in the development of its spores when air fails it. STUDIES ON FERMENTATION. 105 Aspergillus glaucus. Growth with abundant air-supply at the edge of the mycelium crust. Growth with reduced air-supply in the central and deeper parts of the mycelium. Mucor racemosus. Growth with abundant air-supply at the edge of the mycelium tuft. Fig. Growth with reduced air-supply in the central and deeper parts. 21. lOG STUDIES OX FERMENTATItJN. more complicated parts, to which the oxygen cannot penetrate in consequence of its absorption by the surrounding parts, are more granular in appearance as well as larger, and inclined to develop swellings. We can observe no conidia * on these filaments, but we may say that they are on the point of appear- ing, for the spherical segments often tend to assume an appear- ance of close jointing, as when they take the form of those rows of swelling, or cells, which has given rise to the idea of the cliaplets of the conidia-cellules. This is represented in the accompanying sketches (Fig. 21), which we have purposely con- trasted with tvro similar ones which relate to the mucor, of which we shall soon speak. The conidia of these latter are very remarkable, and their fermentative character becomes apparent as soon as their growths are deprived of air. It is scarcely necessary to add that in these vegetations of aspergillus, which were accompanied by a corresponding alcoholic fermentation, it was impossible to find cells of yeast ; and that, notwithstanding this, the liquid was so adapted to ordinary alcoholic fermentation, that, when we added a small quantity of yeast to it, in the course of a few hours, a most active alcoholic fermentation declared itself. We may give some other facts relating to a crop of aspergillus glaucus which was also grown in ordinary hopped wort, and which was left to itself for a year. A two-necked flask, holding 800 c.c. (rather more than 10 fl. oz.) was prepared and impregnated on December 21st, 1873, and was then placed in an oven at a temperature of 25° C. (77° F.). The fungoid growth developed in isolated tufts, which subsequently united, but without entirely covering the surface of the wort. A few tufts also vegetated at the bottom of the liquid ; those on the surface soon became surrounded by large bubbles of gas. On December 12th, 1874, we examined the liquid and the plant, which for a long time had appeared dead. Its mycelium was * By the term conidia is meant certain chains of colls, which are in reality mycelial spores. STUDIES ON FERMENTATION. 107 formed of aged, granulated filaments, with few swellings. The weight of the dry fungoid growth was 050 gramme (about 8 grains) for a total volume of liquid of 122 c.c. (4^ fluid ounces). "We obtained 4*4 c.c. of alcohol of 15°, which was about seven times the weight of the plant. Finallj^, we determined the acidity of the liquid, and found 2 '8 grammes, in equivalents of sulphuric acid, a quantity greatly in excess of the total acidity of an equal volume of wort, a fact which shows us that fermentation caused by aspergillus glaucus is accompanied by the forma- tion of an organic acid, the nature of which it would be interest- ing to determine. M. Gay on has commenced the study of this subject in our laboratory. In concluding our observations on the aspergillus glaucus, we may give the comparative results of two growths that were obtained under precisely similar conditions, in flasks of exactly the same size, but difiering in this respect — that one of them was constantly subjected to a current of pure air that played on the liquid. In the course of a few days, when the fungoid growth in the flask that had been aerated had attained a con- siderable size, in comparison with the other, we broke the flasks in order that we might take out the two growths and compare their weights. After drying them at 100° C. (212° F.) v>^e found : — Growth in the aerated flask . . 0*92 Growth in the closed flask . . 0"16 Ratio of weights, f| = 5-75. Again, although we had taken the precaution of condensing in a U tube, over which cold water played, the vapours carried away by the current of air, the liquid in the aerated flask save no evidence of alcohol. That in the other flask contained a very appreciable quantity, although the weight of fungoid growth in that flask was scarcely a sixth part of what it was in the other. The preceding facts taken altogether, seem to us to demon- strate once more, in the most conclusive manner : — 108 STUDIES ON FERMENTATION. Firstly, That neither 2)enicilliiini nor uspergillua glaiicus can change into yeast, even under conditions that are most favour- able to the life of that ferment. Secondly, That a fungoid growth which vegetates by using the oxygen of the air, and which derives from the oxidating action of that gas, the heat that it requires to enable it to per- form the acts necessary to its nutrition, may continue to live, although with difficulty, in the absence of oxygen ; that, in such a case, the forms of its mycelian or sporic vegetation undergo a change, the plant, at the same time, evincing a great tendency to act as alcoholic ferment, that is to say, decomposing sugar and forming carbonic acid gas, alcohol, and other substances which we have not determined, and which probably vary with different growths. Such, at least, is one interpretation of the facts that we have reviewed. The observations in the following paragraphs and chapters may the more incline our readers to accept it as the true one. § II. — Growth of Mycoderma Vini in a state of Purity — Confirmation of our original Conjectures as to the CAUSE of Fermentation — Mycoderma Vini does not Change into Yeast, although it may give rise to Fermentation. The efflorescence of wine, cider, and beer is pretty generally known.* Fermented liquors cannot be exposed to the air with- out soon becoming covered with a white film, which grows thick and becomes wrinkled in a marked manner in proportion as it is deprived of room wherein to spread horizontally, in accord with the extraordinary multiplication of the cellules that compose it. The rapidity of this multiplication is sometimes astounding. During the heat of summer, when the medium is well adapted to the life of the plant, we maj^ count the number of cells which grow in the course of a few hours by millions. The absorption * See Pasteub, Etudes sur h Vin, 1st Edition, pp. 20 and following. STUDIES ON FERMENTATION. 109 of the oxygen necessary to the activity of this growth, and the heat developed in the film, as well as the liberation of carbonic acid gas, that result from it are considerable. A piece of glass covering the mycoderma, at some distance above it, becomes wet with moisture, that soon accumulates to form large drops of water. The quantity of oxygen absorbed is so great that we never see any other fungoid growth on the surface of this film, although the air is constantly depositing on it, as dust, spores of an entirely diflferent character ; for, notwithstanding that the warm and moist surface is in contact with an atmosphere that is being continually renewed, yet the mycoderma ap- propriates to itself all the oxygen contained in the air. When, however, the vegetation begins to languish, we often find, on the other hand, that the plant becomes associated with other species of mycoderma, notably mycoderma aceti, as well as other fungi, amongst which 2)eniciUium glaucrim generally appears. This is one of the facts which, wrongly interpreted, have led to the belief that mycoderma vini or ceremdce may possibly, or even readily, become transformed into innicillium, and vice versa* As the study of the growth of * Since writing this paragraph, we have found in M. Ch. Eobin's Journal cTAnatomie. et de Physiologie, an article signed by that gentleman, and entitled Sur la Nature cles Fermentations, &c. (July-August, 1875), in which the learned microscopist says: — "The torula cerevisice is derived from the mycoderma cerevisice. My observations leave no doubt on my mind that penicilliuin glaucum is one of the forms evolved from spores or ferments that have preceded it, as M. Trecul showed a long time ago, and that, moreover, the spores of peniciUium, germinating in suitable media, give us the sporical form termed mycoderma.'" We take the liberty to observe that these assertions of M. Eobin's are purely gratuitous. Up to the present time it has been impossible to discover a suitable medium for the proof of these different transforma- tions or polymorphisms. From the time of Turpin, who firmly believed that he had observed these changes, to our own, none of the micro- scopists who have affirmed these transformations have succeeded in adducing any convincing proof of them, and M. Trecul's latest observa- tions, especially as regards penicillium and its transformation into ferment or into the mycoderma of beer, have been positively disproved by ours, supported, as they are, by proofs that we consider irrefutable. 110 STUDIES ON FERMENTATION. mycoderma vini on the surface of saccharine liquids and in their depths, unaccompanied by any other species, has the most important bearing on the theory of alcoholic fermentation, we may pursue it through a few examples with all the detail that it allows of. On June 21st, 1872, we sowed some mycoderma vini in three flasks, with double necks. A, B, C (Fig. 22), containing some Fig. 22. wort. The spores employed for the purpose were obtained from plants growing on sweetened yeast- water in an ordinary closed flask. This had been impregnated with spores from plants grown on wort, which in turn had sprung from spores taken directly from mycoderma vini growing on wine. The several impregnations were effected by means of a plati- num wire, held by forceps, both having been first cleaned by passing through flame, and then smeared with the fungoid films. By this series of growings in closed vessels, which were but momentarily open at the time when we dropped the spores into them, we secured the separation of the mycoderma from all foreign organisms ; and more particularly from germs of myco- STUDIES ON FERMENTATION. Ill derma aceti, which is generally found along with it, but which propagates with difficulty iu neutral saccharine liquids. On the following days films of mycoderma vini had spread over the surface of the liquid in the three flasks. To all appear- ance they were very pure ; and the microscope showed the com- I')lete absence of any mixture of mycoderma aceti, lactic ferment, or other foreign growths.* On June 26th we decanted and distilled the liquid in A without finding any trace of alcohol. We shook up the liquids in B and C, with ail due precautions, so as to submerge their films as much as possible, and then we raised the temperature of the flasks to 26° C. or 28° C. (82° F.). For some days after- wards we saw a constant succession of minute bubbles of carbonic acid gas rising through the liquid, which remained bright under the part of the film that had not fallen in. It had all the appearance of a slow but continuous fermentation. On June 29th we decanted and distilled the liquid in B, and found in it an appreciable quantity of alcohol, which showed itself in the first distillation. The flask C, which was shaken afresh, continued to give signs of fermentation, but, some days later, the evolution of the bubbles ceased. On July 15th, 1873, we examined the flask with its film and its deposit of mijcoderma vini, without finding a trace of any foreign growths, either in the shape of penicilliiim glaucum, or mucor mucedo, or rhi/zopus nigrans, or mijcoderma aceti, or, in short, any of the organisms which could not have failed to appear on the surface of a substratum so peculiarly adapted to their development, had it been in the nature of mycoderma vini * It is a very easy matter to study the liquids and growths in our flasks during the course of a single experiment. We take out the glass stopper that closes the india-rubber tube on the straight-neck, and, by means of a long rod or a glass tube previously passed through the flame, take up a quantity, which we draw out immediately for micro- scopical examination. "We then replace the glass stopper, taking care to pass it through the flame before doing so, to burn up any organic particles of dust that it may have picked up from the table on which we laid it. 112 STUDIES 0>; FERMENTATION. to transform itself into one or other of those common fungoid growths. The liquid, moreover, still remained sweet, and did not contain any cells of actual yeast. We may conclude then that when one or more of these fungi occur, after an interval of some days, in a growth of mycoderma vini conducted in contact with common air, it does so in consequence of that air having, without the knowledge of the observer, impregnated the liquid spontaneously with germs of these foreign organisms. There might perhaps be room for some fear that the conditions of growth in our flasks were not favourable to the simultaneous appearance of these common fungoid growths along with the mt/- coderma vini. On June 24th, 1872, we sowed, in three flasks of sugared yeast- water, prepared as before — in the first, mycoderma vini, together with. jJeniciUium glaucum ; inihQ&QConA, mycoderma vini, together with mucor mucedo ; in the third, mycoderma vini alone. We efiected this by plunging the platinum wire, which we used for impregnating the liquids, into the pure film of mycoderma vini, and then touching with the wire the sporanges of the other fungus. On June 29th, we saw on the surface of our first flask some green patches of jx'nicillium, along with some spots of mycoderma vini ; in the second flask a voluminous mycelium of mucor mucedo, distended by large bubbles, had risen to the surface of the liquid, and was entirely covered by a film of mycoderma vini. As for the liquid in the third flask, there were only a few spots of very pure mycoderma vini. This last flask, after being kept in an oven at 25° C. (77° F.) for several months, still contained nothing but mycoderma vini, unmixed with any other fungoid growth whatever. We may therefore be sure that mycoderma vini, vegetating on the surface of liquids adapted to its nutrition, in contact with air deprived of its germinating dust, will not present the least sign of a transformation into any of these other common fungi, or into yeast, however long may be the duration of its exposure to contact with that pure air. AYc may now return to that feeble and limited production of STUDIES ON FERMENTATION. 113 carbonic acid gas and alcoliol, the formation of which we have shown experimentally to take place at a high temperature, after submerging the film of mycoderma vini* There can be no doubt that we have here a phenomenon similar in every point to that presented by penicillium and mpe)'gillus, which we studied in the preceding paragraph. When the germs or jointed filaments of mycoderma vini, growing on a saccharine substratum in contact with the air, are in the full activity of life, this activit}^ is carried on at the expense of the sugar and other materials in the liquid, in the same way that animals consume the oxygen of the air and evolve carbonic acid. The consumption of the different materials is attended with a proportionate formation of new materials, development of structure, and reproduction of organisms. Under these conditions, not only does the mycoderma vini not form a sufficiency of alcohol for analytical determination, but, if any alcohol exists in the subjacent liquid, the mycoderma con- sumes it, converting it into water and carbonic acid gas, by * We may prove the occurrence of alcoliolic fermentation by the cells of submerged mycoderma vini in a different manner. To do this, after having made all our preparations as before and shaken up the film of mycoderma vini in its liquid, we must attach our flask to a test flask (Fig. 19), and pass the turbid liquid into the latter. On succeeding days we shall detect a very protracted fermentation in the test flask ; there will be a succession of minute bubbles rising from the bottom, but in small number at a time. The fermentation is very evident whilst it lasts, but is rather sluggish, and, although of very long duration, ceases long before the sugar is exhausted. This experiment proves better than any other the non -transformation oi mycoderma vini into other ordinary fungoid growths. For after decanting the liquid into the test flasks, the sides of the experimental flask remain covered with streaks of mycoderma vini along with some of the liquid. Moreover, the flask is refilled with air, and this air is being constantly renewed, in part, by variations of the temperature of the oven, so that the mycoderma remaining on the sides is thus placed under the most favourable conditions for transformation into other fungoid growths, if that were possible. It is still more easy to detach the experimental from the test flask, and to pass pure air into it, once or twice a day, or constantly. In any case, we shall never see anything besides the mycoderma vini spring up within it. I 114 STUDHi;? ON FERMENTATION. fixation of the oxygen of the air.* If, however, we suddenly submerge the mycodemia, we shall obtain a different result. If, on the one hand, the conditions of life of this fungus are incompatible witli the altered circumstances in which it is placed, the plant must perish, just as an animal does when deprived of oxygen. But if, in spite of these changed condi- tions of nutrition, it can still continue in life, we should expect to see marked changes in its organic structure, or chemical metamorphoses. The result of our observations points to the continuance of life, in a distinct though sluggish and fugacious activity, accompanied by the pheoomena of alcoholic fermenta- tion, that is, the evolution of carbonic acid gas, and the pro- duction of alcohol. If we take a drop of liquid charged with disjointed cells of mycoderma, a day or two immediately after the submersion of the film, we shall observe changes, small but appreciable, in the aspect of a great number of these cells ; they will show increase in size, their protoplasm will be in process of modification, and many of them will have put forth little buds. It will be quite evident, however, that these acts of interior nutrition and the changes of tissue resulting from them, proceed with difficulty ; the buds when they form will soon wither, and there will be no multiplication of new cells. These changes will, nevertheless, be accompanied by the decomposition of sugar into alcohol and carbonic acid. In comparing these facts with those which we have pointed out in connection with the cultivation of 2^€nicillii(m and asper- gillus, we are compelled to admit that the production of alcohol and carbonic acid gas from sugar — in one word, alcoholic fer- mentation— is a chemical action, connected with the vegetable life of cells which may difier greatly in their nature, and that it takes place at the moment when these cells, ceasing to have the power of freely consuming the materials of their nutrition by respiratory processes — that is, by the absorption of free oxygen * See Pasteue, Oo77i2Jics rcndus des Seances de r Academic des Scie7ices, t. liv., 1SG2, and t. Iv., 18G2. Etudes sur les Mi/codennes, &e. STUDIES ON FERMENTATION. 