plant in flask B, we subjected it to the same operation to which the
flask C had been subjected on October 20th: that is to say, we poured
the contents of the test-glass back into the connected flask, with the
object of supplying the plant with oxygen.
On December 24th, 25th, 26th, 27th, there was no apparent change.
On December 28th bubbles of gas began to be evolved carrying up clusters
of mycelium to the surface of the liquid. It was evident, therefore,
that the quiescence in the test-glass attached to flask B, was solely
due to deprivation of air, as had happened in the case of the test-glass
attached to flask C, up to the date of October 31st.
On this day, December 28th, we re-decanted the contents of the flask
into the test-glass, and the following day a continuous but feeble
fermentation proceeded. This lasted until January 22nd, although very
sluggish in character; it is evident that these effects were exactly the
same as those which took place in flask C.[76]
We should observe before we proceed further, that we took specimens from
the flasks A, B, C, at different times between June and January, and
that the microscope never revealed the least trace of yeast in them. We
may note besides that, during this interval, we impregnated fresh flasks
of wort with specimens taken from the deposits in the flasks A, B, C,
and that we always obtained reproduction of the _mucor_ and its peculiar
fermentation without the least appearance of ordinary ferment.
The inferences from the results that we have just detailed follow
readily, and are besides of great interest. In the first place, it is
evident that even if the _mucor mucedo_ may be able to produce alcoholic
fermentation, it is totally incapable of changing into yeast. The two
plants are necessarily and radically distinct, and, if different authors
have succeeded in obtaining them mixed one with another in growths of
_mucor_, this intermixture was doubtless the result of a spontaneous
sowing of the yeast, the germs of which abound, particularly in the
particles of dust existing in the atmosphere of any laboratory in which
studies relating to fermentation are pursued.
This, however, is not the most striking inference from the facts which
the cultivation of these organisms revealed. The _mucor_ is evidently a
plant, at the same time _aërobian_ and _anaërobian_. If we had sown the
spore-bearing filaments of _mucor_ on slices of pear, lemon, or similar
fruit, we should have seen the spores germinate, tubes of mycelium
ramifying on the surface of the substratum, and reproducing sporiferous
aerial _hyphae_. In this case the plant would have effected all its
phenomena of nutrition by absorbing oxygen and emitting carbonic acid,
after the manner of animals, as, in our essay on the organic corpuscles
which exist in a state of suspension in the atmosphere, we have shown to
be the case generally with fungoid growths. Under these circumstances,
the only sugar decomposed would have been a quantity equivalent to that
assimilated in forming the cellulose of the young tissues of the fungus,
or in entering into combination, either with the elements of ammonia or
with the sulphur of the sulphates, or the phosphorus of the phosphates,
to form the albuminous substances of the interior of the cells.[77] In
this case the sugar used up would furnish no alcohol, or at least, if
alcohol were formed, it would be decomposed immediately. All _aerial_
growths take place in the same manner; and such is the nature of
nutrition and life in all the larger forms.
In our flasks, on the other hand, the life of the little plant functions
quite differently. Deprived of oxygen, or having at its disposal but an
insufficient quantity of that gas, after a life of activity in contact
with air, it can, nevertheless, live apart from the direct action of
that element, and the combinations to which it gives rise. On the other
hand, we see all the signs of alcoholic fermentation appear; that is, a
notable proportion of sugar, in comparison with the weight of solid
matter assimilated and fixed by the plant, is decomposed into alcohol
and carbonic acid gas; and this decomposition continues as long as life
itself continues in the cells, and they remain submerged, this last
condition being effected by the decantation of the liquid and its
deposit into the test-glass. Along with the disappearance of the
phenomena of vital activity in the cells, the fermentation ceases
absolutely, or at least is no longer visible externally, by reason of
its extreme feebleness. The cells then assume an old, shrivelled,
worn-out appearance, with irregular outlines and granular markings.
Their life is merely suspended, however, not extinct; for if they be
supplied once more with oxygen, and suffered to exist under the
influence of that gas, they will vegetate again, and become capable of
producing fermentation afresh, even after having been excluded from the
air for a considerable time.
Oxygen then presents itself to us as being endowed with a certain
determining stimulus in the matter of nutritive action enabling this
action to be prolonged beyond the point where the direct influence of
oxygen ceases. In time the energy that has been imparted to the cells
will die away, and then also fermentation will cease, to be resumed,
however, when the plant is once more submitted to the revivifying action
of the gas. It seems as though the vital energy derived from the
influence of gaseous oxygen were capable of effecting an assimilation of
oxygen, not in the gaseous state, but existing in some state of
combination, and hence its power of causing the decomposition of sugar.
Looking at the matter in this light, it seems to us that we may discover
in it a fact of general occurrence, that this peculiar action of the
oxygen and the cells is to be seen in all living beings. For indeed is
there any cell which, if suddenly and completely deprived of air, would
perish forthwith, and absolutely? Probably there is not a single one
that would do so. With certain modifications of greater or less amount
the assimilative and excretive acts which have taken place during life
must be carried on after the suppression of oxygen, resulting in
fermentations ordinarily obscure and feeble, but in the case of the
cells of ferments, properly so called, manifesting an activity both
greater in amount and more enduring.
Let us now proceed to compare the weight of alcohol formed by the
_mucor_ during fermentation with the weight of the plant itself.
_First experiment._—One of the double-necked flasks contained at
starting 120 c.c. (about 4 fl. oz.) of wort.
On January 2nd, 1873, we attached this flask to a test-glass, containing
a deposit of _mucor_ ferment (Fig. 19, p. 101), a few drops of which we
poured into the wort in the flask, to impregnate it. On January 3rd we
decanted the wort from the flask into the test-glass; under these
conditions we have seen that the wort must ferment.
On January 18th the fermentation in the test-glass ceased. On July 31st,
1873, we transferred the liquid from the test-glass back to the flask.
On August 4th, 1873, we again decanted this same liquid from the flask
into the test-glass. On December 25th, 1873, we once more removed the
liquid from the test-glass to the flask, and allowed it to remain so
until December 23rd, 1874, on which day we submitted it to examination.
It was found to contain per 100 c.c. (3-½ fl. oz.)
Grains. Grammes.[78]
Total weight of the fungus 5·7 0·37
Absolute alcohol 50·9 3·3
Acidity, estimated in its equivalent of 1·7 0·11
sulphuric acid
Sugar, determined by cupric solution 82·2 5·2
Dextrine (?) 24·6 1·6
The total weight of fungoid growth being 0·37 gramme, and the total
weight of absolute alcohol for the 120 c.c. of fermented liquid being 4
grammes, we had, consequently, from ten to eleven times by weight more
alcohol than fungus.
_Second experiment._—On June 13th, 1872, we sowed two or three
sporiferous heads of _mucor_ in some wort contained in one of the
double-necked flasks. The temperature of our oven varied between 23° C.
and 25° C. (73° F. to 77° F.) The total volume of liquid was 120 c.c.,
as before.
June 15th, mycelium had developed, buoyed up on bubbles of gas.
June 16th, patches of bubbles, due to fermentation, covered the surface
of the liquid.
