Chapter V

. we shall return to observations on this subject.

§ IV.—Wort and Must Exposed to Common Air.

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 fermentation, he introduced some bubbles of oxygen, and fermentation showed itself the following day. Gay-Lussac concluded that the fermentation of must could not commence without the help of oxygen.[38]

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. Trécul 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 alcoholic fermentation often takes place in liquids that are not exposed to contact with air; but we shall prove by experiment that, notwithstanding what may happen during 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 _spontaneous_ generation have based most of the objections which they vainly urge against their opponents upon erroneous interpretations of certain facts relating to the spontaneous impregnation of organic infusions. Taking a very wrong view of the essential conditions of the phenomena, they require that the assertors of the diffusion of the germs of microscopic organisms should be compelled 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 mountains, and so on. “Fermentations,” said one of these gentlemen 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 explanation 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 germs adhering to the vessels and utensils used in experiments, the origin of which may be altogether special, is erroneously imputed to the dust-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 exist 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 fermentation, if we may use such a term, in the atmospheric air.[39]

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, 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 correctly 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, nevertheless, 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 laboratory 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 endeavoured to repeat this experiment of Gay-Lussac’s in such a manner as we believed would confirm our views, by varying the conditions in such a way as would cause it to succeed or fail, according to the circumstances of the manipulation employed.

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 laboratory. 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 washed, 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 immediately 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 we have recently expressed, and with the principles that we have maintained for nearly twenty years, on the subject of the causes of change in organic liquids.

It is by no means our intention to assert that in the atmospheric air there exist no germs of ferment in a state of suspension, 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 off 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 experiments on alcoholic fermentation.

_1st 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.

_2nd bottle._—May 7th, particles of fungoid growth on the surface of the liquid, and also a veil of _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 _vibrios_.

In this case, alcoholic ferments had come into existence, and 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 other organisms which made their appearance—the fungoid growths, _mycoderma 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 alcoholic 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 experiment.

The same day, May 2nd, 1873, we uncorked, with the precautions 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 experiments relating to fermentation were seldom conducted.

_1st bottle._—On May 8th we observed on the surface of the liquid large, frothy pieces of mycelium (_mucor mucedo_ or _mucor racemosus_). The liquid was perfectly bright.[40]

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 _penicillium glaucum_ upon it. No signs of fermentation.

_3rd bottle._—May 8th, patches of fungoid growth on the surface of the liquid. May 30th, thick and abundant fungoid growth, but no fermentation.

_4th 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 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 precautions, 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 appeared 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 where one is perpetually dealing with that kind of growth.[41] If we conduct our experiments with exactness, we very soon learn that all that has been written on the facility with which saccharine musts may be made to ferment, by being rapidly brought into contact with the surrounding air, is greatly exaggerated.

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 with 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 laboratory.

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 that we were obliged to open the neck to avoid an explosion. We append a sketch of its ferment (Fig. 10).

[Illustration: 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 considerable 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 water and then cooled, necks downwards. The beak of the basin, by means of which the liquid was 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.).[42]

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 observations which we made on the liquids and their deposits.

On June 1st the liquid in one of the six flasks which had begun to ferment was covered with a continuous scum of _mycoderma vini_, below which appeared a filamentous network belonging 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, consisting 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. 11).

_aaa._—Thick cells of alcoholic ferment, the size of which is given in our sketch: thus, 13/450 indicates that the corresponding figure is 13/450 millimetre in length (rather more than 1/1000 inch).

_bbb._—Small alcoholic ferment, such as we generally see in the must of acid and sugared fruits—especially in filtered grape must. Its dimensions varied from 1 to 1-½, or 2, 450ths of a millimetre.[43]

[Illustration: Fig. 11.]

_ccc._—“Low” yeast, of a type resembling that existing in other preparations fermenting in the room.

_ddd._—Enlarged, distended spores of _mucor racemosus_. 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.

