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_.
CHAPTER IV.
The Growth of Different Organisms in a State of Purity: Their
_Autonomy_.[49]
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 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 atmosphere in the form of dust,
invisible to the naked eye, or, as such, spread over the surface of the
different materials 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 which he describes and which
he sketches, and all the conclusions 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 embarrassment 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 multiply 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 vini_ in an old wine, especially if it is a little acid,
we shall obtain _mycoderma aceti_.[50] It is from facts of this kind,
wrongly interpreted, that many errors have crept into our knowledge of
the lower organisms, and that we are constantly 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 with 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.—Preliminary 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 whatever, 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 _penicillium_ 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 3/4 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 _penicillium_. No matter how
few spores may be taken up on the end of the platinum wire, we shall
have far more than we require for the impregnation of the liquid. At the
moment of charging the point of the wire 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 replace 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
experiments, the volume of air with which we are concerned being
exceedingly limited. Moreover, our flask being in free communication
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
_penicillium_ 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.[51]
The operator, without knowing it, may frequently sow, besides the
_penicillium_, which is all he can see, spores of _mucor mucedo_ and
_mycoderma vini_, in short, of all the most common fungoid growths.
[Illustration: Fig. 17.]
This process of impregnation, therefore, does not afford us sufficient
safeguards, but by means of the following device we shall render it more
satisfactory. Let us take a series of flasks shaped as in Fig. 17,
containing an organic liquid suitable to the development of fungoid
growths, that is to say, slightly acid—yeast-water, plain or sugared,
the wort of beer, or Raulin’s fluid[52] will answer our purpose; let us
boil the liquid, and having previously 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 nearly all expelled. Having prepared ten
or twenty of these flasks in this manner, when they 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 observations. 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 afterwards
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 conditions, we shall evidently
obtain a field of sporanges quite free from the presence of other
organisms. If we now take off the neck of one of the flasks containing
the pure _penicillium_, and take out some of the germs with our platinum
wire,[53] we shall thus obtain with most certainty spores of
_penicillium_ free from impurities.
Our readers will excuse the length of these details and the minutiæ 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.
[Illustration: Fig. 18.]
On June 17th, 1872, we placed some pure spores of _penicillium_ in a
series of three flasks containing wort (Fig. 18), observing all the
precautions that we have indicated. We shall designate 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.[54] We also shook the flask B, after
observing the precaution of sealing up the slender bent tube.[55] 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
transformation 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 different results. If, for instance, we sow spores of
_penicillium_ grown in free contact with the atmosphere, and
consequently exposed to the particles of dust floating therein, we shall
frequently observe, mixed with the fruiting hyphæ of the fungoid growth,
yeast and _mycoderma vini_ and _torulæ_, or even _bacteria_ and lactic
ferment. Thus we shall be 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 may
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.[56] 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. H. Hoffmann.
“In some cases,” this author writes, “and under favourable
circumstances, I have been able to see the ferment produce filaments,
both small specimens that could be examined immediately under the
microscope, and also large specimens, and I have recognized, amongst
other varieties, _penicillium glaucum_, _ascophora mucedo_, _ascophora
elegans_, and _periconia hyalina_, sometimes isolated, sometimes
intermixed. This result is most easily to be obtained by the following
method:—Pour a small quantity of water into a test-tube, which should
then be placed slantingly: 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. H. Hoffmann have also obtained _penicillium_ from
ferment, by a similar process.”[57]
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 exactness, are also far
from being satisfactory.[58]
M. Trécul, who is almost the only one in France, besides Messrs. Ch.
Robin and Fremy, to participate in these errors, does not confine
himself to affirming the change of the spores of _penicillium_ into
yeast, and _vice versâ_; 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 _mycoderma_; _bacteria_ into
lactic ferment, there becoming immoveable; lactic ferment into alcoholic
ferment; alcoholic ferment into _mycoderma cerevisiæ_; finally,
_mycoderma cerevisiæ_ into _penicillium_.[59]”
M. Trécul does not stop here. He goes on to explain the principal of
these changes, as though his testimony were quite beyond refutation:
“If we use a perfectly filtered wort, containing no granulations, and
prepared at a temperature between 60° C. and 70° C. (140° F. and 160°
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 appearance 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 yeast,
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 of _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 _penicillium_, chosen when young and in full growth,
fermented after a varying 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, by drying 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 the spores will be transformed, if the flasks are
perfectly air-tight.”[60]
Such is the manner in which M. Trécul 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. Trécul’s argument is based on the
successive phenomena which manifest themselves in filtered wort
“containing no granulations.” As M. Trécul reasons, this condition is a
necessity, for he starts with the assertion that the albuminous
substances in the wort become changed into granulations “that will
develop into active bacteria.” This is another of M. Trécul’s illusions.