June 17th, we transferred the liquid to the test-glass.
June 28th, fermentation in the test-flask had ceased.
June 28th, fermentation recommenced, the temperature of the oven being
raised to 27° C. (80° F.).
October 20th, the liquid was transferred back from the test-glass to the
flask.
October 24th, mycelium had developed, supported by big bubbles on the
surface of the liquid in the flask.
October 31st, we retransferred the liquid to the test-glass.
November 1st, a feeble, but continuous fermentation commenced. This was
kept up until January 2nd, 1873, on which day we transferred the liquid,
with its deposit from the test-glass to the flask, when it now seemed to
be quite inert. We left it in this flask until December 24th, 1874,
without its manifesting during this long interval any sign of
fermentation; nor did the fungus appear to grow at all.
We then submitted the liquid to analysis, and found in it, per 100 c.c.—
Grammes.[79]
Total weight of fungoid growth 0·25
Absolute alcohol 3·4
Acidity, estimated in its equivalent of sulphuric 0·12
acid
Sugar, determined by copper solution 6·2
Sugar, determined after treatment by boiling with 1·0
sulphuric acid, and deduction of amount of sugar
already obtained (dextrine)?
The total weight of absolute alcohol for the 120 c.c. of fermented
liquid was 4·1 grammes—that is, the weight of the alcohol was sixteen or
seventeen times that of the plant.
[Illustration: Plate 5. Mucor, Vegetating Submerged, in Deficit of Air.]
The structure of the plant differs considerably when it lives surrounded
by air, and when it is more or less completely deprived of that fluid.
If it has an abundance of air at its disposal, if it vegetates on the
surface of a moist substance or in a liquid in which the air held in
solution may be renewed without being incessantly displaced by carbonic
acid gas, we shall see it develop as an ordinary fungoid growth, with a
mycelium consisting of filaments more or less slender, branching, and
entangled, sending up from the surface of the liquid aerial organs of
fructification. This is the well-known form of vegetation of the common
_mucor_. On the other hand, if we compel the _mucor_ to live in a
saccharine liquid with insufficiency of air, at least for some of its
parts, the mode of vegetation will change completely, as we have seen in
the case of _penicillium_, _aspergillus_, and _mycoderma vini_ when
submerged, but with this difference, that in the case of the _mucor_ the
changes in question, and the activity of nutrition under these new
conditions, are much more marked than in the case of those other
organisms. The spores grow larger and the filaments of mycelium which do
develop are much stronger than those in the normal plant. These
filaments put forth, here and there, other filaments which detach
themselves and vegetate at the side of the others, being terminated or
interrupted by chains of large cells, species of spores which can live
by budding and reproducing cells similar to themselves or by elongating
into filaments.
Plate V. represents the living plant submerged at a little depth, and
having, consequently, still at its disposal a certain quantity of air,
insufficient, however, to supply the oxygen needed for all the acts of
nutrition. In this case the _mucor_ appears very different,
morphologically, from what it is when in free contact with air. Here it
forms short filaments, having a diameter double or triple that of the
filaments of the ordinary mycelium with branches and buds all over, and
what is especially characteristic, forming a network of chains of cells,
sometimes spherical, sometimes oval or pear-shaped, which are the actual
spores. These, as soon as they are detached, bud in their turn, and
reproduce either cells or branching tubes; these cells or the chaplets
which they form being known under the name _mycelian spores_ or
_conidia_. Our plate gives these different aspects very correctly, and
affords us a good idea of the luxuriant state of this remarkable
vegetation.
Plate VI. represents the plant living at a greater depth with less air,
expending, by means of sugar as source of heat, the energy which it
acquired in vegetating under the influence of the oxygen of the air. The
filaments are fewer and older in aspect, and the number of cellular
forms is proportionately larger than in the former case, the budding
giving rise by preference to spherical or oval cells. On a single cell
we often see two, three, four, five, six, and even more buds.
When the buds of the oval or spherical cells detach themselves whilst
young, they often resemble in form and size cells of ordinary yeast, nor
can even considerable experience in this kind of observation always
enable us to distinguish them. Hence we may easily understand how many
have come to believe, with so skilful a botanist as Dr. Bail, in the
transformation of _mucor_ into yeast.
With the forms represented in Plates V. and VI., the plant is more of a
ferment than of a fungoid growth. In such cases the weight of sugar
decomposed in comparison with the weight of new cell-globules formed is
very considerable, an effect which is more marked the less air the plant
has at its disposal. Under the latter conditions, however, vegetation is
slow and laborious, and the ferment very soon assumes an aspect of age,
and we must constantly rejuvenate the cells by bringing them into
contact with oxygen, and subjecting them to the action of limited
quantities of that gas, and so promote their vegetation and prolong
their fermentative activity. This effect we brought about when we
retransferred the liquid and its deposit of _mucor_ from the test-glass
to the flask, thus bringing them into contact with fresh air. We saw
cells that appeared old, dark, and highly granulated, become inflated,
grow more transparent, and fill with a gelatinous protoplasm, the few
granulations which they still exhibited assuming a brilliant appearance
when we succeeded in distinguishing them; and finally, a very active
budding was set up. Under this reviving influence life could continue
once more away from the air, although with difficulty, so that
fermentation would be most intense if the large filaments and their
conidia were constantly being removed from and to the action of air.
The preceding plates show several instances of this rejuvenescence of
the old cells of _mucor_ ferment.
[Illustration: Plate 6. Ferment of Mucor.]
We have omitted to represent amongst the old cells some cells which have
their granulations collected about the centre, with an empty space
between the granulations and the exterior borders.[80] In this state,
cells are generally dead and incapable of any revival. It is impossible
to avoid being impressed by the striking analogies which exist between
all these facts and those presented by cells of yeast.
In concluding our study of the vegetation of _mucor_ as a mould and
_mucor_ as a ferment, we may again remark that the most striking
analogies also exist between the preceding observations and those we
have seen in the case of _penicillium_, _aspergillus_, and _mycoderma
vini_. These latter plants do not furnish alcohol or carbonic acid gas
by direct fermentation of the sugar, as long as we let them vegetate
with plenty of air at their disposal. Once submerged, however, their
vital aspect changes; on the one hand the cells or filaments of the
mycelia evince a tendency to become larger; on the other hand there is a
tendency to greater closeness in these latter, and, consequently, a
transition to the state of _conidia_. Lastly, there is a correlative
budding of cells, accompanied by a formation of alcohol and liberation
of carbonic acid gas; in short, all the ordinary signs of alcoholic
fermentation.
The principal difference in the case of _mucor_ consists in this, that
the vegetation of this latter, under the conditions of insufficient
aeration or none at all, is more decided, both as to extent and
duration.
It may be thought that all the varieties of _mucor_ are capable of
yielding the kind of ferment that we have just mentioned. But this is
not the case; and here we have another striking proof of the great
physiological differences presented by forms of vegetation so intimately
connected with each other that, in botanical classifications, they must
be put as closely as possible together. Of this fact we have the most
striking example in _mycoderma vini_ and the alcoholic ferments,
properly so called, which so closely resemble each other in form and
development that they might be supposed to be identical, at least,
according to our present knowledge, but which differ so widely in their
physiological aspects.