115 — continue to live by utilizing oxygenated matters which, like sugar or such unstable substances, produce heat by their decomposition. The character of ferment thus presents itself to us, not as being peculiar to any particular being or to any particular organ, but as a general property of the living cell. This character is always ready to manifest itself, and, in reality, does manifest itself as soon as life ceases to perform its functions under the influence of free 0x3^ gen, or without a quantity of that gas sufficient for all the acts of nutrition. Thus we should see it appear and disappear concomitantly with that mode of life ; feeble and fugacious in its action when the conditions of this vitality are of a similarly restricted character ; intense, on the other hand, and of long duration and productive of large quantities of carbonic acid gas and alcohol, when the con- ditions are such that the plant or cell can multiply with facility in this novel manner. To this we may attribute all possible degrees of activity in fermentation, as well as the existence of ferments of every variety of form and of very different species. It may readily be imagined that sugar may undergo decompo- sition in a quite different manner from that of which we have spoken, that instead of alcohol, carbonic acid gas, glycerine, and similar substances, it may yield lactic, butyric, acetic, and other acids. It would be only one definite class of cellular organisms, the members of which resembled each other more or less, that decomposed sugar into alcohol and carbonic acid ; others, speci- fically different, would act in a different manner. In short, we may say that the number of these living organisms is a measure of the number of different ferments. Plate IV. represents in its two halves the condition of the mycoderma inni at two different and unequal periods after its submersion. In the left-hand semi-circle, it is evident that many of the figures are swollen, that modification of their protoplasm has taken place, and incipient budding is going on in several of them. A budding of this kind would not wither ; the buds would grow and, detaching themselves, would form new cells capable of budding in their turn. We should have I 2 116 STUDIES ON FERMENTATION. under our eyes all the characteristics of a yeast, which, beyond doubt, would give rise to a very active fermentation, inasmuch as it would belong to the order of phenomena of nutrition and vital energy of which we are speaking. Instead, however, of insisting upon the acceptance of our interpretations, based on a few facts merely, let us go on to accumulate facts, varying the conditions as much as possible. Our examples, taken singly, may seem insufficient to establish the theory that it will be our endeavour to substantiate, but taken together we trust that they will secure our readers' confidence. We may now, perhaps with advantage, introduce two new expressions to embody the preceding facts, by the help of which we may often shorten our subsequent explanations. Since life can continue, under certain conditions, away from contact with the oxygen of the air, and since the altered nutrition is accom- panied by a phenomenon which is of great scientific as well as industrial importance, we may divide living beings into two classes, aerobian, that is those which cannot live without air, and anaerobian, which, strictly speaking, and for a time, can do without it ; these latter would be ferments, properly so called. Again, since we can conceive, in an entire organism, some organ or even a cell capable of existing, at least momentarily, apart from the influence of the air, and endowed at a given moment with the character of a ferment, we may, in like manner, make use of the expression anaerobian cell, in opposition to a cell that is aerobian. As long ago as 1863, in our work on putrefaction, we pro- posed to adopt the preceding expressions, and since then we have had the satisfaction of seeing them used by different authors in France and other countries. One of the principal assertions in this paragraph relates to the non -transformation of mycoclerma vini into other moulds or into yeast.* For a long time, like Turpin and many other observers, although we had no belief in the transformation oi mycoclerma vini mio any * In a subsequent chapter we shall prove that j'east is likewise in- capable uf trausformatiou into mycodernui vini. ^ ,jt>' o ^ ®, / ,«5^ :J^ d I / ©' at 4oo Deyrolle del MVCODERMA ViNI FUNCTIONING AS AN ALCOHOLIC FeRMENT. — RiGHT HaLF, SHOWING Api'EAKA.NCE OF Spores just Sown ; Left Half, their Appearance after an Interval of Submerged Life. ^ @ ^ Pic art sc STUDIES ON FERMENTATION. 117 one of the common moulds, yet we did believe in its transforma- tion into alcoholic ferment. In the course of more elaborate researches, however, we at last discovered that our previous experiments had been vitiated from the same source of error which we have so often had occasion to point out as affecting the observations of our opponents, namely, the fortuitous and spontaneous introduction, unknown to the experimentalist, of germs of the very plant for whose appearance by way of transformation he is seeking. When we consider that every fermented vinous liquor, when put on draught, is liable to efflorescence, it is difficult to avoid the supposition that this efflorescence is primarily due to cells of the yeast that has caused the liquid to ferment, from which cells the liquid could not be completely freed, no matter how bright it might have been, and which come to the surface of the liquid to live after the manner of fungoid growths. "We wished to test this supposition by means of experiments. So great, however, was the resemblance between tlie forms possible to yeast and mycoderma, of which latter efflorescence is really composed, that we quite despaired of being able to solve the question by microscopical examination, that is, by observing the actual conversion of a cell of yeast into a cell of mycoderma. In order, then, to overcome that difficultj'^, we endeavoured to produce an inverse transformation — that of mycoderma into yeast. We imagined that we should doubtless obtain this result by submerging some of the efflorescence of wine or beer in a saccharine liquid well adapted to alcoholic fermentation. By submerging the mycoderma we would do away with the ordinary conditions of life in this kind of fungoid growth ; for we would thus prevent the supply of oxygen from the air, since that oxygen would always be excluded, in the most effectual manner possible, by the portion of mycoderma that would remain on the surface of the liquid, even after the submersion process ; and on the other hand, we would be subjecting our growth to the ordinary conditions of ferment life, which acts at the bottom or in the bulk of liquids fermenting. 118 STUDIES ON FERMENTATION. Our experiments were conducted in the following manner : — In some flat porcelain basins, we grew some pure mycoderma rini * on fermented liquids, such as wine or beer, or on arti- ficially vinous liquids, such as alcoholized yeast-water, taking care to boil these liquids previously to kill any germs of yeast or other organism that they might contain. The basins them- selves, as well as the plates of glass with which they were to be covered, were plunged into boiling water just before they were wanted for use. As soon as the film of mycoderma had become well developed and thick, and even wrinkled — a process requir- ing not more than two or three days during summer heat — we decanted the subjacent liquid, by means of a siphon, so as to leave the film on the bottom of the basin. We then diffused the whole mass of efflorescence in a saccharine liquid that had been boiled and afterwards cooled down in a closed vessel ; generally, we used wort or must preserved by Appert's process. After that, we emptied the mixture of saccharine liquid and efflorescence into long-necked flasks that had likewise been previously heated, as also had the funnels used in the process of transference. It seemed to us that experiments conducted with all these precautions must be free from causes of error. It was true that we were working more or less in contact with atmospheric air, but all that we had to fear for the soundness of the conclusions which we might draw was the presence of germs of alcoholic ferment, and we considered how few of these there are amongst floating particles of dust. Consequentl}^ if we succeeded in observing the advent of yeast in each of the long-necked flasks, accompanied by an active alcoholic fermentation, we thought that we might, without danger of error, admit as a fact the transformation of cells of mj^coderma into cells of yeast. Again, we thought that we should probably find in the forms of the cells of yeast which were directly derived from the cells of * We secured the purity of our mycoderma by the same means that we have already described for the procuring of spores oi peniciUium or other fungoid growths in a state of purity. STUDIES ON FERMENTATION. 119 mycoderma, a more or less elongated structure, which would be a convincing proof of the transformation that we were seeking, if, indeed, such transformation were possible. Strange to say, everything happened in a manner that seemed to realize our expectations. The saccharine worts in the flasks in which we had mixed and submerged the mycoderma, fer- mented in the course of a few days ; the yeast first appeared in elongated shapes ; lastly, we could see under the microscope that many of the cells or jointed filaments of mycoderma were inflated and presented the appearance of undoubted gradations between their natural state and that of the cells of yeast which soon formed part of the deposit in the vessels. In spite of all this, however, we were the victims of an illusion. In experiments conducted as we have just described, the yeast which appears, and which soon sets up an active alcoholic fermentation, is introduced in the first place by atmospheric air, from which germs are constantly falling either upon the film of mycoderma or upon the objects that are employed in the successive manipulations. Two peculiarities in these experiments first opened our eyes to the existence of this cause of error. We sometimes found at the bottom of the flasks in which we had submerged the efflorescence, along with the cells of mycoderma, large, spherical cells of mucor nmcedo or racemosus, ferment-cells that we shall soon learn to recognize in studying this curious fungoid growth. The existence of mucor nmcedo or racemosus, where we had only sown mycoderma mni, was to us a proof that one or more spores of that mucor had been introduced by the surround- ing air. If then, we reasoned, the air can introduce spores of mucor into our field of operations, why should it not introduce cells of yeast, especially in our laboratory ? Again, it sometimes happened that a negative result was obtained. Harassed by doubts about the reality of this transformation, which accorded so well with the physiological theory of fermentation we had been led to adopt, we repeated the experiments many times, and in some cases we failed to detect any appearance whatever of a transformation of mycoderma into yeast cells, although the 120 STUDIES ON FERMENTATION. conditions under which each of the experiments was conducted had been as similar as could be. We were at a loss to account for this inactivity in the cells of the mycoderma. Even in the most favourable cases of the sup- posed fermentation, it was evident that a host of cells of mycoderma vini did not become cells of yeast ; but how could it possibly be admitted that amongst the millions of submerged cells, none were adapted for transformation, if that transforma- tion were at all possible ? Thereupon, to find a way out of the difficulty, we resolved to modify completely the conditions of our experiments, and to apply to the research that we had in view a mode of cultivation that might completely, or nearly so, obviate the sole cause of error that WG suspected, namely, the possible fall of cells or germs of yeast during the manipulations. We secured this by the use of flasks with two tubes, the right hand one of which was closed by means of a piece of india-rubber tubing with a glass stopper, the other one being drawn out in the shape of a swan's neck. The use of these flasks, which was then new to us, permitted us to grow mycoderma and to study it under the microscope without fear of disturbance from exterior particles of dust. This time we obtained the results given in the first part of this paragraph. We no longer observed yeast or alcoholic fermentation following the submersion of the efilorescence, either in the flasks them- selves, or in the test-flasks attached to them, as represented in Fig. 19. We observed, however, that kind of alcoholic fer- mentation of which we have already spoken and which is due to the raj'coderma itself, a fermentative action that is still more instructive than the one which we thought we had determined, and certainly not less calculated to support the theory of fermentation which we have already briefly sketched. In an age when ideas involving transformation of species are so readily accepted, perhaps in consequence of their requiring no rigorous experimental work, it is not without interest to consider that, in the course of our researches upon the growths of microscopic plants in a state of purity, we once were STUDIES ON FERMENTATION. 121 inclined to believe in tlie transformation of one organism into another — the transformation of mycodernia vini or cerevisuv. into yeast, and that, on that occasion, we were altogether wrong, through having ovirselves fallen a victim to the identical source of error which confidence in our theory of germs had led us so frequently to detect as affecting the observations of others. § III. — Growth of Mycoderma Aceti in a State of Purity. The study of mycoderma aceti has not escaped the numerous causes of error which are apt to attend all observations made on microscopic organisms. This little fungus is still believed by many authors to be one of those polymorphous species capable of great modifications, according to the conditions of their culti- vation— it could be, in turns, bacterium, vibrio, yeast, &c. Respecting it, we have seen resuscitated under a modern name, in the course of the last few years, the old hypothesis of Buffon concerning organic molecules, that of Turpin concerning the punctiform globulines of barley, milk, and albumen, and the theory maintained by Dr. Pineau, of Nancy, and by Pouchet concerning proliferous pellicles* M. Bechamp, Professor in the Faculty of Medicine at Mont- pellier, disdaining to adopt the expressions which we have just used, has 'substituted for them that of microzyma, whilst adhering to the opinions and errors represented by the other expressions. This savant designates under the name of microzyma all those punctiform globulines thit are met with in most organic liquids when submitted to the microscope ; and attributes to them, with Turpin, the faculty of playing the part of ferments, as well as of transforming themselves into yeast * Buffon, Histoire de V Homme, i. viii., edition 12uio, 1778; Turpin, Ilemoires de VAcademie des Sciences, t. xvii. ; Dr. PiNEAU, Annahs des Sciences Naturelles, t. iii., 1845 ; Pouchet, Traite de la Oeneration dite Spontanee, p. 335, 1859. See also our Memoire sur les Oenerations dites Sponianees, 1862, pp. 100 and following, in which, we give a restime of some of these theories. 122 STUDIES ON FERMENTATION. and various other organisms. They are contained in milk, blood, eggs, the infusion of barley, and such like ; nay, we may even find them in chalk, and so we have the fine discovery of Microzyma cretae as a distinct species ! Those who, like ourselves, cannot see in these granulations of organic liquids ought besides things whose nature is still undetermined, term them molecidav granules, or, in reference to their Brownian movements, mobile granules. Indefinite expression is the best exponent of imperfect knowledge ; when a precise terminology is invented, without any basis of precise ideas derived from a rigid observation of facts, sooner or later the hypothetical facts disappear, but the terminology prematurely created to explain them, hangs about the Science, and, bearing an erroneous interpretation, retards rather than promotes real progress. We may here introduce a summary of Turpin's system, as given by himself. It forms a complete biogenesis, which leaves far behind it M. Bechamp's theory oi microzymata, M. Fremy^s descriptions of hemi-organism, and M. Trecul's account of the genesis of bacteria and lactic ferment : — " When a mucous substance presents nothing visible through the microscope, as, for example, gelatinous matter, dissolved gum, the white of eggs, or plant-sap, simply thickened on its way to cambmni, we call it organic matter or organizahle matter. We attribute to it the fecundating power of organic life in the simplest degree ; we consider it as material still isolated from organization. We suppose that the invisible molecules, of which this organizable matter is composed, come together, combine and serve through this association in the construction of the different elementary forms of future tissues. " May we not with greater truth believe that organizable matter is of varied origin, formed of innumerable globulines, too minute and transparent as yet to be observed by our present microscopical means, and that these globulines which are always endowed with motion and a special vital centre, are all capable, although many of them do abort, of separate develop- STUDIES ON FERMENTATION. 123 ment either into a formative element of tissue or into a mucedinous plant ? " Organizable matter may, according to its successive states of development or age, and according to the different forms it takes in the tissues, be distinguished by special names : — "1. — We may term matter organizable as long as the globulines composing it are not yet visible to microscopes of existing power. " 2. — We may speak of amorphous or (jlohuline tissue when even the globulines, previously invisible, have increased so as to be seen under the microscope, the term amorphous, or shapeless, being here applied to the association of globulines, and not to the globulines themselves. " 3. — Then we have 'vesicular tissue, when the globulines, con- tinuing to increase, have developed in such a manner as to present a mass of continuous vesicles, still empty or already containing a new generation of globulines, "4. — ljdi%t\y y^ehdiYB filamentous or tubular tissue, when the globulines, instead of vesiculating, form threads or tubes."* * The following is Turpin's application of his theory to the formation of the ferments of fruits {Memoires de V Academie, t. xyii., 1840, p. loo), where also, on p. 171 the above quotation will be found: — Ferments Produced by the Filtered Juice of the Pulp of Different Fruits — " By the word pulp we mean the soft and juicy cellular tissue of the fleshy part, mesocarp or middle layer of the pericarp of certain ripe fruits. This cellular tissue, which is very abundant in the peach and all stone-fi'uit, in the apple and pear, in the orange and grape, and similar fruits, is the same as that which forms the body of a leaf. Being in every case com- posed of a simple agglomeration of contiguous mother- vesicles, which are always filled with globulines that are more or less developed, more or less coloured, and individually endowed with a special vital centre, it is not surprising that its globulines when free and detached from the com- pound organisms to which they belong, and from association with its vegetable life, should, when placed in a suitable medium, themselves vegetate and become transformed, under these new influences, into a mucedine, with filaments and articulations. Such are the very fine, and, consequently, very transparent globulines, which, when left to them- selves in sweetened water, grow and become vesicular, producing other globuUnes in their interior, then bud, vegetate into mucedinous fila- ments, decompose sugar, and produce all the efi'ects that constitute what we term alcoholic fermentation." 