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 atmosphere 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 abundance 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 conclusions 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 employed. Thus, for example, we could draw no

inference as to the nature and relative number of _bacteria_ or _vibrios_, in the case where we employ an acid liquid; for organisms of that kind we must have recourse to neutral or slightly alkaline infusions. On the other hand, liquids having a feeble acid reaction would favour the growth of _mucedines_, _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 _École 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.

Different 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 difference in the number of germs held in suspension in the three different places whence we had taken our air was, therefore, considerable.

The difference in the nature of the germs was equally marked. Those flasks of our first two series which had undergone change presented no trace of _torulæ_, or anything besides fungoid growths, whilst three of the last ten contained _torulæ_ associated with fungoid growths.[44]

[Illustration: Plate 3. Torulæ in Process of Development.]

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 _École 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, lastly, 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 containing about 100 c.c. (about 3-½ fluid ounces) of grape must. These eighteen basins were placed on a table in the room, and left there for five days, 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 _torulæ_—the liquid had remained very bright. With 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 removing 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 contents 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 deposited 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 aforementioned room and also in our laboratory, seven basins prepared as just described. On June 8th all the basins in the room showed signs of fungoid growths, without any trace of _mycoderma vini_, or lines indicative of the presence of _torulæ_, 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.[45] This is another striking proof of the difference between the number of germs of ferment and _torulaceæ_ in the air of our laboratory and that of an ordinary room.

[Illustration: Fig. 12.]

We append drawings of the _torulæ_ found in the six laboratory 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 carried on there, as well as to the power of endurance peculiar to the germs, or the minute vegetative cells of these microscopic 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 distinct forms, marked _a_, _a_; _b_, _b_; _m_, _m_; _n_, _n_ respectively—it must not be supposed that they correspond necessarily to distinct species. From the ends of a compound organism like those in No. III, a little spherical cell may detach itself and then, by a process of budding, give rise to a series of other minute 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 arborescent 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 morphological modifications. We are strongly inclined to believe that each of the cells, or vegetating forms so represented, differing so greatly as they do in aspect, and begotten, all of them, spontaneously 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 transmission, 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 _torulæ_ 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 _torulæ_ in Fig. 12 are not ferments; they do not cause sugar to decompose into alcohol and carbonic acid, any more than the _mycoderma vini_ does; but, nevertheless, there may be an absolute similarity in aspect, development, shape, and size between the alcoholic ferment, properly so called, and these _torulaceæ_.

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 experiments, containing must of grapes and deprived of air. We then closed up the ends immediately after the sudden entrance of the exterior air. One of these flasks developed only one kind of organism, which belonged to the _torulaceæ_. On June 7th this was sufficiently abundant to cover all the sides with a white deposit, and the surface of the liquid appeared quite free of any _mycoderma vini_. To assure ourselves that we were actually dealing with one kind of _torulæ_, 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. We then subjected the plant to microscopic examination. It was quite homogeneous, and formed of a mass of cells, absolutely identical in aspect and size with old cells of ordinary yeast (Fig. 13).

[Illustration: Fig. 13.]

We distilled all the liquid, of which there was about 100 c.c., (3-½ 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.[46]

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 multiplication 1/10,000 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[47] 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 _torula_ 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 liberation 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 presence 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 very 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.

From the previous facts it is obvious that there exist certain productions of various aspects, the germs of which are particularly abundant in the dust of a laboratory where the phenomena 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 vini_, properly so called, when compared with alcoholic ferments. There appears to be no other difference between the _mycoderma vini_ and the _torulæ_ 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.[48]

We have frequently, but without success, endeavoured to bring about the conversion of these unsubmerged _torulæ_ and _mycoderma vini_ into alcoholic ferments; in other words, we have never succeeded in imparting to these _torulæ_ or to _mycoderma 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 conditions 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.

§ VI.—Yeast may become Dry and be Reduced to Dust without losing its Faculty of Reproduction.

In the preceding paragraphs we have given examples of liquids becoming impregnated with self-sown alcoholic 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° C. (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° C. (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.