On November 17th, 1873, we found a very beautiful specimen of _mucor
mucedo_ on a pear, under a glass bell jar. It was a mass of perfectly
straight filaments, simple and isolated, very large in comparison with
those ordinarily met with, each terminating in a sporange, identical to
that of _mucor mucedo_, and proportionately well developed. We are able
to distinguish _mucor racemosus_ from _mucor mucedo_ only by the
circumstance of its having on its sporange-bearing hyphæ lateral
branches which also terminate in sporanges.
We sowed only one of the terminal heads of the large erect hyphae in
some wort, in which it soon produced an abundant mycelium, but without
the least appearance of gas. For a very long time, up to January 7th,
1875, we studied the developments of this organism, which remained all
the time perfectly pure, in consequence of our having cultivated it in
one of our two-necked flasks on pure wort.
The total volume of liquid, which was 130 c.c., (4·57 fl. oz.) contained
2·3 grammes (35·3 grains) of alcohol. In spite of this rather large
proportion of alcohol, a clear sign of undoubted fermentation, the plant
had yielded no _conidia_ at all, nor any cell-globules of ferment. Some
of the filaments, however, were larger than the rest, and exhibited
irregularly-shaped swellings, which in some cases were of enormous size.
Whilst the natural mycelial filaments, by which we mean the vegetating
part of mucor, which were supplied with abundance of air, only measured
3/450 of a millimetre[81] in diameter, the filaments that had grown
probably with an insufficiency of oxygen, and performed the functions of
ferment, measured 8/450, and the swellings as much as 30/450 of a
millimetre in diameter, as represented in Fig. 24.
[Illustration: Fig. 24.]
In concluding this paragraph, we may mention a very able research on
fermentation which we have lately studied, the author of which, Dr.
Fitz, communicated it to the Chemical Society of Berlin in 1873. In
section II., page 48, this author explains his observations in a manner
conformable to our own views, as may be seen from the following passage
of the memoir:—
“In the presence of oxygen, the ferment of _mucor_ develops into a
mycelium and consumes the sugar; in the absence of oxygen, on the other
hand, the spores develop into ferment of _mucor_, that buds and
decomposes the sugar into the products of fermentation.
“The properties of _mucor mucedo_ in a fermentable liquid, in the
presence or in the absence of oxygen, accord perfectly with the theory
of fermentation established by Pasteur in 1861 (_Comptes rendus de
l’Académie des Sciences_, t. lii., p. 1260). According to this theory a
fermentative fungus needs oxygen for its development; if it finds any
free oxygen it utilizes the whole of it, assimilating one part of the
sugar and burning the other; whilst in the absence of free oxygen, the
fungus appropriates what it requires from the sugar.”
Footnote 49:
In the course of this work we shall combat, by means of experimental
proofs which appear to us irrefragable, the opinions which many
writers entertain on the subject of certain transformations of
organisms—that of _penicillium glaucum_ into ferment, or _mycoderma_;
of _bacteria_ into lactic ferment; of ferment into _vibrios_; of
_mycoderma aceti_ into ferment, and so on. Nevertheless, we shall
pronounce no _a priori_ opinion on the question whether the inferior
organisms, which will be the subject of this chapter, and which
include yeast and the ferments properly so called, are perfect beings
in their habitual form, or whether they are susceptible of
polymorphism. It is with this reservation that we employ the word
_autonomy_. If we claim polymorphism for any species, we shall not do
so without furnishing proofs. Some organs detached from higher
organisms, and some beings in a certain phase of their existence, may
reproduce themselves under a special form, with special properties,
when brought into media and under conditions that are unfit for the
production of the plant or animal under its other shape or ordinary
mode of reproduction. Modern Science affords many examples of this,
and certain alcoholic ferments present us with analogous facts; but to
wish to stretch these facts beyond their due significance, and to
admit a polymorphism that cannot be proved, in consequence of a belief
that it is possible, or on the faith of confused observations, is to
indulge in gratuitous assertion from a mere spirit of system.
Footnote 50:
See, on this subject, the author’s _Études sur le Vinaigre_, Paris,
1868, p. 76, note; and especially _Études sur le Vin_, 2nd Edition,
1873, p. 19.
Footnote 51:
Some observations in the preceding chapter enable us to account for
the vast number of germs which are constantly falling on the surface
of everything. We may here allude to the use we have made of flasks,
shaped as in Fig. 17, and holding from 250 c.c. to 300 c.c., which are
a third part filled with an organic liquid, and are closed up when
boiling. They contain no air when cool, and are opened in series of
10, 20, &c., out of doors, and closed up again immediately. The air
rushes violently into the vacuum, and thus we introduce about 200 c.c.
of air, with all the particles of dust contained in that air, into
each flask. It has been proved that a certain number of these flasks
undergo change in the course of time, the number of those changing and
the nature of their changes being in close proportion to the probable
number and nature of the floating germs able to develop in the
particular nutritive liquid used. If we work at great elevations, far
from houses and the dirt of towns and inhabited plains, as we did at
Montanvert, near the _Mer de Glace_, change will seldom occur. The
opposite will be the case if we work in a place like the living-room
of the little, dirty, ill-kept inn at Montanvert. In a laboratory
where fermentation is studied we obtain certain kinds of germs which
often differ from those found in the air of the open country. If we
desire to have organisms in all our flasks, we have only to stir up
the dust on the ground or on surrounding objects at the moment when we
open the flasks. This simple and easy experiment clearly shows us that
it is impossible for a field of sporanges of fungoid growth, existing
in an uncovered vessel or on the surface of a fruit, to escape
becoming mixed with germs that are foreign to the little plant; in
other words, the student who sows spores of _penicillium_, which he
has collected from one place or another on a brush, exposes himself to
serious causes of error.
Footnote 52:
M. Jules Raulin has published a well-known and remarkable work on the
discovery of the mineral medium best adapted by its composition to the
life of certain ordinary fungoid growths; he has given a formula for
the composition of such a medium. It is this that we call here
“Raulin’s fluid” for abbreviation.
Water 1,500
Sugar Candy 70
Tartaric Acid 4
Nitrate of Ammonia 4
Phosphate of Ammonia 0·6
Carbonate of Potassium 0·6
Carbonate of Magnesia 0·4
Sulphate of Ammonia 0·25
Sulphate of Zinc 0·07
Sulphate of Iron 0·07
Silicate of Potassium 0·07
J. RAULIN. Paris, Victor Masson, 1870. _Thèse pour le doctorat._
Footnote 53:
If we do not wish to take the chance of procuring the pure
_penicillium_ by means of these spontaneous sowings, effected by
opening and then closing in the flame a certain number of flasks with
drawn-out points, we may utilize one of the flasks, which, having been
opened and closed again, has notwithstanding developed no organized
forms, as follows:—We impregnate the contained liquid directly, by
dropping into it from a metallic wire spores taken from any growth of
_penicillium_ exposed to the common air; and then from the new field
of sporanges formed by this sowing in the flask that has been
re-closed, we must, later on, take the pure spores that we require.