No doubt we may filter hopped wort to almost perfect 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, undergoes 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 transformed into
active bacteria. Nothing 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. Trécul used wort in his experiments,
he does not tell us if it was hopped or not. Had M. Trécul 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° C. 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. Trécul 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. Trécul’s experiments on the _penicillium_, we
have no doubt that that gentleman has sown germs of yeast or _torulæ_,
which bear so striking a resemblance to yeast, at the same time that he
sowed spores, since he took his spores from sporangia of _penicillium_
that had been exposed to contact with ordinary air. The conditions under
which M. Trécul 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 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, probably, 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
_penicillium glaucum_, we do not suppose that the cause of the
difference between our results and those obtained by M. Trécul can be
attributed to our having operated upon a different variety of
_penicillium_ from that which he used. Supposing that there had been a
great difference between our two varieties, still M. Trécul declares
that he has realized the phenomenon with numerous varieties of this
fungus.
M. Trécul expresses himself as follows: “I have used spores of
_penicillium_ of several varieties in these experiments: Firstly, thick,
green, elliptic spores of a variety of _penicillium_ that grows on
lemons; secondly, elliptic spores of a bluish colour and smaller than
the preceding, of another variety of _penicillium_ found on lemons;
thirdly, spherical spores of the variety termed _penicillium
crustaceum_; fourthly, spores of the _penicillium_ that develops on the
yeast of beer.[61]”
This criticism of M. Trécul’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 descriptions struck us as
being at times insufficient, we resolved to ask him for certain
explanations _vivâ voce_ (November 3rd, 1873), which he gave us with the
greatest willingness.
“Every variety of _penicillium_,” said M. Trécul, “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 1-½ fluid ounces) in capacity, filled quite full with wort, or,
at least, containing 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 introduced into them the
spores which had been prepared as follows: I placed on a piece of glass
some spores of the variety of _penicillium_ 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 transformation into ferment took
place next day.”
Provided with these new data we set to work again and prepared a series
of little flasks which were filled quite full with hopped wort, or
contained but very little air, as M. Trécul 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. Trécul 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 substratum of the spores as taken from a
mouldy lemon. If M. Trécul 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 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 case, 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 previously 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. Trécul, M. H. Hoffmann, and
other naturalists, it is not true that the spores of _penicillium_ can
change into alcoholic ferment.
Regarded from another point of view, growths of pure _penicillium_ will
give us some remarkable results, the interpretation 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 we
may 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 mycelium 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
difficulty, in presence of an insufficiency of oxygen, 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 pure _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 a few days will have become perceptibly heavier.
In distilling 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 _aspergillus 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 alcohol; but
could find none.
[Illustration: Fig. 19.]
The flask B was connected with a test flask (Fig. 19), into which the
liquid, together with its fungoid growth, was transferred from B. The
next day, June 21st, the mycelium which 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 by shaking, but the bubbles formed again
by the next day, and this effect continued for several days;
nevertheless the liberation of gas was not continuous, 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 _aspergillus_ 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 unchanged; the liquid was covered with _mycelium_ loaded with
sporanges and borne up by large old bubbles that had not disappeared.
The following was the analysis of the liquid:—
Alcohol 1·2
Glucose 84·0
Dextrine (?) 32·0
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 quantity of mycelium that had
risen to the surface of the liquid, and obtained a field such as is
represented in Fig. 20. Amongst the ordinary filaments of mycelium
belonging to the plant, which are not represented in our engraving, and
which were not more than 1/300 of a millimetre (nearly 1/7500 in.) in
diameter, we perceived much larger ones, swollen and contorted in the
most singular manner, and measuring as much as 1/50 of a millimetre
across their broadest parts. There was also a multitude of the ordinary
spores of _aspergillus_ 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.[62] Beyond all doubt these
cells and irregularly shaped segments, in vegetating with difficulty,
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 development 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.