[Illustration: Fig. 14.]

On December 18th, we took up with a platinum spatula, previously passed through the flame, a pinch of the yeast-and-plaster powder, and sowed it in a two-necked flask (Fig. 14) 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 19th and 20th the yeast 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).

[Illustration: Fig. 15.]

On November 7th, 1873, we once more sowed some of the powder. This time the yeast was dead; we observed the flask which contained it, day by day, until February 1st, 1874, without detecting the slightest sign of fermentation or development of the yeast that we had sown. On February 1st, we made a microscopical examination of the yeast, and found 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 evidently 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.

[Illustration: Fig. 16.]

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 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 possibility 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.

Footnote 21:

We have heard of liquids even less sensitive than these, which required a temperature of 120° C. (248° F.) or more, but we have had no opportunity of studying them.

Footnote 22:

See Pasteur, _Mémoire sur les Générations dites Spontanées_ (_Annales de Chimie et de Physique_, t. lxiv. 3^e série, année 1862).

Footnote 23:

See my _Mémoire sur les Générations dites Spontanées_, already cited.

Footnote 24:

Jules Duval (of Versailles), _Nouveaux faits concernant la mutabilité des germes microscopiques. Rôle passif des êtres classés 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 l’Académie des Sciences_, Nov. 1874). M. Béchamps had previously fallen into similar errors.

Footnote 25:

On this subject see the observations of M. Coste (_Compte-rendus de l’Académie_, t. lix. pp. 149 and 358, 1864).

Footnote 26:

Chauveau’s experiments were directed to show that the operation _bistournage_, 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 effects 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 introducing septic germs, he concludes that the phenomena always arise from the actual presence of such germs.—D. C. R.

Footnote 27:

See Pasteur, _Mémoire sur les Générations dites Spontanées_, pp. 51 and 52, 1862.

Footnote 28:

DAVAINNE, _Compte-rendus de l’Académie des Sciences_, t. lvii. p. 220, 1863.

COZE and FELTZ, _Recherches cliniques et expérimentales sur les maladies infectieuses_, Paris, J. B. Baillière, 1872. Summary of all their works published before 1865.

Dr. LISTER, Medical and surgical journals, particularly the _Lancet_, 1865-67.

Dr. GUÉRIN, _Compte-rendus de l’Académie des Sciences_, March 23, 1874, and May 28, 1874, also the Report of M. Gosselin, December, 1854.

Dr. SÉDILLOT, _Compte-rendus de l’Académie des Sciences_, November, 1874, t. lxxix. p. 1108.

PASTEUR, _Mémoire sur la fermentation appelée lactique_. (_Annales de Chimie et de Physique_, t. lii. 3^e _série_, 1875.)—_Animalcules infusoires, vivant sans gaz oxygène libre et déterminant des fermentations._ (_Compte-rendus de l’Académie des Sciences_, t. lii. 1861.)—_Recherches sur la putréfaction._ (_Compte-rendus de l’Académie des Sciences_, t. lvi. 1863.)

GOSSELIN, ROBIN, and PASTEUR, _Compte-rendus de l’Académie des Sciences_, January 5, 1874. _Urines ammoniacales._

TRAUBE, _Gazette hebdomadaire de médecine et de chirurgie. Sur la fermentation alcaline de l’urine_, April 8, 1864.

CHAUVEAU, _Putréfaction dans l’animal vivant_. (_Compte-rendus de l’Académie des Sciences_, April 28th, 1873.)

Footnote 29:

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 quantity 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 crystals, 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 colourless and very elastic fibrine, associated with masses of crystals, without the slightest trace of blood-globules. Where our observations were protracted, it sometimes happened that all the fibrine collected into one hyaline mass, which gradually expelled every crystal from its interior.

Footnote 30:

PASTEUR, _Comptes rendus de l’Académie des Sciences_, t. lvi. p. 738, 1863.

Footnote 31:

GAYON, _Comptes rendus de l’Académie des Sciences_, and _Annales Scientifiques de l’École Normale Supérieure_, 1874-75.