This method is quicker and almost as safe.
We should add that, if we wish to use for our purpose spores of
_penicillium_ from a closed flask, in which the plant has fructified,
we must be careful not to leave the plant too long closed up. A few
days after the sowing the growth of the fungus is arrested, in
consequence of all the oxygen being absorbed, and its place being
supplied by a mixture of carbonic acid and nitrogen; and the spores,
if kept too long in this atmosphere, will all perish.
Footnote 54:
To shake the liquid without danger of introducing exterior particles
of dust, we apply the flame of the spirit lamp to the drawn-out neck
of the flask, and close up the open end; we may then shake our flask
without risk. We must afterwards reopen the end of the drawn-out neck
for the purpose of re-establishing communication with the exterior
air.
Footnote 55:
The flask B was closed with the lamp in consequence of one of the
objects of these experiments being to test M. Trécul’s experiments on
the transformation of _penicillium_ into ferment. Strangely enough,
according to M. Trécul, as we shall see later on, the spores of
_penicillium_ refuse to change into ferment, if the vessels in which
they are sown are not “perfectly air-tight.”
Footnote 56:
_Bulletin de la Société Philomathique._
Footnote 57:
HERMANN HOFFMANN, _Études Mycologiques sur la Fermentation_.
_Botanische Zeitung_ and _Annales des Sciences Naturelles_, 4^e série,
t. xiii. p. 24, 1860.
Footnote 58:
_Communication sur l’Origine et le Développement de quelques
Champignons._ Dantzig, 1867.
Footnote 59:
TRÉCUL, _Comptes rendus de l’Académie_, t. lxxiii. p. 1454; December
28, 1871.
Footnote 60:
TRÉCUL, _Comptes rendus de l’Académie_, t. lxxv. p. 1169, November 11,
1872. A proof of M. Trécul’s carelessness in experiments of this kind
is the fact that in studying the fertility of an impregnated wort, he
often obtains different productions. Our experiments give opposite
results. If we sow nothing, we obtain nothing. If we sow a plant, we
obtain a similar plant; or, should there be any difference, the change
may be traced, beyond question, to its origin in the plant sown, and
is the consequence of some alteration in the conditions of our
experiment.
Footnote 61:
_Comptes rendus des Séances de l’Académie des Sciences_, t. lxxv. p.
1220; Nov. 18, 1872.
Footnote 62:
Since writing the above we have experienced some doubt as to whether
the forms of development represented in Fig. 20 are actually those of
the _aspergillus glaucus_, which we supposed our fungoid growth to be.
In some of the later sketches of our observations we find similar
forms, which belong to a bluish kind of _penicillium_, with rather
large spores. Fortunately, this doubt affects our argument in no
essential particular. It matters very little what variety of fungoid
growth it is that gives rise to alcoholic fermentation attended by
peculiarities of shape that only occur in the development of its
spores when air fails it.
Footnote 63:
By the term _conidia_ is meant certain chains of cells, which are in
reality mycelial spores.
Footnote 64:
See PASTEUR, _Études sur le Vin_, 1st Edition, pp. 20 and following.
Footnote 65:
Since writing this paragraph, we have found in M. Ch. Robin’s _Journal
d’Anatomie et de Physiologie_, an article signed by that gentleman,
and entitled _Sur la Nature des Fermentations_, &c. (July-August,
1875), in which the learned microscopist says:—“The _torula cerevisiæ_
is derived from the _mycoderma cerevisiæ_. My observations leave no
doubt on my mind that _penicillium glaucum_ is one of the forms
evolved from spores or ferments that have preceded it, as M. Trécul
showed a long time ago, and that, moreover, the spores of
_penicillium_, germinating in suitable media, give us the sporical
form termed _mycoderma_.”
We take the liberty to observe that these assertions of M. Robin’s are
purely gratuitous. Up to the present time it has been impossible to
discover a suitable medium for the proof of these different
transformations or polymorphisms. From the time of Turpin, who firmly
believed that he had observed these changes, to our own, none of the
microscopists who have affirmed these transformations have succeeded
in adducing any convincing proof of them, and M. Trécul’s latest
observations, especially as regards _penicillium_ and its
transformation into ferment or into the _mycoderma_ of beer, have been
positively disproved by ours, supported, as they are, by proofs that
we consider irrefutable.
Footnote 66:
It is a very easy matter to study the liquids and growths in our
flasks during the course of a single experiment. We take out the glass
stopper that closes the india-rubber tube on the straight-neck, and,
by means of a long rod or a glass tube previously passed through the
flame, take up a quantity, which we draw out immediately for
microscopical examination. We then replace the glass stopper, taking
care to pass it through the flame before doing so, to burn up any
organic particles of dust that it may have picked up from the table on
which we laid it.
Footnote 67:
We may prove the occurrence of alcoholic fermentation by the cells of
submerged _mycoderma vini_ in a different manner. To do this, after
having made all our preparations as before and shaken up the film of
_mycoderma vini_ in its liquid, we must attach our flask to a test
flask (Fig. 19), and pass the turbid liquid into the latter. On
succeeding days we shall detect a very protracted fermentation in the
test flask; there will be a succession of minute bubbles rising from
the bottom, but in small number at a time. The fermentation is very
evident whilst it lasts, but is rather sluggish, and, although of very
long duration, ceases long before the sugar is exhausted.
This experiment proves better than any other the non-transformation of
_mycoderma vini_ into other ordinary fungoid growths. For after
decanting the liquid into the test flasks, the sides of the
experimental flask remain covered with streaks of _mycoderma vini_
along with some of the liquid. Moreover, the flask is refilled with
air, and this air is being constantly renewed, in part, by variations
of the temperature of the oven, so that the _mycoderma_ remaining on
the sides is thus placed under the most favourable conditions for
transformation into other fungoid growths, if that were possible. It
is still more easy to detach the experimental from the test flask, and
to pass pure air into it, once or twice a day, or constantly. In any
case, we shall never see anything besides the _mycoderma vini_ spring
up within it.
Footnote 68:
See PASTEUR, _Comptes rendus des Séances de l’Académie des Sciences_,
t. liv., 1862, and t. lv., 1862. _Études sur les Mycodermes, &c._
Footnote 69:
In a subsequent chapter we shall prove that yeast is likewise
incapable of transformation into _mycoderma vini_.
Footnote 70:
We secured the purity of our mycoderma by the same means that we have
already described for the procuring of spores of _penicillium_ or
other fungoid growths in a state of purity.
Footnote 71:
BUFFON, _Histoire de l’Homme_, t. viii., edition 12mo, 1778; TURPIN,
_Mémoires de l’Académie des Sciences_, t. xvii.; Dr. PINEAU, _Annales
des Sciences Naturelles_, t. iii., 1845; POUCHET, _Traité de la
Génération dite Spontanée_, p. 335, 1859. See also our _Mémoire sur
les Générations dites Spontanées_, 1862, pp. 100 and following, in
which we give a _resumé_ of some of these theories.