[Illustration: Fig. 20.]
If we study the vegetation of _aspergillus 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 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_[63] on these filaments, but we
may say that they are on the point of appearing, for the spherical
segments often tend to assume an appearance 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 _chaplets of the conidia-cellules_. This is
represented in the accompanying sketches (Fig. 21), which we have
purposely contrasted with two 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.
[Illustration: Fig. 21 A. Growth with abundant air-supply at the edge of
the mycelium crust.]
[Illustration: Fig. 21 B. Growth with reduced air-supply in the central
and deeper parts of the mycelium.]
[Illustration: Fig. 21 C. Growth with abundant air-supply at the edge of
the mycelium tuft.]
[Illustration: Fig. 21 D. Growth with reduced air-supply in the central
and deeper parts.]
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 300 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 formed of aged,
granulated filaments, with few swellings. The weight of the dry fungoid
growth was 0·50 gramme (about 8 grains) for a total volume of liquid of
122 c.c. (4-1/4 fluid ounces). We obtained 4·4 c.c. of alcohol of 15°,
which was about seven times the weight of the plant. Finally, 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
formation of an organic acid, the nature of which it would be
interesting to determine. M. Gayon 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
differing 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 considerable 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.) we found:—
Growth in the aerated flask 0·92
Growth in the closed flask 0·16
Ratio of weights, 92/16 = 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 gave 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 demonstrate once
more, in the most conclusive manner:—
Firstly, That neither _penicillium_ nor _aspergillus glaucus_ can change
into yeast, even under conditions that are most favourable 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 perform 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.[64] Fermented liquors cannot be exposed to the air without 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 may count the
number of cells which grow in the course of a few hours by millions. The
absorption 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 different
character; for, notwithstanding that the warm and moist surface is in
contact with an atmosphere that is being continually renewed, yet the
_mycoderma_ appropriates 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 _penicillium glaucum_ generally appears. This is one of the facts
which, wrongly interpreted, have led to the belief that _mycoderma vini_
or _cerevisiæ_ may possibly, or even readily, become transformed into
_penicillium_, and _vice versâ_.[65] As the study of the growth of
_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.
[Illustration: Fig. 22.]
On June 21st, 1872, we sowed some _mycoderma vini_ in three flasks, with
double necks, A, B, C (Fig. 22), containing some 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 platinum 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 _mycoderma aceti_, which is generally found
along with it, but which propagates with difficulty in 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 appearance they were
very pure; and the microscope showed the complete absence of any mixture
of _mycoderma aceti_, lactic ferment, or other foreign growths.[66]
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 all 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 afterwards 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 _mycoderma vini_, without finding a trace of any foreign growths,
either in the shape of _penicillium glaucum_, or _mucor mucedo_, or
_rhyzopus nigrans_, or _mycoderma 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_ 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 _mycoderma vini_. On June
24th, 1872, we sowed, in three flasks of sugared yeast-water, prepared
as before—in the first, _mycoderma vini_, together with _penicillium
glaucum_; in the second, _mycoderma vini_, together with _mucor mucedo_;
in the third, _mycoderma vini_ alone.
We effected 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
_penicillium_, 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.
We may now return to that feeble and limited production of carbonic acid
gas and alcohol, the formation of which we have shown experimentally to
take place at a high temperature, after submerging the film of
_mycoderma vini_.[67] There can be no doubt that we have here a
phenomenon similar in every point to that presented by _penicillium_ and
_aspergillus_, 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 activity 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_ consumes it, converting
it into water and carbonic acid gas, by fixation of the oxygen of the
air.[68] If, however, we suddenly submerge the _mycoderma_, we shall
obtain a different result. If, on the one hand, the conditions of life
of this fungus are incompatible with 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 conditions 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 phenomena of
alcoholic fermentation, that is, the evolution of carbonic acid gas, and
the production 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 _penicillium_ and _aspergillus_, we
are compelled to admit that the production of alcohol and carbonic acid
gas from sugar—in one word, alcoholic fermentation—is a chemical action,
connected with the vegetable life of cells which may differ 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—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 oxygen, 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 conditions 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
decomposition 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, specifically 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
vini_ 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 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 accompanied by a phenomenon which is of great
scientific as well as industrial importance, we may divide living beings
into two classes, _aërobian_, that is those which cannot live without
air, and _anaërobian_, 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 _anaërobian_ cell, in
opposition to a cell that is _aërobian_.