Footnote 32:

Amongst these influences one of the most important, according to M. Fremy, is “_organic impulse_,”—another gratuitous assumption.

Footnote 33:

FREMY, _Comptes rendus de l’Académie des Sciences_, t. lviii. p. 1167, 1864.

Footnote 34:

FREMY, _Comptes rendus de l’Académie des Sciences_, t. lxxiii. p. 1425, 1871. M. Trécul shares M. Fremy’s opinions, and extends them to the development of different fungoid growths.

Footnote 35:

This observation had already been made by Anthon and H. Hoffmann. “If we scrape the surface of a gooseberry with a blunt knife,” says H. Hoffmann, “and put under the microscope the scrapings, which are of a whitish colour, we shall recognize amongst many varieties of shapeless dirt, earthy particles and other things, the same fungoid spores that we find in the expressed juice, but we shall see them there in infinitely larger quantities. Some of them will be of a dusky colour (_Stemphylium_, _Cladosporium_), 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, Botanique_, t. xiii. p. 21, 1860).

Footnote 36:

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 influence, they assume an amber or brownish colour, but this effect is only produced when the liquids are placed under conditions of unalterability. Should fermentation or the development of fungoid growths be possible, scarcely any change of colour will take place. 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.

Footnote 37:

_Comptes rendus de l’Académie, séance du 28 Octobre, 1872._

Footnote 38:

GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 245; read at the Institute, December 3rd, 1810. Long before Gay-Lussac, it had been remarked that atmospheric air had a great influence on fermentation. See M. Chevreul’s articles on the history of chemistry in the _Journal des Savants_.

Footnote 39:

GAY-LUSSAC, _Annales de Chimie_, t. lxxvi. p. 247, _Mémoire cité_, 1810.

Footnote 40:

It is well to notice that under the influence of fungoid growths, properly 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.

Footnote 41:

It has already been observed in our Memoir on spontaneous generation, 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.

Footnote 42:

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.

Footnote 43:

This small ferment is very curious, although it scarcely affects industrial fermentation. It was first described in 1862. (PASTEUR, _Bulletin de la Société chimique_, 1862, page 67, and following: _Quelques faits nouveaux au sujet des levûres alcooliques_.) It has since been described by Dr. Rees under the name _Saccharomyces apiculatus_. (Dr. Rees, Leipzig, 1870: _Sur les champignons de fermentation alcooliques_. See also Dr. Engel, _Thèse 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 will soon become associated with another, thicker in appearance and more elongated, which also is one of the ferments peculiar to the fermentation of grape must.

Footnote 44:

We may remind the reader that in 1862, in our _Mémoire sur les Générations dites spontanées_, we applied the expression of _torula_ to all the little cellular plants of spontaneous growth, excepting _mycelium_, propagated 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.

Footnote 45:

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 spontaneous 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 collecting 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 ferment 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.

Footnote 46:

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 always assume the form of little drops or _striæ_ or, better still, oily tears, when alcohol is present in the distillate. The distillation should be effected with a small long-necked retort and a Liebig’s condenser. We must carefully watch the neck of the retort at the moment of boiling; should the liquid contain 1/1000 part of its volume of alcohol, we shall observe the indications given above for a short, but appreciable time. 1/10,000 of alcohol is difficult to judge, but with care and practice we may do it without failing. 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 1/10,000 of 1 c.c. of alcohol in a total volume of 100 c.c.

Footnote 47:

Yeast when macerated in water imparts to it certain soluble nitrogenous materials. The solution so obtained, filtered from yeast globules, is known as _yeast water_.—D. C. R.

Footnote 48:

It is possible that this greasiness 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 _torulæ_ are found; fermented liquids more readily give birth to the forms of _torulæ_ and _mycoderma vini_ which exist as a scum. In all probability, however, there is no radical difference between these two kinds of little cellular plants of aerial growth, the floating _torulæ_ and _mycoderma vini_.

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