Footnote 72:
The following is Turpin’s application of his theory to the formation
of the ferments of fruits (_Mémoires de l’Académie_, t. xvii., 1840,
p. 155), where also, on p. 171 the above quotation will be
found:—_Ferments Produced by the Filtered Juice of the Pulp of
Different Fruits_—“By the word pulp we mean the soft and juicy
cellular tissue of the fleshy part, mesocarp or middle layer of the
pericarp of certain ripe fruits. This cellular tissue, which is very
abundant in the peach and all stone-fruit, in the apple and pear, in
the orange and grape, and similar fruits, is the same as that which
forms the body of a leaf. Being in every case composed of a simple
agglomeration of contiguous mother-vesicles, which are always filled
with globulines that are more or less developed, more or less
coloured, and individually endowed with a special vital centre, it is
not surprising that its globulines when free and detached from the
compound organisms to which they belong, and from association with its
vegetable life, should, when placed in a suitable medium, themselves
vegetate and become transformed, under these new influences, into a
mucedine, with filaments and articulations. Such are the very fine,
and, consequently, very transparent globulines, which, when left to
themselves in sweetened water, grow and become vesicular, producing
other globulines in their interior, then bud, vegetate into mucedinous
filaments, decompose sugar, and produce all the effects that
constitute what we term _alcoholic fermentation_.”
Footnote 73:
BÉCHAMP, _Recherches sur la Nature et l’Origine des Ferments (Annales
de Chimie et de Physique, 4^e série_, t. xxiii., and _Comptes rendus
de l’Académie des Sciences_, Oct. 23, 1871).
Footnote 74:
We need scarcely here observe, having done so on previous occasions,
that whenever we opened our flasks to obtain specimens, we made use of
a fine tube, previously passed through the flame of a spirit lamp, and
that we also passed this flame over the surface of the india-rubber,
glass stopper, &c., to consume the organic particles of dust which
floating about might introduce themselves at the moment when we opened
the right-hand tube of the flask.
Footnote 75:
Ever since the year 1861 (see p. 92), this question of the possible
transformation of the ordinary fungi, especially _penicillium_ and
_mucor mucedo_, into yeast has engaged our attention. The results
attained have been entirely negative; but hitherto only the
conclusions of our work have been published, some account of which was
given at the meeting of the _Société Philomathique_ of March 30th,
1861. The following extract is from the _Bulletin_ of the
society:—“Meeting of March 30th, 1861. At this meeting a paper was
read by M. Pasteur ‘On the supposed changes in the form and vegetation
of yeast-cells, depending on the external condition of their
development.’ It is well-known that Leuwenhoeck was the first to
describe the globules of yeast, and that M. Cagnard-Latour discovered
their faculty of multiplying by budding. This interesting vegetable
organism has been the subject of a host of researches by chemists and
botanists. The latter, from the days of Turpin and Kutzing, have
almost unanimously regarded yeast as a form of development of various
inferior vegetable types, especially _penicillium_. The studies of
this subject which seem to have won most favour during the last few
years are those of MM. Wagner, Bail, Berkeley, and H. Hoffmann. The
researches of these botanists seem to strengthen and confirm the
original observations of Turpin and Kutzing. M. Pouchet has, quite
recently, expressed the same ideas, and has determined certain points
in connection with them with much precision of detail. M. Pasteur has
long studied this important question, which is so intimately connected
with the essential nature of yeast and with those phenomena of the
polymorphism of the inferior types of vegetable life, to which most of
the remarkable works of M. Tulasne relate; he has, however, arrived at
results that are altogether negative, and he declares that he was
unable to detect the transformation of yeast into any of the
_mucedines_ whatsoever, and, inversely, that he could never succeed in
producing the smallest quantity of yeast from ordinary _mucedines_.”
These same results we communicated to the _Société Chimique_ of Paris,
at a meeting held April 12th, 1861. Throughout the investigation of
which we have just indicated the conclusions, we insisted on the
necessity of cultivating the separate organisms in a state of purity
in all researches relating to these inferior forms of life, if we
desire to attain to sure inferences about them; and the method of
working, which we recommended, did not differ essentially from that
adopted in the present work. Since then the study of these growths has
been conducted with the utmost precautions; and other apparatus,
perhaps as safe as those which we employ and better adapted than ours
for the study of polymorphism of species, have been invented by
botanists of great skill—M. de Bary, in Germany, and M. Van Tieghem,
in France.
Footnote 76:
We found, after the lapse of another year, in December, 1873, that the
ferment of the _mucor_ in the test glass might still be easily
revived; that it was able to propagate, both in the mycelium and in
the cellular form, in wort, and that it might produce a fermentation,
more or less active, according to the condition of aeration; in short,
that it was capable of producing all the characteristic phenomena
described. By means of the method of cultivation that we employ, our
study, which was continued for years, was pursued without the least
fear of any foreign fungoid growths being introduced into the vessels,
although they remained constantly open, and the air in them was being
perpetually renewed by the action of diffusion and variations of
temperature. In 1875 nothing remained alive in our flask, and further
revival became impossible.
Footnote 77:
We do not here take into account certain phenomena of oxidation of
which the fungoid growths are the seat, and which remind us of those
that are presented in so remarkable a degree by _mycoderma vini_ and
_mycoderma aceti_.
Footnote 78:
[There are 15·43 grains in the gramme.]
Footnote 79:
[For English equivalent see Experiment 1, p. 135.]
Footnote 80:
The figure given below supplies this omission. The cells that are
isolated or are in chains, _b.b.b._, show this state of the old cells.
The cells _a.a.a._ are younger, and may be more easily revived. We may
see by the dimensions of some of these how greatly, in certain cases,
the cells of _mucor_ resemble cells of yeast; nevertheless, in the
state of the contents and the aspect of the outlines, there are always
some differences sufficiently appreciable to strike the practised
observer.
[Illustration: Fig. 23.]
The figures adjoining the cells indicate fractions of a millimetre. (A
millimetre may be taken as ½5-in.)
Footnote 81:
[0·000089 in., 0·00026 in. and O·00089 in. respectively.]
CHAPTER V.
The Alcoholic Ferments.
§ I.—On the Origin of Ferment.
Amongst the productions that appear spontaneously, or, we should rather
say, without direct impregnation, in organic liquids exposed to contact
with the air, there is one that more particularly claims our study. It
is that one which, by reason of its active energy as an agent of
decomposition, has been distinguished and utilized from the earliest
times, and is considered as the type of ferments in general; we mean the
ferment of wine, beer, and more generally, of all fermented beverages.
Yeast is that viscous sort of deposit which takes place in the vats or
barrels of must or wort that is undergoing fermentation. This kind of
ferment presents for consideration a physical fact of the most
extraordinary character. Take a morsel of the substance and put it in
sweetened water, in must, or in dough, which always contains a little
sugar; after a time, the length of which varies, a few minutes often
sufficing, we see these liquids or the dough rise, so to speak. This
inflation of the mass, which is due to a liberation of carbonic acid
gas, may cause it to overflow the vessels containing it, if their
capacity is not considerably greater than the volume of the matters
fermenting. It is equally remarkable that these phenomena are natural
and spontaneous; that is to say that the must, the wort, and the dough
are able to rise, as we have termed it, when left to themselves, without
the least addition of foreign substances. The only difference that may
occur in these phenomena is a certain amount of retardation, in cases
where the yeast does not reach the saccharine matters in a perfectly
natural form, inasmuch as it then requires a certain time to get itself
together before it can begin to act.