124 STXTDIES ON FERMENTATION. Such are the purely hypothetical and exploded ideas which MM. Freray, Trecul, Bechamp, H. Hoffmann, Hallier, and others would revive in our own day, in opposition to a theory so clear and so well supported by facts as that of germs floating in the air, or spread over the surface of objects, as fruits, dry or green wood, and so on. M. Bechamp believes that he has discovered that another of vinegar, introduced into various saccharine liquids, in the pre- sence of carbonate of lime, generates bacteria, which, with the sugar or dregs, produce butyric, lactic, and acetic acids, and that this same mother of vinegar, without the addition of the carbon- ate of lime, " generates, on the other hand, the fine cells, which produce the normal alcoholic fermentation of cane sugar." Further, M. Bechamp advances the hypothesis that mother of vinegar is a conglomeration of microzymata, and, as he fails to see in the experiments on which he bases the conclusions which we have just given, that bacteria and ferment cells are the result of spontaneous impregnation, having no connection with the presence of mother of vinegar, on which he experimented, he arrives at this conclusion : "In the experiments which I have just described, things happened as though the microzyma, under some peculiarly favourable conditions, had been the parent both of the bacteria and the cells." * . . . . The object of the following experiments was the study of these assumed transformations of the mycoderma aceti in sac- charine liquids, in the presence and in the absence of carbonate of lime. We prepared some two-necked flasks, containing as a growing medium a liquid composed of one- third of Orleans vinegar, and two-thirds of a white wine used by vinegar-makers in Orleans. This liquid is peculiarly adapted to the development of ?>iycoc?erw(7 aceti. On December 13th, 1872, we sowed the little plant in a state * 3360HAMP, Recherches sur la Nature et VOrigine des Ferments [Annales de Chimie et de Physique, 4" serie, t. xxiii., and Oomptes rendus de VAcademie des Sciences, Oct. 23, 1871). STUDIES ON FERMENTATION. 125 of purity, by means of a piece of platinum wire, in the manner already explained in connection with propagation of other fungoid growths On December 19th a young and thin film of mycoclenna aceti covered the surface of the liquid. AVe then poured out the liquid through the right-hand tube, at the same time heating the end of the bent tube, to purify the air that passed into the flask. The whole film of mycoderma aceti re- mained adhering to the interior sides of the flask during this decanting. The question then was how to conve}^' this film of the little plant into a saccharine liquid of a particular kind. We effected this easily by the following means : After having emjDtied the flask, as just described, instead of re-closing the india-rubber nozzle on the end of the right-hand tube, we attached it to a test-flask containing the saccharine liquid on which we wished to operate. This had been previously boiled in the test-flask, and when we attached the neck of the test-flask, previously slightly drawn out and curved, to the india-rubber tube, the liquid was still very warm. We per- mitted the liquid in the test-flask to cool down, and, then, taking up the test-flask, we decanted its contents into the other flask, in which, as we have already said, the film of mycodenna aceti had been left. In this way the film became partly sub- merged, partly spread over the surface of the new liquid. Ex- periments were made with two saccharine liquids, must and wort. In the case of the latter, from December 22nd the whole surface of the liquid was covered by a film of mycoderma aceti, which even spread up the moist sides of the flask above the level of the liquid. In the case of the must, on the other hand, the plant for some time did not seem to be developing ; on December 24th, however, it was visibly spreading over the surface of the must. The following days we frequently shook up the films to separate them, and spread them over the subjacent liquid. There were no signs of alcoholic fermentation. On December 30th we introduced several grammes (50 or 60 grains) of carbonate of lime into each of the flasks, an opera- tion of little difficultv, which we effected in a manner similar to 126 STUDIES ON FERMENTATION. that just described. We substituted for the test-flask another flask — or, better still, a simple glass tube — containing carbonate of lime that had been subjected to great heat in the flask or tube, and there left to cool down. When cold, we poured the powdered carbonate of lime into the liquid in the flask, in this way avoiding the possibility of any error from the introduction with the carbonate of lime of any foreign germ. In neither case did we obtain alcoholic fermentation, nor was there any appearance of lactic fermentation, or bacteria, or vibrios, properly so called. The flasks remained in the oven, at a temperature of about 25° C. (77° F.), until the end of January, 1873, when we made a microscopical examination of their deposits, exercising greater care and precaution than we had adopted in the case of those examinations which we had made from time to time in the course of the experiment to assure our- selves of the nature of the organisms present.* The result was that we never found anything besides the mycoderma aceti, which had developed, although with great difficulty, on the sur- face of the liquids neutralized with carbonate of lime. The beaded filaments had, under these circumstances, only become a little larger than they had been in the unsweetened acid liquids. Mycoderma aceti, then, grown on sweetened acid or neutral liquids, grown in the absence or in the presence of carbonate of lime, undergoes no transformation into bacteria or vibrios or yeast, if only we operate with pure germs, free from the dust floating in the air, and from that which, unknown to the operator, may be introduced by means of the vessels and materials employed. It may be asked, do we, therefore, abso- lutely, reject the theory of the polymorphism o^ mycoderma aceti ? * We need scarcely here observe, having done so on previous occasions, that whenever we opened our flasks to obtain specimens, we made use of a fine tube, previously passed through the flame of a spirit lamp, and that we also passed this flame over the surface of the india-rubber, glass stopper, &c., to consume the organic particles of dust which floating about might introduce themselves at the moment when we opened the riirht-hand tube of the flask. STUDIES ON FERMENTATION. 127 On the contrary, we have endeavoured to prove the existence of this polymorphism again and again in a variety of ways. We have been mostly concerned with physiological polymorphism ; that is, our eflforts have been directed to ascertain if mijcoder))ia aceti might be, for example, the aerobian form of a ferment from which it differed physiologically, as, for instance, lactic ferment, which, in shape, sometimes bears a striking resemblance to mycoderma aceti. "We have not succeeded in discovering any- thing of the kind up to the present time. What, in view of the positive proofs to the contrary, we do absolutely reject in the matter of this mycoderma, is the theory of polymorphisms, advocated by M. Bechamp and other authors, which, in our judgment, can only be founded on incomplete and erroneous observations, § IV. — Growth of Mucor Racemosus in a state of Purity — Example of Life more active and lasting when removed from the influence of Air. Side by side with the facts explained in the last para- graph, the study of varieties of the genus mucor, grown in natural or artificial saccharine liquids, is of great importance to the establishment of the physiological theory of fermentation, which we shall explain later on. There is a ver}^ remarkable work on the subject of this mucedinous fungus by a German botanist, M. Bail, who, in 1857, declared that mucor mucedo caused alcoholic fermentation, and could change into ordinary yeast. The first assertion, relating to the alcoholic fermentation that this fungoid growth which is everywhere so abundant may cause, is quite correct ; the second which relates to its faculty of changing into yeast is erroneous.* * Ever since the year 1861 (see p. 92), this question of the possible transformation of the ordinary fungi, especially peniciUium and mucor mucedo, into yeast has engaged our attention. The results attained have been entirely negative ; but hitherto only the conclusions of our work have been published, some account of which was given at the meeting of the Societe PMlomatliique of March 30th, 1861. The following extract is 128 STUDIES ON FERMENTATION. On June 13th, 1872, we sowed by the help of a platinum wire in some wort, contained in two-necked flasks, A, B, and C, several of the minute sporange-bearing filaments of mncor along with the heads containing the spores. On June 14thj there was no mycelium visible to the naked eye in the liquids. from the Bulletin of the society:—" Meeting of March 30th, 1861. At this meeting a paper was read by M. Pasteur ' On the supposed changes in the form and vegetation of yeast-cells, depending on the external con- dition of their development.' It is well-known that Leuwenhoeck was the first to describe the globules of yeast, and that M. Cagnard-Latour discovered their faculty of multiplying by budding. This interesting vegetable organism has been the subject of a host of researches by chemists and botanists. The latter, from the days of Turpin and Kutziug, have almost unanimously regarded yeast as a form of develop- ment of various inferior vegetable tyjDes, especially peniciUium. The studies of this subject which seem to have won most favour during the last few years are those of MM. Wagner, Bail, Berkeley, and H. Hoffmann. The researches of these botanists seem to strengthen and confirm the original observations of Turpin and Kutzing. M. Pouchet has, quite recently, expressed the same ideas, and has determined certain points in connection with them with much precision of detail. M. Pasteur has long studied this important question, which is so intimately connected with the essential nature of yeast and with those phenomena of the polymorphism of the inferior tjqies of vegetable life, to which most of the remarkable works of M. Tulasne relate ; he has, however, arrived at results that are altogether negative, and he declares that he was unable to detect the transformation of yeast into any of the mucedines whatsoever, and, inversely, that he could never succeed in producing the smallest quantity of yeast from ordinary mucedines." These same results we communicated to the Societe Chimique of Paris, at a meeting held April 12th, 1861. Throughout the investigation of which we have just indicated the conclusions, we insisted en the necessity of cultivating the separate organisms in a state of purity in all researches relating to these inferior forms of life, if we desire to attain to sure inferences about them ; and the method of working, which we recom- mended, did not differ essentially from that adopted in the i>resent work. Since then the study of these growths has been conducted with the utmost precautions ; and other apparatus, perhaps as safe as those which we employ and better adapted than ours for the study of j^olymorphism of species, have been invented by botanists of great skill — M. de ISary, in Germany, and M. Van Tieyhem, in France. STUDIES ON FERMENTATION. 129 On June 15tli ra3'celium was very abundant, and was borne up by bubbles of gas. In addition to this there were a few scattered patches of bubbles on the surface of the liquid, showing that fermentation had commenced. On June 16th fermentation continued to show itself by the frothy state of the crusts of mycelium buoyed up by the bubbles of gas. On June 17th we attached B and C separately, as indicated in Fig. 19 (p. 101) to test-flasks, into which we transferred nearly all their contents. Some clusters of entangled filaments of mycelium remained on the surface of the liquids in the test-flasks. On June 18th a very slow fermentation commenced in the test-flasks; it continued for some days without becoming more active. A little bubble would slowly rise from the bottom of the vessel, succeeded after a short interval by another, and so on. The temperature of the oven was 24° C. (75° F.). On June 22nd we raised it to 28° C. (82° F.). The fermentation became more rapid, a constant succession of bubbles rose quickly from the bottom of the test-flasks ; still there was none of the vivacity of an alcoholic fermentation produced by yeast. On June 25th the fermentation was in much the same con- dition, if anything rather less active. On June 28th temperature 25° C. (77° F.) ; fermentation had stopped. On June 29th we raised the temperature to 27° C. (81° F.) again, and some slight revival of fermentation manifested itself. The increase in temperature, therefore, as might have been expected, exercises a considerable influence on this kind of fermentation. The vessels were then left to themselves, and during the course of three months they did not show the least sign of fermentation ; moreover, we did not observe, either on the interior walls of the empty flasks, or on the surface or through- out the body of the liquid in the test-glasses, any fungoid pro- duction or organism whatever different from mucor itself. The same observations apply to the vessel A ; in this case the K 130 STUDIES ON FERMENTATION. liquid that remained in the flask was covered with a gelatinous and frothy mycelium. On October 20th, 1872, after a lapse of three months and a half, we poured the liquid from the test-flask attached to flask C back again to that flask. The test-flask connected with flask B we left untouched alongside the other flasks to serve as a means of comparison. On October 21st, 22nd, 23rd, we observed nothing; on succeed- ing days, however, some patches of bubbles appeared on the surface of the liquid in flask C, and clusters of mycelium buoyed up by the bubbles of gas which they imprisoned. Life had resumed its course, and with life fermentation had recommenced. What had been the cause of this change in the condition of the liquid, after an absolute quiescence of three months ? There can be but one answer to this question : for in the other vessels there was no corresponding movement, or sign of life to be detected. In this vessel, however, an aeration of the plant had evi- dently taken place, consequent on the decautation and contact with the atmosphere of the flask, which communicated with the exterior air through the curved tube. This aeration had been absent or ineffective before decantation, in consequence of the great depth of liquid in the test-flask, the surface of which, too, was covered by a mass of mycelium filaments, itself effectually opposing any aeration of the liquid. Moreover, the surface of the liquid in the narrow neck of the test-flask had necessarily been covered by a layer of carbonic acid gas. We may investigate more thoroughly the influence of aeration, and its relation to the resumption of life in the mycelium of mucor, by restoring the liquid to its previous condition of depth and so cutting off" again contact with the air. For this purpose, on October 31st we decanted Once more the liquid and its deposit from the flask into the test-glass. The same evening a slight but continuous fermentation, with forma- tion of froth, appeared on the surface of the liquid in the neck of the test-glass. Fermentation although never vigorous, con- tinued the following davs, and until December 20th. STUDIES ON FERMENTATION. 131 Between December 20th and 23rd, it ceased altogether to mani- fest itself by liberation of gas. As for the flask B, during all this time it had remained quite inactive and in the same state in which it had existed since June 29th, although the oven had on several days been heated to 28° C. (82° F.). On December 23rd, 1872, wishing to assure ourselves of the state of the plant in flask B, we subjected it to the same opera- tion to which the flask C had been subjected on October 20th : that is to say, we poured the contents of the test-glass back into the connected flask, with the object of supplying the plant with oxygen. On December 24th, 25th, 26th, 27th, there was no apparent change. On December 28th bubbles of gas began to be evolved carrying up clusters of mycelium to the surface of the liquid. It was evident, therefore, that the quiescence in the test-glass attached to flask B, was solely due to deprivation of air, as had happened in the case of the test-glass attached to flask C, up to the date of October 31st. On this day, December 28th, we re- decanted the contents of the flask into the test- glass, and the following day a continuous but feeble fermentation proceeded. This lasted until January 22nd, although very sluggish in character ; it is evident that these efiects were exactly the same as those which took place in flask C* We should observe before we proceed farther, that we took * We found, after the lapse of another year, in December, 1873, that the ferment of the mucor in the test glass might still be easily revived ; that it was able to jDropagate, both in the mycelium and in the cellular form, in wort, and that it might produce a fermentation, m.ore or less active, according to the condition of aeration ; in short, that it was capable of producing all the characteristic phenomena described. By means of the method of cultivation that we employ, our study, which was continued for years, was pursued without the least fear of any foreign fungoid growths being introduced into the vessels, although they remained constantly open, and the air in them was being perpetually renewed by the action of diffusion and variations of temperature. In 1875 nothing remained alive in our flask, and further revival became impossible. K 2 132 STUDIES ON FERMENTATION. specimens from the flasks A, B, C, at difierent times between June and January, and that the microscope never revealed the least trace of yeast in them. We may note besides that, during this interval, we impregnated fresh flasks of wort with specimens taken from the deposits in the flasks A, B, C, and that we always obtained reproduction of the mucor and its peculiar fermentation without the least appearance of ordinary ferment. The inferences from the results that we have just detailed folio w readily, and are besides of great interest. In the first place, it is evident that even if the mucor mucedo may be able to produce alcoholic fermentation, it is totally incapable of changing into yeast. The two plants are necessarily and radically distinct, and, if difierent authors have succeeded in obtaining them mixed one with another in growths of mucor, this intermixture was doubtless the result of a spontaneous sowing of the j^east, the germs of which abound, particularly in the particles of dust existing in the atmosphere of any laboratory in which studies relating to fermentation are pursued. This, however, is not the most striking inference from the facts which the cultivation of these organisms revealed. The mucor is evidently a plant, at the same time aerobian and anaerobian. If we had sown the spore-bearing filaments of mucor on slices of pear, lemon, or similar fruit, we should have seen the spores germinate, tubes of mycelium ramifying on the surface of the substratum, and reproducing sporiferous aerial hyphae. In this case the plant would have eflected all its phenomena of nutrition by absorbing oxygen and emitting carbonic acid, after the manner of animals, as, in our essay on the organic corpuscles which exist in a state of suspension in the atmosphere, we have shown to be the case generally with fungoid growths. Under these circumstances, the only sugar decomposed would have been a quantity equivalent to that assimilated in forming the cellulose of the young tissues of the fungus, or in entering into combination, either with the elements of ammonia or with the sulphur of the sulphates, or STUDIES ON FERMENTATION. 133 tlie phosphorus of the phosphates, to form the albuminous sub- stances of the interior of the cells.