As long ago as 1863, in our work on putrefaction, we proposed 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 _mycoderma vini_ into other moulds or into
yeast.[69]
[Illustration: Plate 4. Mycoderma Vini functioning as an Alcoholic
Ferment.—Right Half, showing Appearance of Spores just Sown; Left Half,
their Appearance after an Interval of Submerged Life.]
For a long time, like Turpin and many other observers, although we had
no belief in the transformation of _mycoderma vini_ into any one of the
common moulds, yet we did believe in its transformation 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
the 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 difficulty, 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.
Our experiments were conducted in the following manner:—In some flat
porcelain basins, we grew some pure _mycoderma vini_[70] on fermented
liquids, such as wine or beer, or on artificially 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 themselves, 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 requiring 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. Consequently, 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 mycoderma 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 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, fermented 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 mucedo_ or _racemosus_, ferment-cells that we
shall soon learn to recognize in studying this curious fungoid growth.
The existence of _mucor mucedo_ or _racemosus_, where we had only sown
_mycoderma vini_, was to us a proof that one or more spores of that
mucor had been introduced by the surrounding 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 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 supposed
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 transformation 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 we
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 efflorescence, either in the flasks
themselves, or in the test-flasks attached to them, as represented in
Fig. 19. We observed, however, that kind of alcoholic fermentation of
which we have already spoken and which is due to the mycoderma 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 inclined to believe in the transformation
of one organism into another—the transformation of _mycoderma vini_ or
_cerevisiæ_ into yeast, and that, on that occasion, we were altogether
wrong, through having ourselves 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 cultivation—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_.[71]
M. Béchamp, Professor in the Faculty of Medicine at Montpellier,
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
that 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
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 _molecular 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. Béchamp’s
theory of _microzymata_, M. Fremy’s descriptions of _hemi-organism_, and
M. Trécul’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 _cambium_, we call
it _organic matter_ or _organizable 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 development 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 _globuline 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, continuing 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.—Lastly we have _filamentous_ or _tubular tissue_, when the
globulines, instead of vesiculating, form threads or tubes.”[72]
Such are the purely hypothetical and exploded ideas which MM. Fremy,
Trécul, Béchamp, 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. Béchamp believes that he has discovered that _mother of vinegar_,
introduced into various saccharine liquids, in the presence 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 carbonate of lime, “generates, on the other
hand, the fine cells, which produce the normal alcoholic fermentation of
cane sugar.” Further, M. Béchamp 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.”[73] ...
The object of the following experiments was the study of these assumed
transformations of the _mycoderma aceti_ in saccharine 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 _mycoderma aceti_.
On December 13th, 1872, we sowed the little plant in a state 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 _mycoderma aceti_ covered the surface of the
liquid. We 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_ remained
adhering to the interior sides of the flask during this decanting. The
question then was how to convey this film of the little plant into a
saccharine liquid of a particular kind. We effected this easily by the
following means: After having emptied 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 permitted 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 _mycoderma aceti_ had been left. In
this way the film became partly submerged, partly spread over the
surface of the new liquid. Experiments 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 operation of little
difficulty, which we effected in a manner similar to 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
ourselves of the nature of the organisms present.[74] The result was
that we never found anything besides the _mycoderma aceti_, which had
developed, although with great difficulty, on the surface 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,
absolutely, reject the theory of the polymorphism of _mycoderma aceti_?
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 efforts have
been directed to ascertain if _mycoderma aceti_ might be, for example,
the _aërobian_ 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 anything 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. Béchamp 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 paragraph, 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 very 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.[75]
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 _mucor_ along with the heads containing
the spores.
On June 14th, there was no mycelium visible to the naked eye in the
liquids.
On June 15th mycelium 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 condition, 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 throughout the body of the liquid in the test-glasses,
any fungoid production or organism whatever different from _mucor_
itself.
The same observations apply to the vessel A; in this case the 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 succeeding 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
evidently taken place, consequent on the decantation 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 formation of froth, appeared on
the surface of the liquid in the neck of the test-glass. Fermentation
although never vigorous, continued the following days, and until
December 20th.
Between December 20th and 23rd, it ceased altogether to manifest 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.).