It is necessary, indeed, that sugar should be present; for if we
abstracted by some means or other from the must or dough all the sugar
contained in it, without touching the other constituents, the addition
of yeast would produce no gas. Everything would remain quiet until the
moment when signs of a more or less advanced putrefaction showed
themselves. Yeast is one of the most putrescible of substances, and it
is worthy of notice that its alteration is also the consequence of the
formation of one or more ferments, very different, however, from that of
which we are speaking. As for the nature of yeast, the microscope has
taught us what it is. That marvellous instrument, although still in its
infancy, enabled Leuwenhoeck, towards the close of the 17th century, to
discover that yeast is composed of a mass of cells. In 1835
Cagnard-Latour and Schwann took up Leuwenhoeck’s observations, and by
employing a more perfect microscope, discovered that these same cells
vegetate and multiply by a process of gemmation. Since then the physical
and chemical phenomena already mentioned, such as the raising of the
mass, the liberation of carbonic acid gas, and the formation of alcohol,
have been announced as acts probably connected with the living processes
of a little cellular plant, and subsequent researches have confirmed
these views.
In introducing a quantity of yeast into a saccharine wort, it must be
borne in mind that we are sowing a multitude of minute living cells,
representing so many centres of life, capable of vegetating with
extraordinary rapidity in a medium adapted to their nutrition. This
phenomenon can occur at any temperature between zero and 55° C. (131°
F.), although a temperature between 15° C. and 30° C. (59° F. and 86°
F.) is the most favourable to its occurrence.
As regards the rapidity of the budding, the following observations will
give some idea of what it is in the case of one of the ferments of
natural must. The temperature was between 12° C. and 13° C. (55° F.).
“On October 12, 1861, at ten o’clock in the morning, we crushed some
grapes, without filtering the juice that ran from them; afterwards, at
different times during the day, we examined the juice under the
microscope, until at last, although not before seven o’clock in the
evening, we detected a couple of cells, as represented in Fig. 25, _a_.
[Illustration: Fig. 25.]
From that time we kept these contiguous cells constantly in view. At
7.10 we saw them separate and remove to some little distance from each
other (Fig. 25, _b_). Between 7 and 7.30 we saw, on each of these cells,
a very minute bud originate and grow little by little. These buds
developed very near the point of contact, where the disjunction had just
taken place. By 7.45 the buds had increased greatly in size (Fig. 25,
_c_). By 8 they had attained the size of the mother-cells. By 9 each
cell of each couple had put forth a new bud (Fig. 25, _d_). We did not
follow the multiplication of the cells any farther, having seen that in
the course of two hours two cellules had furnished eight, including the
two mother-cells.”[82]
An increase like this, which would have been more rapid at a temperature
between 15° and 25° (59° and 77° F.), and still more so between 25° and
30° (77° and 86° F.), may indeed seem surprising. It is really, however,
nothing to what sometimes occurs. In choosing proper conditions of
temperature and medium, of state and nature of yeast, it has sometimes
happened that the bottom of a vessel has become covered with a white
deposit of yeast cells, in the course of not more than five or six hours
after we had sown a quantity of yeast so small as to effect no change at
all in the transparency of the liquid contained in the vessel after it
had been shaken up. Such a rapidity of vegetation reminded us of those
exotic plants which are said to grow several feet in height in the
course of twenty-four hours.
[Illustration: Fig. 26.]
Budding commences in the form of a simple protuberance on the cell—a
kind of little boss, as represented in Fig. 26, No. 1. This protuberance
goes on increasing, and assumes a spherical or oval form. At the same
time, there is a tendency in the points of attachment in the young cell
to meet—a kind of strangulation occurs (Fig. 26, No. 2). The junction
takes place a little sooner or later, according to the species (Fig. 26,
No. 3); the two individuals then separate (Fig. 26, No. 4). In certain
cases a single cell may give rise to several protuberances, and,
consequently, to several daughter-cells. Where there is only one
protuberance or bud we generally see it originate at the thick end and a
little on one side of the apex of the oval outline, which, in a greater
or less degree, characterizes the cells of the majority of ferments.
Certain authors have maintained that the method of budding which we have
just described, and which we think was first promulgated by
Mitscherlich, is merely an illusion, and that the cells of yeast break
up and scatter their granular contents, and that these scattered
granules eventually attach themselves to the cells, growing there, and
so giving the appearance of buds or daughter-cells. This error has been
revived quite recently.[83] Nothing can be less admissible. We could
count the number of yeast cells which we have seen undergo this process
of rupture in the course of some ten years of observation, every day of
which, we may say, thousands of these cells have passed under our eyes.
This breaking up of the cells is really of the most rare occurrence, and
may always be explained by some abnormal circumstance affecting the
yeast; being indeed a mechanical accident, not a physiological fact. We
may easily convince ourselves of this by growing some yeast in a
saccharine wort, filtered perfectly clear, and, consequently, deprived
of all granular amorphous deposit that might deceive the observer. The
cells will be observed to bud and multiply without exhibiting the most
minute appearance of granulation, or disruption; moreover, there will
always be cells of all sizes, ranging from the smallest visible up to
the largest. This very simple piece of observation may be made in all
the alcoholic ferments, and with any wort capable of fermenting, and in
its presence the hypothesis, which we have been repudiating, cannot hold
its own.
In Plate VII. (left side) there is represented a field of yeast,
magnified 400 times. We see a mass of disjointed cells, such as appear
after fermentations without sufficient aliment; of the kind represented
some are nearly spherical, others oval or cylindrical, more or less
elongated. If we mix a little of this yeast, of about the size of a
pin’s head, with wort, and put the wort into a small, shallow,
flat-bottomed basin, having a surface of about 1 square decimetre (10
sq. ins.) exposing it to the surrounding temperature, we shall find next
day the bottom of the basin covered with a fine white deposit, of the
forms of which we give a sketch in the right half of the plate. In this
it will at once be observed that the cells sown have lost their interior
granulations, having become more transparent and filled with a
gelatinous protoplasm. The principal difference between the two halves
of the plate consists in this, that whereas the cells in the left half
are isolated and granular, those in the right half are more inflated,
more transparent, and provided with buds, which may be seen in every
stage of development, from their first appearance till they become as
large as the parent cells. They continue to grow until they detach
themselves; then they bud in their turn, so that the same figure may
furnish examples of cells of the first, second, and third generations.
In the right half, the protoplasm contained in the cells exhibits
circular spots or vacuoles, which may be made to appear lighter or
darker than the rest of the cell by slight movements of the object-glass
of the microscope. These spots are due to a migration of the protoplasm
towards the sides; they commonly occur in yeast cells the vitality of
which, from deficient nourishment, has become suppressed—the shrivelled
appearance which they then assume being due to their being forced to
live upon themselves, so to speak. However, by introducing such cells
into a nutritive and aerated liquid the vacuoles quickly disappear.