* In this case the sugar used up would furnish no alcohol, or at least, if alcohol were formed, it would be decomposed immediately. All aerial growths take place in the same manner ; and such is the nature of nutrition and life in all the larger forms. In our flasks, on the other hand, the life of the little plant functions quite differently. Deprived of oxygen, or having at its disposal but an insufficient quantity of that gas, after a life of activity in contact with air, it can, nevertheless, live apart from the direct action of that element, and the combinations to which it gives rise. On the other hand, we see all the signs of alcoholic fermentation appear ; that is, a notable proportion of sugar, in comparison with the weight of solid matter assimi- lated and fixed by the plant, is decomposed into alcohol and carbonic acid gas ; and this decomposition continues as long as life itself continues in the cells, and they remain submerged, this last condition being effected by the decantation of the liquid and its deposit into the test-glass. Along with the disappearance of the phenomena of vital activity in the cells, the fermentation ceases absolutely, or at least is no longer visible externally, by reason of its extreme feebleness. The cells then assume an old, shrivelled, worn-out appearance, with irregular outlines and granular markings. Their life is merely suspended, however, not extinct ; for if they be supplied once more with oxygen, and suffered to exist under the influence of that gas, they will vegetate again, and become capable of pro- ducing fermentation afresh, even after having been excluded from the air for a considerable time. Oxygen then presents itself to us as being endowed with a certain determining stimulus in the matter of nutritive action enabling this action to be prolonged beyond the point where * AVe do not here take into account certain phenomena of oxidation of ■which the fungoid growths are the seat, and which remind us of those that are presented m so remarkable a degree by mycoderma vini and mycoderma aceti. 134 STUDIES ON FERMEKTATION. the direct influence of oxygen ceases. In time the energy that has been imparted to the cells will die away, and then also fermentation will cease, to be resumed, however, when the plant is once more submitted to the revivifying action of the gas. It seems as though the vital energy derived from the influence of gaseous oxygen were capable of effecting an assimilation of oxygen, not in the gaseous state, but existing in some state of combination, and hence its power of causing the decomposition of sugar. Looking at the matter in this light, it seems to us that we may discover in it a fact of general occurrence, that this peculiar action of the oxygen and the cells is to be seen in all living beings. For indeed is there any cell which, if suddenly and completely deprived of air, would perish forthwith, and absolutely ? Probably there is not a single one that would do so. With certain modifications of greater or less amount the assimilative and excretive acts which have taken place during life must be carried on after the suppression of oxygen, resulting in fermentations ordinarily obscure and feeble, but in the case of the cells of ferments, properly so called, manifesting an activity both greater in amount and more enduring. Let us now proceed to compare the weight of alcohol formed by the mucor during fermentation with the weight of the plant itself. Mrst experiment. — One of the double-necked flasks contained at starting 120 c.c. (about 4 fl. oz.) of wort. On January 2nd, 1873, we attached this flask to a test-glass, containing a deposit of mucor ferment (Fig. 19, p. 101), a few drops of which we poured into the wort in the flask, to impregnate it. On January 3rd we decanted the wort from the flask into the test-glass ; under these conditions we have seen that the wort must ferment. On January 18th the fermentation in the test-glass ceased. On July 31st, 1873, we transferred the liquid from the test-glass back to the flask. On August 4th, 1873, we again decanted this same liquid from the flask into the test- glass. On December STUDIES OX FERMENTATION. 135 25th, 1873, we once more removed the liquid from the test- glass to the flask, and allowed it to remain so until Decem- ber 23rd, 1874, on which day we submitted it to examination. It was found to contain per 100 c.c. (3| fl. oz.) Grains. Grammes.* Total weight of the fundus .. .. 5 7 .. 0'37 Absolute alcohol . . . . . . 50-9 . . 3*3 Acidity, estimated in its equivalent of sulphuric acid .. .. .. 1'7 .. 0*11 Sugar, determined by cupric solution . . 82'2 .. 5*2 Dextrine (?) 24-6 . . 1-6 The total weight of fungoid growth being 037 gramme, and the total weight of absolute alcohol for the 120 c.c. of fermented liquid being 4 grammes, we had, consequently, from ten to eleven times by weight more alcohol than fungus. Second experiment. — On June 13th, 1872, we sowed two or three sporiferous heads of nmcor in some wort contained in one. of the double-necked flasks. The temperature of our oven varied between 23° C. and 25° C. (73° F. to 77° F.) The total volume of liquid was 120 c.c, as before. June 15th, mycelium had developed, buoyed up on bubbles of gas. June 16th, patches of bubbles, due to fermeatation,, covered the surface of the liquid. June 17th, we transferred the liquid to the test-glass.. June 28th, fermentation in the test-flask had ceased. June 28th^ fermentation recommenced, the temperature of the oven being raised to 27° C. (80° F.). October 20th, the liquid was transferred back from the test- glass to the flask. October 24th, mycelium had developed, supported by big bubbles on the surface of the liquid in the flask. October 31st,. we retransferred the liquid to the test-glass. [* There are 15-43 grains in the gramme.] 136 STUDIES ON FERMENTATION. November 1st, a feeble, but contiimous fermentation com- menced. This was kept up until Januar}' 2nd, 1873, on which day we transferred the liquid, with its deposit from the test- glass to the flask, when it now seemed to be quite inert. We left it in this flask until December 24th, 1874, without its mani- festing during this long interval any sign of fermentation ; nor did the fungus appear to grow at all. We then submitted the liquid to analysis, and found in it, per 100 CO. — Grammes.* Total weight of fungoid growth . . . . 0'25 Absolute alcohol. . . . . . . . . . 3"4 Acidity, estimated in its equivalent of sulphuric acid . . ., . . . . . . . . 0'12 Siigar, determined by copper solution . . . . 62 Sugar, determined after treatment by boiling with sulphuric acid, and deduction of amount of sugar already obtained (dextrine) ? 1"0 The total weight of absolute alcohol for the 120 c.c. of fermented liquid was 4"1 grammes — that is, the weight of the alcohol was sixteen or seventeen times that of the plant. The structure of the plant differs considerably when it lives surrounded by air, and when it is more or less completely deprived of that fluid. If it has an abundance of air at its disposal, if it vegetates on the surface of a moist substance or in a liquid in which the air held in solution may be renewe<l without being incessantly displaced by carbonic acid gas, we shall see it develop as an ordinary fungoid growth, with a mycelium consisting of filaments more or less slender, branching, and entangled, sending up from the surface of the liquid aerial organs of fructification. This is the well-known form of \cge- tation of the common mucor. On the other hand, if we compel the mucor to live in a saccharine liquid with insufl&ciency of air, at least for some of its parts, the mode of vegetation will change completely, as we have seen in the case oi penicilUum, asperyilluSy [* For English equivalent see Experiment I, p. 135.] PI V 4oo Larkertauer del Picart sc MucoR, Ve(;etating Submerged, in Deficit of Air. STUDIES ON FERMENTATION. 137 and mycoderma tini when submerged, but with this difference, that in the case of the mucor the changes in question, and the activity of nutrition under these new conditions, are much more marked than in the case of those other organisms. The spores grow larger and the filaments of mycelium which do develop are much stronger than those in the normal plant. These fila- ments put forth, here and there, other filaments which detach themselves and vegetate at the side of the others, being ter- minated or interrupted by chains of large cells, species of spores which can live by budding and reproducing cells similar to themselves or by elongating into filaments. Plate V. represents the living plant submerged at a little depth, and having, consequently, still at its disposal a certain quantity of air, insufficient, however, to supply the oxygen needed for all the acts of nutrition. In this case the mucor appears very difierent, morphologically, from what it is when in free contact with air. Here it forms short filaments, having a diameter double or triple that of the filaments of the ordi- nary mycelium with branches and buds all over, and what is especially characteristic, forming a network of chains of cells, sometimes spherical, sometimes oval or pear-shaped, which are the actual spores. These, as soon as they are detached, bud in their turn, and reproduce either cells or branching tubes ; these cells or the chaplets which they form being known under the name mycelian spores or conidia. Our plate gives these different aspects very correctly, and affords us a good idea of the luxuriant state of this remarkable vegetation. Plate YI. represents the plant living at a greater depth with less air, expending, by means of sugar as source of heat, the energy which it acquired in vegetating under the influence of the oxygen of the air. The filaments are fewer and older in aspect, and the number of cellular forms is proportionately larger than in the former case, the budding giving rise by preference to spherical or oval cells. On a single cell we often see two, three, four, five, six, and even more buds. 138 STUDIES ON FERMENTATION. When the buds of the oval or spherical cells detach them- selves whilst young, they often resemble in form and size cells of ordinary j^east, nor can even considerable experience in this kind of observation always enable us to distinguish them. Hence we may easily understand how many have come to believe, with so skilful a botanist as Dr. Bail, in the transforma- tion of mucor into yeast. With the forms represented in Plates V. and VI., the plant is more of a ferment than of a fungoid growth. In such cases the weight of sugar decomposed in comparison with the weight of new cell -globules formed is very considerable, an effect which is more marked the less air the plant has at its disposal. Under the latter conditions, however, vegetation is slow and laborious, and the ferment very soon assumes an aspect of age, and we must constantly rejuvenate the cells by bringing them into contact with oxygen, and subjecting them to the action of limited quantities of that gas, and so promote their vegetation and prolong their fermentative activity. This effect we brought about when we retransferred the liquid and its deposit of mucor from the test-glass to the flask, thus bringing them into contact with fresh air. We saw cells that appeared old, dark, and highly granulated, become inflated, grow more transparent, and fill with a gelatinous protoplasm, the few granulations which they still exhibited assuming a brilliant appearance when we succeeded in distinguishing them ; and finally, a very active budding was set up. Under this reviving influence life could continue once more away from the air, although with difficulty, so that fermentation would be most intense if the large fila- ments and their conidia were constantly being removed from and to the action of air. The preceding plates show several instances of this rejuvenes- cence of the old cells of mucor ferment. We have omitted to represent amongst the old cells some cells which have their granulations collected about the centre, with an empty space between the granulations and the exterior 4oo Lacl<cpbcvuer del Picart sc Ferment of Mlcok, STUniES ON FERMENTATION. 139 borders.* In this state, cells are generally dead and incapable of any revival. It is impossible to avoid being impressed by the striking analogies which exist between all these facts and those presented by cells of yeast. In concluding our study of the vegetation of mucor as a mould and mucor as a ferment, we may again remark that the most striking analogies also exist between the preceding observations and those we have seen in the case of penicillkun as})ergillus, and mycoderma vini. These latter plants do not furnish alcohol or carbonic acid gas by direct fermentation of the sugar, as long as we let them vegetate with plenty of air at their disposal. Once submerged, however, their vital aspect changes ; on the one hand the cells or filaments of the mycelia evince a tendency to become larger ; on the other hand there is a tendency to greater closeness in these latter, and, consequently, a transition to the state of conidia. Lastly, there is a correlative budding of cells, accompanied by a * The figure given below supplies this omission. The cells that are isolated or are in chains, b.b.b., show this state of the old cells. The cells a.a.a. are younger, and may be more easily revived. We may see by the dimensions of some of these how greatly, in certain cases, the cells of mucor resemble cells of yeast ; nevertheless, in the state of the contents and the aspect of the outlines, there are always some differences sufficiently appreciable to strike the practised observer. 4(3 Fig. 23. The figures adjoining the cells indicate fractions of a millimetre. (A millimetre may be taken as gV'iiiO 140 STUDIES ON FERMENTATION'. formation of alcoliol and liberation of carbonic acid gas; in short, all the ordinary signs of alcoholic fermentation. The principal difference in the case of mucor consists in this, that the vegetation of this latter, under the conditions of insufficient aeration or none at all, is more decided, both as to extent and duration. It may be thought that all the varieties of mucor are capable of yielding the kind of ferment that we have just mentioned. But this is not the case ; and here we have another striking proof of the great physiological differences presented by forms of vegetation so intimately connected with each other that, in botanical classifications, they must be put as closel}' as possible together. Of this fact we have the most striking example in mycoderma vhn and the alcoholic ferments, properly so called, which so closely resemble each other in form and development that they might be supposed to be identical, at least, according to our present knowledge, but which differ so widely in their physiological aspects. On November 17th, 1873, we found a very beautiful specimen oi mucor muceclo on a pear, under a glass bell jar. It was a mass of perfectly straight filaments, simple and isolated, very large in comparison with those ordinarily met with, each terminating in a sporange, identical to that of mucor mucedo, and proportion- ately well developed. We are able to distinguish mucor racemosns from mucor mucedo only by the circumstance of its having on its sporange-bearing hypha) lateral branches which also terminate in sporanges. We sowed only one of the terminal heads of the large erect hyphae in some wort, in which it soon produced an abundant mycelium, but without the least appearance of gas. For a very long time, up to January 7th, 1875, we studied the developments of this organism, which remained all the time perfect!}' pure, in consequence of our having cultivated it in one of our two-necked flasks on pure wort. The total volume of liquid, which was 130 c.c, (4"57 fl. oz.) contained 2"3 grammes (35-3 grains) of alcohol. In spite of STUDIES ON FERMENTATION. 141 this rather large proportion of alcohol, a clear sign of undoubted fermentation, the plant had jdelded no conidla at all, nor any cell-globules of ferment. Some of the filaments, however, were larger than the rest, and exhibited irregularly-shaped swellings, which in some cases were of enormous size. Whilst the natural mycelial filaments, by which wx mean the vegetating part of mucor, which were supplied with abundance of air, only measured -5^ of a millimetre* in diameter, the filaments that had grown probably with an insufficiency of oxygen, and performed the functions of ferment, measured ^-f^,,, and the swellings as much as ^-f^ of a millimetre in diameter, as represented in Fig. 24. Fig. 24. In concluding this paragraph, we ma}'- mention a very able research on fermentation which we have lately studied, the * [0-000089 in., 0-00026 in. and 0 00089 in. respectively.] 142 pTtTDIES ON FERMENTATION. author of wliich, Dr. Fitz, communicated it to the Chemical Society of Berlin in 1873. In section II., page 48, this author explains his observations in a manner conformable to our own views, as may be seen from the following passage of the memoir : — " In the presence of oxygen, the ferment of miicor develops into a mycelium and consumes the sugar ; in the absence of oxygen, on the other hand, the spores develop into ferment of miicor, that buds and decomposes the sugar into the products of fermentation. " The properties of 7nncor mucedo in a fermentable liquid, in the presence or in the absence of oxygen, accord perfectly with the theory of fermentation established by Pasteur in 1861 (Compfes rendus de I'Academie des Sciences, t. Hi., p. 1260). According to this theory a fermentative fungus needs oxygen for its development ; if it finds any free oxygen it utilizes the whole of it, assimilating one part of the sugar and burning the other ; whilst in the absence of free oxygen, the fungus appropriates what it requires from the sugar." 143 CHAPTER V. The Alcoholic Ferments. § I. — On the Origin of Ferment. Amongst the productions that appear spontaneously, or, we should rather say, without direct impregnation, in organic liquids exposed to contact with the air, there is one that more particularly claims our study. It is that one which, by reason of its active energy as an agent of decomposition, has been dis- tinguished and utilized from the earliest times, and is considered as the type of ferments in general ; we mean the ferment of wine, beer, and more generally, of all fermented beverages. Yeast is that viscous sort of deposit which takes place in the vats or barrels of must or wort that is undergoing fermentation. This kind of ferment presents for consideration a ph3^sical fact of the most extraordinary character. Take a morsel of the substance and put it in sweetened water, in must, or in dough, which always contains a little sugar ; after a time, the length of which varies, a few minutes often sufficing, we see these liquids or the dough rise, so to speak. This inflation of the mass, which is due to a liberation of carbonic acid gas, may cause it to overflow the vessels containing it, if their capacity is not considerably greater than the volume of the matters fermenting. It is equally remarkable that these pheno- mena are natural and spontaneous ; that is to say that the must, the wort, and the dough are able to rise, as we have termed it, when left to themselves, without the least addition of foreign 144 STUDIES ON FERMENTATION. substances. The only difference that may occur in these pheno- mena is a certain amount of retardation, in cases where the yeast does not reach the saccharine matters in a perfectly natural form, inasmuch as it then requires a certain time to get itself together before it can begin to act. It is necessary, indeed, that sugar should be present ; for if we abstracted by some means or other from the must or dough all the sugar contained in it, without touching the other constituents, the addition of yeast would produce no gas. Every- thing would remain quiet until the moment when signs of a more or less advanced putrefaction showed themselves. Yeast is one of the most putrescible of substances, and it is worthy of notice that its alteration is also the consequence of the formation of one or more ferments, very different, however, from that of which we are speaking. As for the nature of yeast, the micro- scope has taught us what it is. That marvellous instrument, although still in its infancy, enabled Leuwenhoeck, towards the close of the 17th century, to discover that yeast is composed of a mass of cells. In 1835 Cagnard-Latour and Schwann took up Leuwenhoeck's observations, and by employing a more per- fect microscope, discovered that these same cells vegetate and multiply by a process of gemmation. Since then the physical and chemical phenomena already mentioned, such as the raising of the mass, the liberation of carbonic acid gas, and the forma- tion of alcohol, have been announced as acts probably connected with the living processes of a little cellular plant, and subsequent researches have confirmed these views. In introducing a quantity of yeast into a saccharine wort, it must be borne in mind that we are sowing a multitude of minute living cells, representing so many centres of life, capable of vegetating with extraordinary rapidity in a medium adapted to their nutrition. This phenomenon can occur at an}' tempera- ture betweeen zero and 55° C. (131° F.), although a temperature between 15° C. and 30° C. (59° F. and 86° F.) is the most favourable to its occurrence. As regards the rapidity of the budding, the following observa- STUDIES ON FERMENTATION. 145 tlons will give some idea of what it is in the case of one of the ferments of natural must. The temperature was between 12° C. and 13° C. (55° F.). "On October 12, 1861, at ten o'clock in the morning, we crushed some grapes, without filtering the juice that ran from them; afterwards, at different times during the day, we examined the juice under the microscope, until at last, although not before seven o'clock in the evening, we detected a couple of cells, as represented in Fig. 25, a. I t ^ t % \ a Fig. 25. From that time we kept these contiguous cells constanth^ in view. At 7.10 we saw them separate and remove to some little distance from each other (Fig, 25, h). Between 7 and 7.30 we saw, on each of these cells, a very minute bud originate and grow little by little. These buds developed very near the point of contact, where the disjunction had just taken place. By 7.45 the buds had increased greatly in size (Fig. 25, c). By 8 they had attained the size of the mother-cells. By 9 each cell of each couple had put forth a new bud (Fig. 25, d). We did not follow the multiplication of the cells any farther, having seen that in the course of two hours two cellules had furnished eight, including the two mother-cells. '^' * An increase like this, which would have been more rapid at a temperature between 15° and 25° (59° and 77° F.), and still more so between 25° and 30° (77° and 86° F.), may indeed seem surprising. It is really, however, nothing to what sometimes occurs. In choosing proper conditions of temperature and medium, of state and nature of yeast, it has sometimes happened that the bottom of a vessel has become covered with a white de- posit of yeast cells, in the course of not more than five or six hours * Extract from a Note which I inserted in 1862 in the Bulletin de la Societe chimique of Paris. L 146 STUDIES ON FERMENTATION. after we had sown a quantity of yeast so small as to effect no change at all in the transparency of the liquid contained in the vessel after it had been shaken up. Such a rapidity of vegeta- tion reminded us of those exotic plants which are said to grow several feet in height in the course of twenty-four hours. Budding commences in the form of a simple protuberance on the cell — a kind of little boss, as represented in Fig. 26^ No. 1. 0888 12 3 4- EiG. 26. This protuberance goes on increasing, and assumes a spherical or oval form. At the same time, there is a tendency in the points of attachment in the young cell to meet — a kind of stran- gulation occurs (Fig. 26, No. 2). The junction takes place a little sooner or later, according to the species (Fig. 26, No. 3) ; the two individuals then separate (Fig. 26, No. 4). In certain cases a single cell may give rise to several protuberances, and, consequently, to several daughter-cells. Where there is only one protuberance or bud we generally see it originate at the thick end and a little on one side of the apex of the oval outline, which, in a greater or less degree, characterizes the cells of the majority of ferments. Certain authors have maintained that the method of budding which we have just described, and which we think was first j)romulgated by Mitscherlich, is merely an illusion, and that the cells of yeast break up and scatter their granular contents, and that these scattered granules eventually attach themselves to the cells, growing there, and so giving the appearance of buds or daughter-cells. This error has been revived quite recently.* Nothing can be less admissible. We could count the number * Schiitzenberger, in his work on " Fermentation," following Dr. de Yaureal. Pans, 1875, p. 278. [See pp. 61, 62 English version in International Scientific Series (H. S. King & Co., London, 1876). This appears to be the only reference to this subject in the English copy.— D. C. E.] STUDIES OlS FERMENTATION. • 147 of yeast cells which we have seen undergo this process of rupture in the course of some ten years of ohservation, every day of which, we may say, thousands of these cells have passed under our eyes. This breaking up of the cells is really of the most rare occurrence, and may always be explained by some abnormal circumstance affecting the yeast ; being indeed a mechanical accident, not a physiological fact. We may easily convince ourselves of this by growing some yeast in a saccharine wort, filtered perfectly clear, and, consequently, deprived of all granular amorphous deposit that might deceive the observer. The cells will be observed to bud and multiply without exhibit- ing the most minute appearance of granulation, or disruption ; moreover, there will always be cells of all sizes, ranging from the smallest visible up to the largest. This very simple piece of observation may be made in all the alcoholic ferments, and with any wort capable of fermenting, and in its presence the hypothesis, which we have been repudiating, cannot hold its own. In Plate VII. (left side) there is represented a field of yeast, magnified 400 times. We see a mass of disjointed cells, such as appear after fermentations without sufficient aliment ; of the kind represented some are nearly spherical, others oval or cylindrical, more or less elongated. If we mix a little of this yeast, of about the size of a pin's head, with wort, and put the wort into a small, shallow, flat-bottomed basin, having a surface of about 1 square decimetre (10 sq. ins.) exposing it to the surrounding temperature, we shall find next day the bottom of the basin covered with a fine white deposit, of the forms of which we give a sketch in the right half of the plate. In this it will at once be observed that the cells sown have lost their interior granulations, having become more transparent and filled with a gelatinous protoplasm. The principal difi'erence between the two halves of the plate consists in this, that whereas the cells in the left half are isolated and granular, those in the right half are more inflated, more transparent, and pro- vided with budS; which may be seen in every stage of development, L 2 148 STUDIES ON FERMENTATION. from their first appearance till they become as large as the parent cells. They continue to grow until they detach themselves ; then they bud in their turn, so that the same figure may furnish examples of cells of the first, second, and third generations. In the right hdlf, the protoplasm contained in the cells exhibits circular spots or vacuoles, which may be made to appear lighter or darker than the rest of the cell by slight movements of the object-glass of the microscope. These spots are due to a migration of the protoplasm towards the sides ; they commonly occur in yeast cells the vitality of which, from deficient nourishment, has become suppressed — the shrivelled appearance which they then assume being due to their being forced to live upon themselves, so to speak. However, by introducing such cells into a nutritive and aerated liquid the vacuoles quickly disappear. In the ordinary yeast, as met with in breweries, the majority of cells show one or more of these vacuoles ; if, however, we place a little of this yeast in an aerated wort, and watch under the microscope the changes that occur in the cells, we shall witness, often in the course of a few seconds, a kind of turgescence, a greater tension of the cell- walls, which seem to grow thinner, and a complete disappearance of the vacuoles. At the same time the interior gelatinous matter will become filled with fine granulations that are scarcely visible, but which at a certain distance appear brilliant. At the same time protuberances begin to show themselves, and next day the budding will have already become very active. The newly-formed cells will have such a delicacy of aspect and contour as to be scarcely discernible in the field of the micro- scope. There will also be a tendency to ramification in the budding, which appearance will be more or less marked accord- ing to the kind of alcoholic ferments present, as we shall see presently, attaining its maximum in each case when the cells have been revived after exhaustion by rest and want of food. In the latter case, the process of rejuvenescence may be pro- tracted ; but this is not the case with cells of commercial yeast, which is always used within a few days of its formation. PI VH ^233^^ ^ 1F« ^"^t @ ^^ %?' ?5 0Q, @®^ ^ ^ ^^^ @ o, (^L^^ ^3=- ^©^^ > ^ ^r. ^ =^^- s (^ 9 Q, %:.. 4oo Bcypolle del Picart sc Yeast-cem.s — Worn out and Dissociated (i,ei-t), after Revival in a Sweet Wort (right). /tn/J t^rntf Oroj Pijrt STUDIES ON FERMENTATION. 119 And thus, as I said a little ago, speaking of these cells, they often manifest the first signs of their budding in a few seconds. In our preceding remarks we have expressly assumed that there are many kinds of alcoholic ferment. This is, beyond doubt, the case, as we have given incontestable proofs, first in 1862, in the BaUetin de la Societe chimiqne of Paris, and later on, in 1864 and 1866, in a Note in the Comptes rendus, on the diseases of wines, as well as in our " Studies on Wine." Moreover, we know that brewers have long recognized two distinct methods of fermentation — " high " fermentation and " low " fermentation — and two corresponding yeasts. It is true that the differences presented by these fermentations were believed to be caused by the different conditions under which they took place, and that it was supposed that we might change " high " yeast into " low " 3'east, or inversely, by subjecting the first to a low temperature, or the second to a high one. In our observations of 1862, which we have just mentioned, we dis- covered that must gives rise to several yeasts ; that the ferment of " high " beer cannot develop except with great difficulty in must, whilst one of the ferments of the grape grows rapidly and luxuriantly in wort ; that it is easy to isolate the smallest of the ferments of the grape from its congeners, by subjecting filtered must to fermentation ; and finally, that the secondary fermentations of wines which remain sweet furnish a remark- able ferment, very diS'erent in aspect to the ferment of beer. We have not given specific names to these different fer- ments, any more than we have to the other microscopic organisms which we have had occasion to study. This was not from any disregard for names, but from a constant fear that, since the physiological functions of these minute forms was the exclusive object of our study, we might be led to attach too much importance to exterior characters. We have often found that forms, having nothing apparently in common, belong to one and the same species, whilst similarity of form may associate species far apart. We shall give some fresh 150 STUDIES ON FERMENTATION. examples of this fact in the present paragraph. A German naturalist, Dr. Rees, who has discovered new proofs of the diversity of alcoholic ferments, putting aside, perhaps rightly, such scruples, has attached specific names to the different kinds of ferments, in his brochure published in 1870, which we have already cited (p. 71). Indeed, we have often ourselves, for brevity's sake, made use of the names proposed by Dr. Rees.* In a Note inserted in the Bulletin de la SociefS chimique de Paris, in 1862, we figured a ferment of small dimensions, which develops spontaneously in must, filtered or unfiltered, and which is very different from the ordinary ferment of wine. It is the first to make its appearance in the fermentation of the grape, and may even appear alone if the must has been pre- viously well filtered, doubtless because its germs, being smaller than those of other ferments, pass through the filter more easily and in greater number. Fig. 27, extracted from our o Fig. 27. Note of 1862, represents this ferment, together with some spherical cells of high yeast, with the object of giving a more exact idea of the relative dimensions of these two ferments and their dissimilarities. Dr. Rees has named it saccharomyces apiculatus. * The principal result of Dr. Eees' labours consists in the discovery of a sponilation peculiar to yeast cells, that is to say, to a formation in the interior of these cells, and under particular conditions — such as when the growth occurs on slices of cooked potatoes, carrots, &c. — of two, three, or four smaller cells, which, when placed in fermentable liquids, act like the germinating spores of ferments. The mother-cell may be regarded as an ascus, and the daughter-cells as ascospores, and so the genus sncchnromyces may be classified among the group of fungi termed ascomycetes. These facts have been frequently confirmed, notably by STUDIES ON FERMENTATION. 151 The same savant has given the name of saccharomyces pastoriamis to the yeast of the secondary fermentations of sweet liquids, such as wine that has remained sweet after its prin- cipal fermentation. We have described this yeast in a Note published in 1864, on the diseases of wine, from which we give the following extract : — * Fig. 28. " Fig. 6 (Fig. 28 in this work) represents a very interesting Dr. Engel, professor of the Faculty of Medicine, at Nancy. Previously to Dr. Rees' discovery, M. de Seynes {Comptes rendus, t. Ixvii., 1868) had described an endogenous formation of spores in mycoderma vini, particularly in the elongated cells, followed by the rupture of tho mother-cell, and subsequent absorption of cell-walls and other contents after the issue of the endospores, which we have just termed ascospores. We ourselves had also previously called attention to those refractive corpuscles which appear amongst vibrios as probably being reproductive corpuscles, and we had hkewise witnessed the reabsorption of the parts surrounding them. The plate on page 228 of our " Studies on the Silk- worm Disease " represents the phenomena in question. * See Comptes rendus de VAcademie des Sciences, vol. Iviii. p. 144. 152 STUDIES ON FERMENTATION. variety of alcoholic ferment. It happens pretty often, espe- cially in the Jura, where the vintage takes place about October 15th, when the season is already cold and little favourable to fermentation, that the wine is still sweet at the moment when it is put into casks. This is especially the case in good years, when the sugar is abundant and the proportion of alcohol high, a circumstance which prevents the completion of fei*- mentation when effected at a low temperature. The wine remains sweet in cask sometimes for several years, undergoing a continuous but feeble alcoholic fermentation. In such wines we have always observed the presence of this peculiar ferment. In form it consists of a principal stem, forming nodes at various points, from which short branches arise, ending in spherical or ovoid cells. These cells readily detach themselves, and act as spores of the plant. It is rarely, however, that we see so perfect a vegetation as we have represented, because the different parts fall to pieces, as we have shown in the left half of the figure." What is the origin of cellular plants of this remarkable type ? Where and how are the ferments of the grape gene- rated ? In Chapter III. § 3 we were on our way to a solution of this question. It has been shown that fermentation cannot take place in the juice of crushed grapes if the must has not come into contact and been mixed with particles of dust on the surface of the grapes, or of the woody part of the bunch. It would, however, be sufficient that a vintage vat, of any capacity whatsoever, should receive the particles of dust existing on a single bunch in some cases, on even a single grape, for the whole mass to enter into fermentation. What, then, we must ask ourselves, is the nature of these particles of dust ? On September 27th, 1872, we picked from a vine, in the neighbourhood of Arbois, a bunch of grapes, of the variety called le noirin. The bunch selected, without any injury to a single grape, was brought to our laboratory in a sheet of paper that had been previously scorched in the flame of Picart sc Fektile Mould-cells from the Outek Surface of Grapes. STUDIES ON FKKMENTATION. 153 a spirit lamp, and the grapes were cut off with a pair of fine scissors, which had also been passed through the flame. By- means of a badger-hair brush, thoroughly purified in water, each grape to which a portion of its peduncle remained attached, was washed in a little pure water. The successive washing of a dozen grapes in 3 c.c, of water was sufficient to make the water turbid ; we then examined it under the microscope. Each field contained many little organized bodies, accidentally associated, now and again, with some very scarce crystalline spicules. As a rule, the organisms consisted of simple, trans- parent, colourless cells ; some, indeed, of larger size had a yellowish brown colour, and were detached or united in irregular masses ; and, lastly, there were club-shaped or bottle- shaped vessels, full of spores ready to germinate. We repeated this experiment with bunches of other varieties of grape, and also submitted to examination water in which the outer sur- faces of gooseberries, plums, and pears had been washed ; the result was in each case the same, that is, we found a great number of the same colourless cells, and the same irregular masses of darker cells, which latter, however, we must not con- found with the masses of dead cells sometimes found covering: parts of the epidermis of certain fruits. As we had purposely left each fruit attached to part of its peduncle, we wished to ascertain if these corpuscles proceeded from the grapes or from the wood of the peduncle. For this purpose we washed separately the surface of the grapes and the woody part of the bunch. The water in which the latter was washed was visibly more charged with the minute organisms than that in which the grapes was washed, although the latter was by no means free from them. Plate VIII. represents these corpuscles as they exist on the surface of fruits, magnified 500 times. The groups, b, h, h, ... , c, c, ... are of a brown colour, more or less dark, or of a reddish yellow ; the cells a, a, ... are transparent. Amongst them are some spores of ordinary fungoid growths, and several cells which are probably the issue of a germination 154 STUDIES ON FERMENTATION. that had commenced in certain groups which have a hard, yellowish appearance, and which are provided with what seems to be a double case — b, b, b, ..., c,c, ..., a result of the moisture of the woody part of the bunch, or of rain that fell just before the commencement of our observations. It is an easy matter to trace the germination of these diflPerent varieties of cells with the microscope. We put a drop of the water in which the woody part of a bunch of grapes has been washed into a small quantity of wort, previously boiled and filtered bright. Plate IX. presents a series of developments observed in the case of simple or grouped cells. A, D, G, and J. The process is as follows : The yellowish-brown cells soften and grow larger in the nutritive medium, and gradually become almost transparent and colourless. At the same time we see some very young buds appear on their margins ; these rapidly increase in size, and detaching themselves to make room for others, move off as young cells that after a time bud in their turn. The rapidity with which these cells bud and multiply is often extraordinary. The group A and the cell D produced the groups C and F within twenty-four hours, passing through the intermediate stages represented in groups B, E. The cells A and D did not give rise to any filamentous growths, at least whilst under our observation. Some groups of cells, however, put forth, from the first, long filaments, having cross-partitions and resembling the mycelium in ordinary fungoid growths. Together with these, and along their whole length, was an abundance of cells, often in clusters, as represented by Fig. G, the whole of which growth took place in less than twenty-four hours.