[Illustration: Plate 7. Yeast-cells—Worn out and Dissociated (left),
after Revival in a Sweet Wort (right).]
In the ordinary yeast, as met with in breweries, the majority of cells
show one or more of these vacuoles; if, however, we place a little of
this yeast in an aerated wort, and watch under the microscope the
changes that occur in the cells, we shall witness, often in the course
of a few seconds, a kind of turgescence, a greater tension of the
cell-walls, which seem to grow thinner, and a complete disappearance of
the vacuoles. At the same time the interior gelatinous matter will
become filled with fine granulations that are scarcely visible, but
which at a certain distance appear brilliant. At the same time
protuberances begin to show themselves, and next day the budding will
have already become very active. The newly-formed cells will have such a
delicacy of aspect and contour as to be scarcely discernible in the
field of the microscope. There will also be a tendency to ramification
in the budding, which appearance will be more or less marked according
to the kind of alcoholic ferments present, as we shall see presently,
attaining its maximum in each case when the cells have been revived
after exhaustion by rest and want of food. In the latter case, the
process of rejuvenescence may be protracted; but this is not the case
with cells of commercial yeast, which is always used within a few days
of its formation. And thus, as I said a little ago, speaking of these
cells, they often manifest the first signs of their budding in a few
seconds.
In our preceding remarks we have expressly assumed that there are many
kinds of alcoholic ferment. This is, beyond doubt, the case, as we have
given incontestable proofs, first in 1862, in the _Bulletin de la
Société chimique_ of Paris, and later on, in 1864 and 1866, in a Note in
the _Comptes rendus_, on the diseases of wines, as well as in our
“Studies on Wine.” Moreover, we know that brewers have long recognized
two distinct methods of fermentation—“high” fermentation and “low”
fermentation—and two corresponding yeasts. It is true that the
differences presented by these fermentations were believed to be caused
by the different conditions under which they took place, and that it was
supposed that we might change “high” yeast into “low” yeast, or
inversely, by subjecting the first to a low temperature, or the second
to a high one. In our observations of 1862, which we have just
mentioned, we discovered that must gives rise to several yeasts; that
the ferment of “high” beer cannot develop except with great difficulty
in must, whilst one of the ferments of the grape grows rapidly and
luxuriantly in wort; that it is easy to isolate the smallest of the
ferments of the grape from its congeners, by subjecting filtered must to
fermentation; and finally, that the secondary fermentations of wines
which remain sweet furnish a remarkable ferment, very different in
aspect to the ferment of beer.
We have not given specific names to these different ferments, any more
than we have to the other microscopic organisms which we have had
occasion to study. This was not from any disregard for names, but from a
constant fear that, since the physiological functions of these minute
forms was the exclusive object of our study, we might be led to attach
too much importance to exterior characters. We have often found that
forms, having nothing apparently in common, belong to one and the same
species, whilst similarity of form may associate species far apart. We
shall give some fresh examples of this fact in the present paragraph. A
German naturalist, Dr. Rees, who has discovered new proofs of the
diversity of alcoholic ferments, putting aside, perhaps rightly, such
scruples, has attached specific names to the different kinds of
ferments, in his _brochure_ published in 1870, which we have already
cited (p. 71). Indeed, we have often ourselves, for brevity’s sake, made
use of the names proposed by Dr. Rees.[84]
[Illustration: Fig. 27.]
In a Note inserted in the _Bulletin de la Société chimique de Paris_, in
1862, we figured a ferment of small dimensions, which develops
spontaneously in must, filtered or unfiltered, and which is very
different from the ordinary ferment of wine. It is the first to make its
appearance in the fermentation of the grape, and may even appear alone
if the must has been previously well filtered, doubtless because its
germs, being smaller than those of other ferments, pass through the
filter more easily and in greater number. Fig. 27, extracted from our
Note of 1862, represents this ferment, together with some spherical
cells of _high_ yeast, with the object of giving a more exact idea of
the relative dimensions of these two ferments and their dissimilarities.
Dr. Rees has named it _saccharomyces apiculatus_.
The same savant has given the name of _saccharomyces pastorianus_ to the
yeast of the secondary fermentations of sweet liquids, such as wine that
has remained sweet after its principal fermentation. We have described
this yeast in a Note published in 1864, on the diseases of wine, from
which we give the following extract:—[85]
[Illustration: Fig. 28.]
“Fig. 6 (Fig. 28 in this work) represents a very interesting variety of
alcoholic ferment. It happens pretty often, especially in the Jura,
where the vintage takes place about October 15th, when the season is
already cold and little favourable to fermentation, that the wine is
still sweet at the moment when it is put into casks. This is especially
the case in good years, when the sugar is abundant and the proportion of
alcohol high, a circumstance which prevents the completion of
fermentation when effected at a low temperature. The wine remains sweet
in cask sometimes for several years, undergoing a continuous but feeble
alcoholic fermentation. In such wines we have always observed the
presence of this peculiar ferment. In form it consists of a principal
stem, forming nodes at various points, from which short branches arise,
ending in spherical or ovoid cells. These cells readily detach
themselves, and act as spores of the plant. It is rarely, however, that
we see so perfect a vegetation as we have represented, because the
different parts fall to pieces, as we have shown in the left half of the
figure.”
What is the origin of cellular plants of this remarkable type? Where and
how are the ferments of the grape generated?
In Chapter III. § 3 we were on our way to a solution of this question.
It has been shown that fermentation cannot take place in the juice of
crushed grapes if the must has not come into contact and been mixed with
particles of dust on the surface of the grapes, or of the woody part of
the bunch. It would, however, be sufficient that a vintage vat, of any
capacity whatsoever, should receive the particles of dust existing on a
single bunch in some cases, on even a single grape, for the whole mass
to enter into fermentation.
[Illustration: Plate 8. Fertile Mould-cells from the Outer Surface of
Grapes.]
What, then, we must ask ourselves, is the nature of these particles of
dust? On September 27th, 1872, we picked from a vine, in the
neighbourhood of Arbois, a bunch of grapes, of the variety called _le
noirin_. The bunch selected, without any injury to a single grape, was
brought to our laboratory in a sheet of paper that had been previously
scorched in the flame of a spirit lamp, and the grapes were cut off with
a pair of fine scissors, which had also been passed through the flame.
By means of a badger-hair brush, thoroughly purified in water, each
grape to which a portion of its peduncle remained attached, was washed
in a little pure water. The successive washing of a dozen grapes in 3
c.c. of water was sufficient to make the water turbid; we then examined
it under the microscope. Each field contained many little organized
bodies, accidentally associated, now and again, with some very scarce
crystalline spicules. As a rule, the organisms consisted of simple,
transparent, colourless cells; some, indeed, of larger size had a
yellowish brown colour, and were detached or united in irregular masses;
and, lastly, there were club-shaped or bottle-shaped vessels, full of
spores ready to germinate. We repeated this experiment with bunches of
other varieties of grape, and also submitted to examination water in
which the outer surfaces of gooseberries, plums, and pears had been
washed; the result was in each case the same, that is, we found a great
number of the same colourless cells, and the same irregular masses of
darker cells, which latter, however, we must not confound with the
masses of dead cells sometimes found covering parts of the epidermis of
certain fruits.