* But apart from contact with the air, there was a complete absence of life. The figures H, I, J, K, represent other aspects of developing cells and filaments. The cells II are spherical ; the cells I have numerous buds, as also have those marked K. These * The plates referred to in this paragraph were exhibited at a meeting of the Academy of Sciences, November 18, 1872, and commented npon by the perpetual secretary, M. Dumas. A C?/}/ B / ^^^ r^ ■^ .<y^ ^4 0 /y c/ ^■^6^° o -. G " -J C^Q Q O- ? OgP ?5^ v> ^^J"^ ^ ^§^9%^ ToUe del 5oo Various Examples of the Mode of Growth of Mould-cei.ls from the Outer Surface of Gkai'ES. STUDIES ON FERMENTATION. 155 different forms were all produced in the course of twenty- four hours by the cell which may be observed in the centre of the group J. In connection with this same group, J, we may remark that on September 30, 1872, at 10 a.m., we witnessed the detachment of three oval cells at the points a, h, c ; by 10.45 other buds of the size represented in our engraving had formed in their place ; b}' about five o'clock that same afternoon these buds, a, b, c, having become transformed into cells, fell off in their turn.* * For these observations, we employed small glass cells, whicli we made out of some St. Gobain glass by punching holes through it, and then cementing on one side one of the little glasses used for covering objects in microscopical examinations. In this manner we made small troughs, in which we placed some wort that had been boiled, and a drop of the water in which grapes had been washed. To prevent evaporation we covered the cells with a sheet of glass. We examined the liquid in these cells by inclining our microscope to the angle required, (a) Fig. 29. We also made use of cells similar to those employed by MM. Van Tieghem and Lemonnier {h) in their researches on mucorines (Fig. 30). B aIK TF" B Fig. 30. An apparatus similar to that employed by M. Duclaux in 1853 (c) would do equally well. We should be able to work with even greater facility if we employed bulbs like some which we ordered in Germany, some twelve years ago, of the well-known glass-blower, Geissler. We have heard that these bulbs now sold by that maker are much used by German microscopists. They consist of a tube blown out into a flat (a) In our essay on acetic fermentation, published in 1864, -we have already described this apparatus, -which we employed to foUow the multiplication of the jointed filaments of myco- derma aceti. See Pasteur, Etudes sur le vinaigre, p. 64, Paris, 1868. (ft) Van Tieghem and Lemonnier, Annales des Sciences naturelles, 5th series, Botanique, t. xvii. 1873. (c) Duclaux, Comptts rendus des stances de I'Academie des Sciencts, t. Ivi. p. 122i. 156 STUDIES ON FERMENTATION. It may be asked, wliat proof have we that amongst the filamentous and cellular growths which spring from the small, dark bodies existing in the particles of dust adhering to the sur- face of fruits, and which we here see bud and multiply with such marvellous rapidity, the ferment or ferments of vintage do actually exist ? A very simple experiment will prove conclu- sivel}' that this is the case. When in the course of twenty-four or forty-eight hours, by contact with saccharine must, and in presence of excess of air, the revival and development of the cells has taken place on the bottom of the little troughs employed in our observations ; if then we fill up the trough with the same must, so that there remains no free air under the cover-glass, within a very short time — an hour, half-an-hour, or often less — we shall see bubbles of gas rise from the bottom, accompanied by an increase in the deposit of cells. This will be bulb, the sides of which, in the centre, come sufficiently close together to enclose but a verj^ thin layer of liquid, and to admit of microscopical Fig. 31. examination. We may fill these tubo-bulbs completely with liquid, to the exclusion of air or we may surround the central drop with air. STUDIES ON FERMENTATION. 157 the must fermenting after the submersion of the cellular plants. It follows that the cells, or groups of cells, of a dark colour which cover the grapes, or the woody part of the clusters, are actual germs of the cells of yeast ; more correctly speaking, that germs of yeast-cells exist amongst these groups, for it would not be consistent with truth to say that the various germinating forms present in the dust on the surface of grapes must all of them give rise to actual corresponding ferments. Thus the flask-shaped spores c, c, ... in Plate VIII., are reproductive organs of alternaria tenuis, which have probably nothing in common with alcoholic ferment or ferments, properly so called, except their outward form. "VVe may repeat, however, and it is a point of great importance to bear in mind, that the cells of yeast originate from some or other of the little, brownish, organized bodies, which the microscope reveals in such numbers amongst the particles of dust existing on the surface of fruits. The impossibility, which we have already demonstrated (Chapter III., § 3), of making grape juice ferment apart from the action of external particles of dust, and the knowledge which we have just acquired, that the particles of dust on the surface of the grapes and woody peduncles, at the moment when the grapes have attained maturity, contain certain reproductive cells which give rise to certain ferments, naturally lead us to the investigation of another point, which concerns the period at which these germs make their appearance on the difierent parts of the vine plant. The two following experiments tend to prove that the ferment can only appear about the time when the grapes attain maturity, and that it disappears during the winter, not to reappear before the end of the following summer. I. In the month of October, 1873, we procured from a vine- yard in the canton of Arbois some of the woody parts of very ripe clusters of grapes, taking the precaution to cut off all the grapes, one by one, with a very clean pair of scissors, whilst still on the vine ; we then wrapped up the woody parts of the clusters, thus deprived of their grapes, in thin paper, to convey .them to Paris. Our only object at that time was to secure for 158 STUDIES OX FERMENTATION. use in our subsequent studies the ferment-bearing dust found in October on the woody part of the vine, and, more particularly, on the clusters themselves, as already stated. After our return to Paris, and during the course of our experiments in October and November, it sufficed to wash a few scraps of the bunches in a little pure water, in order to obtain the grape-ferment in abundance ; but later on in the winter we were astonished to find that the same procedure yielded no ferment, only some moulds. The bunches which, when put into boiled and filtered must, in October, very readily caused that must to ferment, at the end of winter could no longer produce the same effect, however favourable might be the temperature to which we raised the must. The particles of dust on the bunches had, therefore, become sterile, as sources of alcoholic ferments. II. On February 17th, 1875, we purchased of Chevet, a dealer in provisions, two bunches of white grapes, which were perfectly' sound, presenting not the slightest trace of injury or bruise. We took an iron pot full of mercury, which had been heated to 200° C. (392° F.), and then covered over its surface with a sheet of paper that had also been subjected to flame. When the mercury had cooled down we placed several of Chevet's grapes, singly and in small bunches, on the surface of the metal, and, after having enclosed them in a glass cylinder that had been previously heated with and by means of the mercury, we crushed them in this vessel, in contact with air, by means of a strong, crooked iron wire that had been passed through the flame of a spirit lamp. The object of all these precautions was to prevent any cause of error, such as might have resulted from the accession of particles of dust associated with the mercury, or floating about our laboratory. We then placed our cylindrical jar in an oven, at a temperature of 25° C. (77° F.) ; but though the experiment was continued for several days following, no fermentation manifested itself. At last, to assure ourselves that the pulp and liquid were, notwithstanding tliis, well adapted to fermentation, we intro- duced into the test-flask an almost imperceptible quantity STUDIES ON FERMENTATION. 159 of yeast. This readily developed, and promptly produced fermentation.* It seems possible, therefore, that the germs of ferment may not exist on bunches of sound grapes during winter, and that the well-known experiment of Gay-Lussac on the influence of air on the fermentation of the must of crushed grapes cannot succeed at all times. The following observations will aSbrd more than sufiicient proof of this statement, being, after all, but an easy method of carrying out Gay-Lussac's experiment, without having recourse to the use of mercury. It may already be inferred from the preceding facts that there must be, in the course of the year, between the end of winter and autumn, a period when the vegetation of the cellules from which yeast proceeds undergoes a revival. When does this period occur ? In other words, how long after winter does sterility of the plant continue, until it is again capable of yield- ing ferment? To ascertain this, we conducted numerous experi- ments during the summer and autumn of 1875 and the winter of 1876. Having to conduct them in a vine-growing country — in the vineyards of Arbois, Franche-Comte — at a distance from our laboratory, we were compelled to adopt a simple form * In experiments of this kind there is always a slight increase in the volume of air in the jar. This increase may be very perceptible even when the experiment made with fresh grapes, in August, for instance, causes no fermentation due to the action of yeast. After the oxygen of the air has been absorbed and replaced by carbonic acid gas, either by direct oxidation or by the action of moulds, the grapes, although crushed, act like fruits plunged into carbonic acid gas {a), and this effect is even more marked in the case of imperfectly crushed grapes. The reason is, that the crushing is never so perfect as to injure all the cells of the parenchyma. We may easily convince ourselves that the experiment on the liberation of carbonic acid gas and the formation of alcohol by grapes and fi-uits in general when plunged into carbonic acid succeeds very well in the case of fragments of fruits or grapes, and succeeds better the less the parts are crushed. (a) Spe paragraph : Fermentation in saccharin? fruits immersed in carbonic acid gas, Chap, vi , § 2, p. 266. 160 STUDIES ON FERMENTATION. of apparatus for our experiments, which, besides being very convenient, was at the same time sufficiently exact for the object we had in view. Into common test-tubes we poured some preserved must ; we then boiled it, with the object of destroying all the germs that it might contain, and then, having passed the flame of a spirit lamp over the upper sides of the tubes, we closed them with corks which had been held in the flame until they began to carbonize (Fig. 32). Having provided ourselves with a ilMMl ii'i illllliiHl rgi Fig. 32. series of tubes prepared in this manner, we carried them to a vine, and there dropped into some of them grapes, into others bunches, from which we had taken all the grapes, by cutting their peduncles ; into others, fragments of leaves or the wood of the branches. The corks were again passed through the flame and replaced successively in each tube. Some of the grapes we dropped in whole, some we crushed at the bottom of the tubes with an iron rod that had previously been passed through the flame ; others, again, at the same moment that we introduced them into the tubes, were cut open with scissors, likewise passed previously through the flame, so that a portion of their interior juice might mix with the must in the tube. Our experiments gave the following results : — As long as the grapes were green, about the end of July and during the first fortnight of August, we obtained no fermentation in our must. Between the 20th and 25th of August a few tubes underwent fermentation, by the action of the little apiculated STUDIES ON FERMENTATION. 161 ferment ; and in the course of September the number of tubes that fermented increased progressively. In each series of tubes, however, we always found a few in which there was a complete absence of fermentation. Here are a few actual examples. In the beginning of September we placed grapes in thirteen tubes, into some whole, into others crushed ones, taken from bunches of the variety known as the ploussard, the fruit being already sufficiently ripe to be very pleasant to the taste. All the tubes of this series failing to give us any trace of fermentation, or anything besides ordinary moulds — which indeed appeared in all our experiments, whether there was or was not fermentation — we began a new series of experiments, under similar conditions, on September 28th, as follows : — Nos. 1, 2, 3 and 4 tubes containing one uncrushed grape. !No. 5 tube containing two uncrushed grapes. No. 6 tube containing two crushed grapes. No. 7 tube containing two crushed grapes, in 2 c.c. of water previously boiled. No. 8 tube with a fragment of a bunch from which grapes had been cut, and occupying the entire depth of liquid. No. 9 tube with a fragment of wood from a branch. Nos. 10, 11, and 12 tubes with a fragment of leaf. On September 29th and 30th there was no appearance of fermentation in any of the flasks, but all contained flakes of fungoid mycelium. On the 1st of October fermentation more or less marked and active occurred in 2, 3, 4, and 5, in which uncrushed grapes were, accompanied by a general turbidity of the liquid, and a suspension of the development of the fungoid growths. It was still absent in 1, 6, and 7, of which the first contained an entire, the latter crushed grapes. No. 8, containing the woody part of the bunch, was in active fer- mentation. Nos. 9, 10, 11, and 12, with fragments of branch or leaves, showed no signs of fermentation. The following day No. 1 was fermenting ; but from October 5th onwards there was no alteration in the number of fermenting tubes. M 162 STUDIES ON FERMENTATION'. In this series we determined the presence of the small apiculated form of yeast (S. apicnlatus) in the tubes that fer- mented, only once finding it associated with saccharomyces pastorianus. We need hardly say that the grapes which we employed were perfectly ripe, the vintage having already commenced in some of the Jura cantons. This experiment shows that, even when the grapes are per- fectly matured, it by no means follows that each individual grape must carry germs of ferment, and that some grapes may be crushed, in some instances several together may be crushed, without being able to set up a fermentation. In the presence of these novel facts, those who support the hypothesis of the trans- formation of the albuminous matter contained in the juice of grapes into yeast will no doubt admit the untenabiKty of their opinions, since their hypothesis requires that every grape or number of grapes, when crushed, should ferment, in contact with air. On the same day we prepared another series of tubes, using grapes of a variety called the trousseau. Nos. 1, 2, 3, and 4 tubes containing one whole grape. Nos. 5 and 6 tubes containing some of the wood of a branch. No. 7 tube containing some of the wood of a branch from which the grapes had been detached. In the course of the following days fermentation took place in 4, 5, and 7. In this case three out of four of the uncrushed grapes did not cause the must in which they were placed to ferment ; whilst the same must fermented in one of the two tubes containing wood of the branch, and in the other remained unchanged ; and, lastly, the tube containing the woody peduncles of the bunch fermented. We have already remarked that it was more particularly the wood of the bunch that was charged with germs of ferment. The truth of this assertion was proved by the following series of experiments. STUDIES ON FERMENTATION. 163 On October 2nd, 1875, we charged at the vineyard twenty- four tubes, all of which were about a third filled with pure must that had been previously boiled. Nos. 1, 2, 3, 4, 5, and 6 tubes containing one crushed grape. No. 7 tube containing two crushed grapes. No. 8 tube containing one crushed grape. Nos 9, 10, 11, and 12 tubes containing some wood of a branch of the vine. Nos. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 tubes containing a fragment of the wood of a bunch from which the grapes had all been detached and removed. In the course of the following days some of these tubes began to ferment ; but in others only fungoid mycelia were visible. On October 7th the following tubes were fermenting : the second of the first eight containing whole or crushed grapes; not one of the four that contained wood of the branches ; whilst, on the other hand, of the tubes containing wood of the bunches, 15, 17, 20, 21, 22, 23, and 24 were all in full fermentation. In short, fermentation, and therefore germs of yeast, were present in one single tube out of eight containing grapes ; in none of the four tubes containing wood of the vine branch ; but in seven out of the dozen containing wood of the bunches. There was no subsequent change in the number of tubes that fer- mented. The same day that we arranged this series of tubes we prepared twenty-four other similar ones, using the wood of bunches preserved from the vintage of the preceding year. Not one of these twenty-four tubes showed the least sign of fer- mentation, although they contained grape juice in presence of the wood of the bunches ; this was a further confirmation of the sterility of the germs of ferment in the case of bunches of grapes preserved for a sufiicient time. The next question to be considered was, what length of time after the vintage do the germs on the surface of the woody part of bunches of grapes preserve the faculty of producing M 2 16 1 STllHES ON FERMEXTATIOX, yeast ? The following experiments were undertaken to dotermme this point. We have just seen that on October 2 fragments of the woody peduncles introduced with must, on the spot, caused that must to ferment in seven cases out of twelve. In order that we misrht test the wood of these same bunches durino' winter we took care to wrap some fragments up in paper previously passed through the flame. We afterwards took occasion to test portions of these fragments as follows : — On December 21st, 1875, we conducted an experiment with twelve. On the following days all began to show flakes of mycelium, or numerous multiplying cells of mycoderms, tornlcB, and dematium ; and only four subsequently produced yeast and alcoholic fermentation. From this we may con- clude that, three months after the vintage, a large number of the germs of yeast spread over the woody part of the bunches lose their vitality, through desiccation by the surrounding air, since two-thirds of the examples taken had become sterile by that time. On January 21st we conducted a similar experiment with twelve other tubes. A.t a temperature between 20° and 25° C. (68° to 77° I'.) fermentation occurred in only two of them. On March 2nd we undertook another experiment, again using twelve tubes, and again fermentation occurred in two tubes. By the beginning of April the sterility was absolute. At that period of the year (April and May) we made numerous experiments of this kind, using the woody parts of fresh bunches of grapes and white grapes preserved from the last Ajutage, plenty of which were still to be had in a state of freshness at the provision store. We also operated on some wood obtained from a vinej'ard at Meudon. In a great number of cases no fermentation occurred ; it even happened that a whole bunch of fresh black grapes, very ripe, which were bought at Chevet's on April 16th, and which had been grown in a hot- house, after having been crushed, did not ferment at all. STUDIES ON FERMENTATION, 165 Up tp March not one of the tubes containing the wood of dry bunches brought from the Jura, which had fermented, showed any signs of apiculahis or of pastorianuSj or anything besides the ordinary low yeast of wine, saccharomyceH eliipsoideus* It would be a study of much interest to determine if yeast exists on other species of plants besides the vine. During the winter we could discover it on no others. Once during the winter, experimenting on box, we obtained fermentation in one of our tubes which contained must In a great number of other experiments we obtained nothing besides moulds and growths of dematium, alternaria, and torulacc(e. Our observations in Chap. Ill § 6, taken in connection with those which we have just made, prove that the yeasts of fermentation, after being dried, preserve the faculty of germination longer than the germ-cells which are scattered over the dead wood of the vine. As might be expected, a microscopical examination of the particles of dust scattered over the surface of the fruit and woody peduncles of grapes, reveals great differences in the number of these fertile particles at different periods of the vege- tation of the grapes. As long as the grapes are green and the vine in full activity we find scarcely an}-, or, at all events, very few spores which seem to belong to ordinary fungoid growths. Towards autumn, however, when the grape is ripening and the leaves becoming yellow, fungoid growths and numerous pro- ductions of great fertility accumulate on the vine, on the leaves, the branches, and the bunches. At this period we find the water in which the grapes and the woody parts of the bunches are washed swarming with difierent kinds of organ- ized corpuscles ; it is at this period, too, that the ferment- * Dr. Eees has given the name saccharomyres elUpsoideus to the ferment of wine represented in Plates VIII. to XI. of our " Studies on Wine," which we have termed the ordinary ferment of wine, from its being the most abundant of the ferments found at the end of the fermentation that produces the wine. 166 STUDIES ON FERMENTATIOX. yielding moulds attain that phase of their vegetation in wliieh, when mixed with the juice of grapes, they produce fermenta- tion. In the Jura district a peculiar kind of wine, called straw wine {vin de imUlc), is manufactured, which seems to contra- dict what we have said as to the advent of sterility towards the end of winter in the 5'east-germs formed on the surface of preserved bunches of grapes. This sti'aw wine is made of grapes preserved for long after the vintage on straw. Fromi what we have said it might be supposed that fermentation could not occur under these circumstances. We have, in fact, no doubt that it is often really produced by quite different yeast-germs from those which cause fermentation in the vintage gathered in autumn. Fermentation as effected in the manufacture of straw wine is probably due to yeast-dust spread over the utensils of the vine-grower, and derived from the preceding vintage. We have seen (Chap. III. § 6) that yeast may be dried and reduced to powder, and yet preserve its faculty of germination for several months. It would be useful, however, to submit this surmise to the test of experi- ment, and it would be easy to do so provided we took care to crush the grapes so preserved in very clean vessels, previously heated to a temperature of 100° C. (212° F.), having first rejected every bunch containing injured grapes, which might have fermented or given occasion to the development of yeast. Fermentation, we believe, would not then take place. Another consequence results from the various facts that we have brought out in relation to the origin of the wine-ferments, which is, that it would be easy to cultivate one or more vine- stocks so that the grapes gathered from them, and crushed to extract their juice, would be unable to ferment spontaneously emn in autumn. For this purpose it would be sufficient to keep the bunches out of contact with particles of dust during the vegetation of the bunches and the ripening of the grapes, and then to effect the crushing in vessels thoroughly freed from germs of alcoholic ferments. Moreover, every fruit and PI X ioo Deypolle del Picart sc ■-•E OF THE FeKMEXTS OF AciD FrUITS AT THE COMMENCEMENT OF FERMENTATION IN ITS Natural Medium. STUDIES ON FERMENTATION. 167 every vegetable might be submitted to important investiga- tions of this kind, the results of which, in our opinion, could hardly be doubtful. The following observations, which relate to the polymor- phism of saccharomyces pasto nanus, seem to me to have an important bearing on the history of alcoholic ferments, as presenting a close analogy between the species of ferment and fungoid growths of a higher order, for example, such fungi as demaiium, which are generally found on dead wood ; and we would say that between the vine and other shrubs there is only this difference, that amongst the dematium forms of the vine there occur one or more which are anaerobian, at a certain period of the year, whilst, on the other hand, the dematia, alfernaria, &c., of other shrubs are more generally aerobian. There would be nothing surprising in this result, considering that amongst the mucors, for instance, we find both aerobian and anaerobian forms, and that there are like- wise torulae-ferments or anaerobian forms, as well as torulae- forms exclusively aerobian. When mccliaromyces pastoriamis begins to develop from its natural germs, such as are scattered over the surface of acid fruits, it takes the form of elongated jointed filaments, branch- ing, often pear-shaped, and more or less voluminous. In proportion as the oxygen held in solution in the liquid dis- appears and the buddings are repeated, the length and diameter of the filaments and cells diminish, and such is the transforma- tion that we might, at last, suppose that we were dealing with a difiierent ferment of smaller dimensions. Plate X. represents this ferment, at the commencement of fermentation in cherry juice. In the course of a short time there is nothing to be seen but cells of comparatively small size, disjointed and round or oval, and filaments comparatively short and slender. This appearance is indicated in our drawing by the cells a, a, a. As these latter forms multiply with great rapidity, we soon have to search widely over the microscopic field before we find any of the long forms from which they 168 STUDIES ON FERMENTATIOX. spring. Instead of the forms given in Plate X., we have only those represented in Plate XL In other words, the aspect of these ferments changes daily, from the very commencement of fermentation. Thus the yeast would appear to grow smaller, coincidently with the progress of fermentation passing from a condition in which it consists of large cells and long ramified filaments, to a condition in which the cells are small and the filaments short. These changes are principally due to an alteration in the method of budding and in the life-processes of the yeast, which speedily exhibits itself when the air supply is reduced, and not through any intermixture of foreign ferments. So, at least, all our observations up to the present time lead us to believe. As soon as the oxygen has been absorbed the cells which form are oval or globular, and the filaments do not lengthen or become so plump. This is, however, not the only cause of these changes in form and aspect, although the presence of air, in greater or less quantity, has a marked influence on the earlier developments of yeast ; there is another circumstance to be taken into account, difficult indeed to state shortly, but which is demonstrated clearly by the microscope, and is connected with the actual state of the germ cells. As a general rule the budding of a cell is not an identical process when the cell is quite young, and when it has become exhausted from want of nourishment. Between these two conditions there is a difierence which may be compared with that which exists, for example, between a newly- formed grain which would not germinate, and the same grain matured by rest, if we may use the expression, that is, which has been kept long enough for its germination to be possible. In other words, and as far as our subject is concerned, we are not to expect that, by reviving our old yeast cells and putting them to grow with abundance of air, in a saccharine nutritive medium, we shall obtain the appearance of the earlier developments of the germ-cells on the surface of sweet and acid fruits. We see this clearly in Plate VII., the right-hand half of which represents the recruited budding of cells, such as those «<:^=s. .■^^' ■s^. ^ OnB O' QV ^ Cb w £/ .>^^. ,eO ii? (5[ K^ ^^ ■^ fi ^r-Cr-, CSE^ v/ Ccaai^ ,(? !^/? 400 E.Helle sc Saccharomyces Pastorianus, in course ok Regular Growth. STUDIES ON FERMENTATIOl'f. 1C9 represented in the left-hand half, in a medium peculiarly adapted to their vitality, and in the presence of much air. As regards the length and size of filaments and cells, there is little appreciable difference between the two sides. The principal difference consists in the relative freshness and the budding going on in individuals in the right-hand half. There is a simple means of transforming the small, disjointed forms of the yeast as it occurs in a deposit, at the end of a fermentation, back into the long, tubular, pear-shaped forms peculiar to the germination of the germ-cells, which exist amongst the particles of dust spread over the surface of fruits. Plate X. illustrates the result of the process. For this purpose we must eflfect as complete an exhaustion as possible of the ferment saccharomyces pastorianus, by leaving it to itself for a very long time, without aliment, in contact with pure air, in a damp state ; or, better still, in presence of sweetened water. We cultivate some yeast in wort, in one of our two-necked flasks, and then carefully decanting the fermented liquid through the right-hand neck, leave the deposit of yeast on the sides of the flask. The glass stopper which closes the india- rubber tube must be replaced, and the moist yeast be left thus, in contact with pure air. The cells will steadily continue their activity, and so gradually age, without meanwhile losing their vitality. We use the word age, as we have already observed, because the period of rejuvenescence in the case of such a yeast is so much the slower the longer the plant has remained in that state. Under these conditions the yeast rarely dies. It becomes attenuated and shrivelled but still preserves its vitality, that is, the power of reproducing itself after a lapse of several months or even several years. In the end, however, it dies, a fact which is proved by the cells, when sown in a nutri- tive medium, remaining inert. To exhaust yeast, without destroying it, sweetened water is preferable. Having decanted the beer, we substitute in its place water sweetened with 10 per cent, of pure sugar. By 170 STUDIES ON FERMENTATION. effecting tlie substitution in the following manner, we escape the risk of introducing germs from floating particles of dust, which would nullify all experiments of this kind. We prepare, then, a flask containing sweetened water, free from all foreign germs, which we attach to the other flask \_i.e., two of M. Pasteur's flasks with two necks, one straight and wide, the other bent and narrow (Fig. 8)]. This is done by taking the india-rubber tube off the flask containing j'east, and removing the glass stopper from the other india-rubber tube attached to the flask containing the sweetened water ; then, introducing the right-hand neck of the yeast flask into the india-rubber tube connected with the other, we raise the latter flask so as to pour the sweetened water on to the yeast. At the same time an assistant passes the flame of a spirit-lamp over the bent part of the curved tube attached to the water flask, with the object of destroying the vitality" of the germs in the floating particles of dust, which enter the flask in proportion as it is emptied into the other. The sweetened water, which is thus brought into contact with yeast of greater or less freshness, soon begins to ferment. Fermentation accomplished, the vinous liquid is decanted and replaced by fresh sweetened water, which ferments in its turn, although even at this stage with greater difficulty than the first ; this second dose is again decanted, and again replaced by fresh sweetened water, and this process is repeated three or four times. The yeast becomes weaker and weaker, and eventually is unable to cause any fermentation in sweetened water poured on it. This exhaustion of yeast in sweetened water may be produced more quickly by the following means : — It is sufficient to sow a mere trace of pure yeast in a large quantity of sweetened water, say 100 c.c. (nearly four fluid ounces), that is, instead of pour- ing the contents of a bottle of sweetened water upon the whole deposit of yeast in the flask which contained the fermented wort, we simply take a little yeast, by means of a fine tube, from the deposit at the bottom of the flask, and introduce it into the flask of sweetened water. This large proportion of STUDIES ON FERMENTATION. 171 liquid is itself sufficient to exhaust the small quantity of yeast, quickly checking the feeble fermentation which it had induced, so feeble indeed as frequently not to be detected by the eye, from the fact that the amount of liquid present is more than sufficient to dissolve any bubbles of carbonic acid gas that might otherwise have been liberated. It is a remarkable fact that the yeast, which during its pro- tracted stay in the sweetened water becomes enfeebled to such a degree that it can no longer excite the least fermentation in that water, but will remain in its presence for an indefinite time in a state of inert dust, does not die. In some of my experiments the yeast has remained alive in the sweetened water for more than two years.* It is almost unnecessary to point out that these results are altogether out of keeping with the various properties that are usuallj^ attributed to j^east.f In these experiments we may use yeast-water J instead of water sweetened with sugar. Into some flasks of pure yeast- water we put a little yeast, taking all precautions to prevent the introduction of foreign germs. No fermentation results, there being no sugar present ; the yeast, however, begins to bud, and this budding is more or less marked according to the quantity * The alcoliolic ferments in general, subjected to these weakening influences, have not all the same power of resistance. That one which seems to possess this power in the highest degree is the saccharomyces pastorianus, which ferment we had in view in writing the above. t The term exhaustion {epuisement), which we have just used, was, perhaps, not altogether felicitously chosen. No doubt wo exhaust the cells of yeast when we sow an imponderable weight of them in a large quantity of sweetened water ; it might, however, be better to say that in such a case we adopt a particular method of preserving the vitality of the cells, without suffering them to die of exhaustion, or to multiply by budding. We may remark that the yeast, in this case, exists in a state of latent life, which resembles that of cells on the surface of fruit. The cells on the surface of fruits, bunches, or barks, can no more find around them sufficient aliment for their propagation than can our yeast-cells in a great excess of sweetened water. We would not, however, say of the spores on the surface of fruits, or their woods, that they are in a state of exhaustion ; the term would be misapplied. X See foot note p. 79.— D. C. R. 172 STUDIES ON FERMENTATION. of carbohydrate food which we introduce along with the specimen. An interior chemical action also goes on, causing a gradual change in the aspect of the yeast. The plasma of the cells collects about the centres, assuming a yellowish-brown colour, becoming granular, and forming within the cells masses more or less irregular in shape, very rarely spherical. We may observe here that these conditions seem to be peculiarly adapted to show the character of the interior sporula- tion of the cells discovered by Dr. Rees. Notwithstanding this we have never succeeded in finding it distinctly, under these circumstances. The fact which should claim all our attention, we repeat, is, that this exhausted, shrivelled-up, aged-looking yeast preserves its faculty of germination for several years ; that, moreover, this faculty may be aroused by placing it in aerated nutritive media, in which case it will exhibit all the peculiarities which, under similar conditions, characterize some of the germ-cells found on the surface of our sweet domestic fruits. In other words, this yeast, instead of multiplying, as it always does in the course of several growths in saccharine musts, in the form of cells which detach themselves readily as soon as they have nearly attained the form and size of the mother-cells, begins to shoot out into such beautiful forms as those of dematiain pullulans, producing like that ferment long, well-grown, branch- ing filaments, as well as plump and frequently pyriform cells, as represented in Plate X. The following figures (33 to 37) and descriptions of the ob- servations to which they relate will furnish fresh proofs of our assertions. In these figures we see saccharomyces jmstorianus, which has been exhausted in sweetened water or in yeast-water, undergo revival in saccharine musts, give rise to elongated, branching, pear-shaped forms, such as belong to the original ferments of fruits, and afterwards assume the most minute forms that we find in fermentations progressing or completed. Let us examine Fig. 33. The history of this growth is as follows : — STUDIES ON FERMENTATION. 173 Some spontaneous yeast which, after repeated cultivation, had acquired the aspect represented in Plate XI. — which aspect the saccharomyces pastorianus generally assumes under these circum- stances— was exhausted in sweetened water, and subsequently revived in must at 10° C. to 11° C. (51° F.). At this tempera- ture germination was not very marked before the end of eight days ; at a temperature of 20° C. (68° F.), it only took three days, under similar conditions. The sketch includes but one EiG. 33. of the long branches from which the ferment cells and the budding joints took rise, but there were a great number more. Some of the forms represented in the figure bear a striking resemblance, it appears to us, to some of those of dematium, in Plate IX. ; and even we may trace out the several peculiarities of form which distinguish the figures in the latter plate. The next figure (34) represents the earliest forms of ger- mination of another speciuien of saccharomyces jjastorianus in 174 STUDIES ON FERMENTATION. wort, after it hud been exhausted by four successive growths in sweetened water. We here see the large ferment-form which appears at the commencement of fermentation, in acid fruits, such as cherries and gooseberries (Plate IX.), associated with smaller forms, which follow it and emanate from it, in proportion as the process of budding is repeated. The field was covered with this minute form, and we had to search about considerably before we could find any of the large cells and the long, branching, jointed filaments which we have sketched. The reason of this was, that these large, extended 0. ^.^^Q -% Fig. 34. filaments only appear at the beginning, when there is still an abundance of air, giving place, after repeated budding, to minute cells or short filaments, the ever-increasing number of which soon hides the others from sight. \% 0 a i ^ Fig. 35. Fig. 35 represents saccharomyces pastoriamis again, as it appears after having been exhausted by two years preserva- STUDIES ON FERMENTATION. 175 tion in yeast-water, in contact with pure air. Strange to say, it has lost its elongated appearance, and would appear to have originated from a round ferment. The cells are much exhausted, and most of them seem to have a double border ; their interior is very granular and of a yellowish colour. One might readily take the specimen to be a dead old ferment, which, however, it by no means is. Fig. 36 represents the germination of this ferment, which had previously been revived in a flask of wort, at the tempera- ture of the air, in May, 1875. The following are the details of our observation : — We sowed a trace of the exhausted yeast (Fig. 35) in a flask of wort on May 16th. The sketch (Fig. 36) was made on May 19th, but on the 18th there was a sensible revival. It will be seen how much the little ferment had developed in the course of three days from the time when the process com- menced. If we had waited a few days longer before taking our sample, we should probably have had difiiculty in finding any cells or filaments of the large ferment form, as there would have been so few of them in comparison with the others. Co Fig. 36. In the above figure we should remark the chain of large cells and long-jointed processes, a, h, c, d : d is one of the cells that we sowed ; it has become transparent, and its contents, which are slightly granular, have lost their brownish tint ; c is a large cell which sprang from the preceding one ; its outline 176 STUDIES ON FERMENTATION. is clear, and it is full of fine, yellowish granules, which present a perfect resemblance to the large ferment-cells of fruits, pro- ceeding from the germ-cells on the surface of those fruits, when it begins to appear in sweet juices ; 6 is a long filament, sprung from the preceding cell ; and, last of all, a is a joint and its hud, in which the border is not yet very clearly defined ; it has scarcely any granules, and is finer than the others, be- longing, in short, to the small ferment form represented in Plate XI. Here, then, we see the transition of the large ferment to the small, on the same branch, after two generations from the germination of the germ-cell d. This observation cor- roborates the opinion maintained by us, that in Figs. 33, 31, 36, as in Plate X., we have not a mixture of two ferments, the one consisting of large, elongated filaments, the other of small cells, but one and the same ferment, the difierences in the form and size of which depend on particular conditions. The smallest ferment-form very soon becomes the only one visible, and it preserves its peculiar appearance in successive growths from inability to return to the full, elongated, filamentous forms before undergoing a prolonged exhaustion. The ferment of miicor would probably afibrd similar indications : it would be very interesting to find out. The following is one of the most curious of the forms pre- sented by saccharomijces pastorianus, occurring after exhaustion in a sweet mineral liquid. The ferment, taken from a closed vat, in which it had been used for beer, was sown in the mineral liquid on July 4th, 1873. The following days the ferment developed feebly, but perceptibly, and gradually^ in- creased in bulk. The flask was left to itself in an oven at 25° C, (77° F.) until December 3rd, when we ascertained that all the sugar had fermented. We then sowed a trace of the deposit, which had become abundant, in a flask of pure wort. On December 4th there was no perceptible change. On December 5th, however, fermentation was in active progress ; a large quantity of froth covered the surface of the liquid, and a considerable deposit of ferment had already taken STUDIES ON FERMENTATION. 177 place at the bottom. We made a microscopical exarainatioii of this deposit, a sketch of which we append (Fig. 37). The dark, double-bordered cells are those which were sown but did not rejuvenesce. We may notice in different places several 1 Div. = iio^li of millimetre (tT'jTo*^ of in.). Fig. 37. of these same cells, recognizable by their granular contents, which they are beginning to lose^ to make room for ger- minating cells and joints, often numerous. For instance, in the group at the bottom of our figure one of the cells is in course of rejuvenescence and germination, and has given rise to no fewer than six cells, filaments, or groups of filaments. In different fields of our microscope we met with a crowd of branches, more or less ramified, and chains of cells, of greater or less length, of which we have sketched a few. In propor- tion as the budding of these branches is repeated, the cells and joints become more readily disunited, grow small, and assume the appearance of saccharomyces pastorianus in ordinary N 178 STUDIES ON FERMENTATION. growths, almost as represented in Plate XI. At first, when the old, exhausted cells begin to germinate, their appearance rather resembles that of dcmatiumj)uUulanH, as seen in the germination of many of the corpuscles on the surface of clusters of grapes or fruits, or their woody parts, some specimens of which are to be found in Plate IX. We may briefly summarize the leading facts demonstrated in the above paragraph. We have seen that there are different alcoholic ferments. In the fermentation of natural saccharine juices, which, especially when acid, so readily undergo a decided alcoholic fermentation, the ferments originate in certain germ-cells, which are spread in the form of minute spherical bodies of a yellow or brown colour, isolated or in groups, over the exterior surface of the epidermis of the plant, and which are gifted with an extraordinary power of budding with ease and rapidity in fermentable liquids. The presence of atmospheric oxygen is indispensable to the germination of these germ-cells, a fact which explains Gay-Lussac's obser- vation that atmospheric oxygen is necessary for the com- mencement of spontaneous fermentation in must.* Of these * M. Bechatnp {Comptes rendus, November IStli, 1872) asserts that the air has no direct influence on the piroduction of ferment or on the process of alcoholic fermentation. That experienced chemist deduces this erroneous assertion from experiments on sweetened water, to which bunches of grapes, petals of corn-poppies and petals of rohinia pseudo-acacia had been added. As may be seen in our " Studies on Wine " (p. 7, 1st edition, 18G6), these experiments conducted by M. Bechamp in 1872 were merely a reproduction of those made long before with vine leaves, petals of elder-flowers, leaves of sorrel, &c., by the Marquis de Bullion, Fabroni, and other experimentalists. M. Bechamp has modified his later experi- ments by not adding the bunches of grapes, leaves, &c., to the sweetened water before having introduced carbonic acid gas into the liquid. Fer- mentation having still taken place in spite of this change, M. Bechamp wrongly concluded that air has no direct influence on the prod nction of yeast on an alcoholic fermentation. The introduction of the carbonic acid gas could not remove all the air imparted to the sweetened water by the objects placed in it, and it was this air which remained adhering to these objects that permitted the production of fermentation. We may avail ourselves of the opportunity here presented to add that, in this same STUDIES ON FERMENTATIO>f. 179 various ferments one deserves special mention — namely, the variety termed saccharomi/ces pastorianus. As is the case with all ferments, when we gather it from the deposits produced in must that has been fermented by its action, it is composed entirely of oval or spherical cells or of short joints. When again placed in a similar must it buds, like all the ordinary ferments, and the buds detach themselves from the joints or mother-cells as soon as they have attained the size of these latter, from which time in the new deposit is reproduced the original ferment-form from which it sprung, and so on. Under certain conditions of exhaustion, however, which may be easily obtained, and which we have already accurately described, the cells undergo an absolute change as regards their capabilities of budding and germinating. Each cell, modified in its structure by the conditions we have mentioned, shows a tendency to shoot out all around its surface, with astonishing rapidity, into a mul- titude of buds, from many of which spring branching chains, covered in parts, and more especially at the internodes, with cells and jointed filaments, which fall off and bud in their turn, soon to present the forms of the yeast deposit. In this way saccharomyces pastorianus seems to afford a kind of bond of union between the race of ferments on the one hand, and certain kinds of ordinary fungoid growths on the other. Of these latter the plant which De Bary has named dematium, and which is generally found on the surface of leaves or dead wood, more especially, however, on the wood of the vine at the end of autumn, the time of the vintage, presents a striking example. There seems every reason to believe that at this period of the year one or more of the varieties of dematium furnish cells of yeast, or even that the ordinary aerobian varieties of Note of November, 1872, M. Bechamp commences by making various assertions concerning the forms assumed by cells of the alcoholic ferment of the grape when in process of fermentation. This question was dis- cussed by us ten years before, and our conclusions supported by sketches, in a Note which appeared in the Bulletin de la Societe chimique de Paris, for 1862. N 2 180 STUDIES ON FERMENTATION. dematium produce at a certain stage of their vegetation, in addition to aerobian cells and torulae, other cells and torulae which are anatirobian, that is, alcoholic ferments. In this manner we arrive at the confirmation of an idea entertained by most authors who have studied yeast closely — namely, that it must be an organ detached from some more complex vegetable form. We may also add that in the case of saccharomyces the chains of filaments, both tubular and fusiform, and septate cells more or less pyriform originating in them, when attentively observed, remind us forcibly of the filamentous chains and spoi-e-balls, or conidia of mucor racemosus when submerged, so that one might suppose that the spore-ferment of our dematium is itself an organ detached from some still more complex vegetable form, in the same way that conidia-ferment of mucor racemosus belongs to that more complex fungoid growth. In the following passage De Bary uses, for the first time, the words dematium puUulans (Hofmeister, vol. ii. p. 182, 1866). The German naturalist begins by citing the opinions of Bail, Berkeley, and H. Hofimann, the first of whom main- tains that mucor mucedo becomes transformed into the yeast of beer, the second that yeast is a peculiar state of penicillium, and the third that it may be generated by fungi of ver}- difierent nature, and especially by pcjiicil/ium glaucum and mucor mucedo. He goes on to say : " I have taken great pains to repeat the experiments of Bail, Berkeley, and II. Ilofi'mann, but I have never been able to confirm the results which they have stated, either in the case of growths in micro- scopic cells or in experiments performed in test-tubes with the pxirest possible substances — specially prepared solutions or must of wine and spores of penicillium, mucor mucedo, hotrylis cinerca, &c." On this point M. De Bary arrives at exactly the same results which we communicated to the Societe Philomathique and the Socieie Chimique of Paris, as already given in Chap. IV. § 4, p. 128, note. M. De Bary goes on to say : '' In researches of this kind STUDIES ON FERMENTATIOX. 181 it is difficult to eliminate two sources of error. On the one hand, it is beyond doubt that cells of ferment are actually scattered over everything, and that, consequently, they may easily get into the experimental liquid along with the spores that we sow, and so occasion mistakes.* On the other hand, there are a great many fungi which develop budding pro- cesses similar to yeast, but incapable of producing fermen- tation, which yet in some cases spring directly from spores as well as from mycelium, especially -we may instance exo- ascus. This last observation is especially applicable to the extraordinarily numerous variety of fungi which rank under the Dematiei and Sphaeriacei, and which I shall term, for convenience of naming, dematium pullulatn. We shall conclude this paragraph with a remark that has doubtless presented itself to the minds of our readers, which is, that it would be impossible to carry out the experiments we have described if we could not make sure of dealing: with pure ferments, or, at least, with mixtures the components of which are sufficiently well known for us to assign to each the effect produced by it in the total phenomena observed. It would be extremely difficult to continue growths of yeast- deposit in sweetened water or in a moist atmosphere if the little plant were mixed with spores of other fungoid growths, a variety of ferment-forms, and germs of bacteria, vibrios, or * The gei-ms of ferments are less widely diffused thau M. de Bary sup- poses, as may be seen from our observations in Chap. III. See, too, our Memoir of 1862, 8ur Its Oenerations dites Spontanees, p. 49. It is only iu a laboratory devoted to researches on fermentation, or places such as vaults, cellars, and breweries, that the air holds appreciably in suspension cells of ferments, ready to germinate in saccharine media. If we except these particular circumstances, ferment is not very largely diffused, save on the surface of fruits and the wood of the trees which bear them, and perhaps, also, on some other plants. The particles of dust held in suspension in any atmosphere whatever rarely produce fermentation in pure must even when we take all possible precautions, so that the action be not overlooked ; for true fermentation may be hidden by fungoid growths, when there is much air and but a small quantity of saccharine liquid present. 182 STUDIES ON FERMENTATION. infusoria in general. All these foreign organisms would tend to develop just in proportion as the conditions of the media were more or less favourable to their growth, and, in a very few days, our flasks would be filled with swarms of beings which, in most cases, would entirely conceal the facts relating to those forms, the separate study of which it was our object to follow out. We shall have occasion, therefore, to examine, in a subsequent paragraph, the preparation of ferments in a state of purity. At present we may state that yeast, which in its ordinary condition is a mass of cells so liable to change that its preservation in a moist state is impossible, manifesting in the course of a few days during the winter, and in twenty-four hours during the heat of summer, all the signs of incipient putrefaction, thereby losing its distinc- tive characteristics, is nevertheless capable, when pure, of enduring the highest atmospheric temperatures for whole years without showing the least signs of putrid change or con- tamination with any other microscopic organisms, and without the Cells losing their power of reproduction. In the pre- sence of facts like these, the theory of spontaneous generation must seem chimerical. The hypothesis of the possible trans- formation of yeast into penicilliiim g/nucum, bacteria, and vibrios, or conversely, which the theories of Turpin, H. Hoffmann, Berkeley, Trecul, Hallier, and Bechamp involve, is equally refuted by these facts. ^ II. — On " Spontaneous " Ferment. The expression spontaneous ferment may be applied to any ferment that appears in a fermentable liquid without having been purposely sown in it. In this respect the ferments men- tioned in the preceding paragraph, those of all saccharine juices of fruit which ferment when left to themselves — the ferments of wine, for example — are spontaneous. The term, however, is not altogether appropriate, because, after all, the process is the same as if an actual sowing had been made, since, STUDIES ON FERMENTATION. 183 as we have showu, it is absolutely necessary for the juice to come into contact with the surfaces of the fruit, so that the ferment may be mixed with it, and so produce subsequent fermentation. Therefore, although we may apply the term spontaneous ferment to the ferments of fruits, we intend that expression to apply in this paragraph solely to those ferments that are generated in a saccharine liquid, in which, by previous boiling, we have destroyed all ferment germs, and which, nevertheless, enters into fermentation after being exposed in free contact with air. In such a case it is entirely from the particles of dust floating in the air that the ferment germs that appear in the liquid are derived. Such are typical spontaneous fermentations, and it is of the ferment so obtained that we are about to speak. In the course of the researches which we undertook in order to ascertain whether mycodei'nia vini, or vinous efflorescence, became transformed, in the case of beer, into actual alcoholic fer- ment— researches which were the more protracted and varied in consequence of their leading to the condemnation as erroneous, on the faith of new and more precise experiments, such as those given in Chap. IV. § 2, of that transformation, in which we had for long believed — we had occasion to observe several spontaneous fermentations of this kind in various saccharine liquids. We then proceeded to describe our method of con- ducting the experiments. Having brought about the develop- ment of a film of mycoderma vini or cerevisicB on the surface of a liquid, fermented or not, we submerged that film in wort, which we afterwards put into long-necked flasks, in which alcoholic fermentation generally took place in the course of a few days. This fermentation in no way resulted from the trans- formation of the cells constituting the efflorescence into ferment. The mycodermic film merely acted as a receptacle of true ferment germs, wafted thither with the particles of dust floating in the air of the laboratory, which germs developed in the liquid into actual alcoholic ferments amongst the cells of the submerged mycoderma. By conducting experiments in this manner we 184 STUDIES ON FERMENTATION. brought about several spontaneous fermentations, the germs of which could have been introduced by notliing but the particles of dust in the air. These fermentations, which we were obliged to follow very carefully with the microscope from the time when they first manifested themselves, on account of the transformation that we were seeking, which transformation we thought might possibly be that of the cells of mycoderma vini into cells of ferment, generally gave us during the first days of fermentation the large, elongated, branch}'- ferment repre- sented in Plate X., which was succeeded by the small ferment represented in Plate XI.* Here let me describe one of these experiments. In the begin- ning of March, 1872, we grew some mycoderma vini, obtained from wine, on some wort contained in a shallow basin. On March 6th we submerged the efilorescence and put it all together, liquid and film, into a long-necked flask. On March 9th we detected in- cipient fermentation, and on March 12th we took a sketch of the yeast of the deposit,