As we had purposely left each fruit attached to part of its peduncle, we
wished to ascertain if these corpuscles proceeded from the grapes or
from the wood of the peduncle. For this purpose we washed separately the
surface of the grapes and the woody part of the bunch. The water in
which the latter was washed was visibly more charged with the minute
organisms than that in which the grapes was washed, although the latter
was by no means free from them.
Plate VIII. represents these corpuscles as they exist on the surface of
fruits, magnified 500 times. The groups, _b_, _b_, _b_, ..., _c_, _c_,
... are of a brown colour, more or less dark, or of a reddish yellow;
the cells _a_, _a_, ... are transparent. Amongst them are some spores of
ordinary fungoid growths, and several cells which are probably the issue
of a germination that had commenced in certain groups which have a hard,
yellowish appearance, and which are provided with what seems to be a
double case—_b_, _b_, _b_, ..., _c_, _c_, ..., a result of the moisture
of the woody part of the bunch, or of rain that fell just before the
commencement of our observations.
It is an easy matter to trace the germination of these different
varieties of cells with the microscope. We put a drop of the water in
which the woody part of a bunch of grapes has been washed into a small
quantity of wort, previously boiled and filtered bright. Plate IX.
presents a series of developments observed in the case of simple or
grouped cells, A, D, G, and J. The process is as follows: The
yellowish-brown cells soften and grow larger in the nutritive medium,
and gradually become almost transparent and colourless. At the same time
we see some very young buds appear on their margins; these rapidly
increase in size, and detaching themselves to make room for others, move
off as young cells that after a time bud in their turn. The rapidity
with which these cells bud and multiply is often extraordinary. The
group A and the cell D produced the groups C and F within twenty-four
hours, passing through the intermediate stages represented in groups B,
E. The cells A and D did not give rise to any filamentous growths, at
least whilst under our observation. Some groups of cells, however, put
forth, from the first, long filaments, having cross-partitions and
resembling the mycelium in ordinary fungoid growths. Together with
these, and along their whole length, was an abundance of cells, often in
clusters, as represented by Fig. G, the whole of which growth took place
in less than twenty-four hours.[86] But apart from contact with the air,
there was a complete absence of life.
[Illustration: Plate 9. Various Examples of the Mode of Growth of
Mould-cells from the Outer Surface of Grapes.]
The figures H, I, J, K, represent other aspects of developing cells and
filaments. The cells H are spherical; the cells I have numerous buds, as
also have those marked K. These different forms were all produced in the
course of twenty-four hours by the cell which may be observed in the
centre of the group J. In connection with this same group, J, we may
remark that on September 30, 1872, at 10 A.M., we witnessed the
detachment of three oval cells at the points _a_, _b_, _c_; by 10.45
other buds of the size represented in our engraving had formed in their
place; by about five o’clock that same afternoon these buds, _a_, _b_,
_c_, having become transformed into cells, fell off in their turn.[87]
It may be asked, what proof have we that amongst the filamentous and
cellular growths which spring from the small, dark bodies existing in
the particles of dust adhering to the surface of fruits, and which we
here see bud and multiply with such marvellous rapidity, the ferment or
ferments of vintage do actually exist? A very simple experiment will
prove conclusively that this is the case. When in the course of
twenty-four or forty-eight hours, by contact with saccharine must, and
in presence of excess of air, the revival and development of the cells
has taken place on the bottom of the little troughs employed in our
observations; if then we fill up the trough with the same must, so that
there remains no free air under the cover-glass, within a very short
time—an hour, half-an-hour, or often less—we shall see bubbles of gas
rise from the bottom, accompanied by an increase in the deposit of
cells. This will be the must fermenting after the submersion of the
cellular plants. It follows that the cells, or groups of cells, of a
dark colour which cover the grapes, or the woody part of the clusters,
are actual germs of the cells of yeast; more correctly speaking, that
germs of yeast-cells exist amongst these groups, for it would not be
consistent with truth to say that the various germinating forms present
in the dust on the surface of grapes must all of them give rise to
actual corresponding ferments. Thus the flask-shaped spores _c_, _c_,
... in Plate VIII., are reproductive organs of _alternaria tenuis_,
which have probably nothing in common with alcoholic ferment or
ferments, properly so called, except their outward form. We may repeat,
however, and it is a point of great importance to bear in mind, that the
cells of yeast originate from some or other of the little, brownish,
organized bodies, which the microscope reveals in such numbers amongst
the particles of dust existing on the surface of fruits.
The impossibility, which we have already demonstrated (Chapter III. §
3), of making grape juice ferment apart from the action of external
particles of dust, and the knowledge which we have just acquired, that
the particles of dust on the surface of the grapes and woody peduncles,
at the moment when the grapes have attained maturity, contain certain
reproductive cells which give rise to certain ferments, naturally lead
us to the investigation of another point, which concerns the period at
which these germs make their appearance on the different parts of the
vine plant. The two following experiments tend to prove that the ferment
can only appear about the time when the grapes attain maturity, and that
it disappears during the winter, not to reappear before the end of the
following summer.
I. In the month of October, 1873, we procured from a vineyard in the
canton of Arbois some of the woody parts of very ripe clusters of
grapes, taking the precaution to cut off all the grapes, one by one,
with a very clean pair of scissors, whilst still on the vine; we then
wrapped up the woody parts of the clusters, thus deprived of their
grapes, in thin paper, to convey them to Paris. Our only object at that
time was to secure for use in our subsequent studies the ferment-bearing
dust found in October on the woody part of the vine, and, more
particularly, on the clusters themselves, as already stated. After our
return to Paris, and during the course of our experiments in October and
November, it sufficed to wash a few scraps of the bunches in a little
pure water, in order to obtain the grape-ferment in abundance; but later
on in the winter we were astonished to find that the same procedure
yielded no ferment, only some moulds. The bunches which, when put into
boiled and filtered must, in October, very readily caused that must to
ferment, at the end of winter could no longer produce the same effect,
however favourable might be the temperature to which we raised the must.
The particles of dust on the bunches had, therefore, become sterile, as
sources of alcoholic ferments.
II. On February 17th, 1875, we purchased of Chevet, a dealer in
provisions, two bunches of white grapes, which were perfectly sound,
presenting not the slightest trace of injury or bruise. We took an iron
pot full of mercury, which had been heated to 200° C. (392° F.), and
then covered over its surface with a sheet of paper that had also been
subjected to flame. When the mercury had cooled down we placed several
of Chevet’s grapes, singly and in small bunches, on the surface of the
metal, and, after having enclosed them in a glass cylinder that had been
previously heated with and by means of the mercury, we crushed them in
this vessel, in contact with air, by means of a strong, crooked iron
wire that had been passed through the flame of a spirit lamp. The object
of all these precautions was to prevent any cause of error, such as
might have resulted from the accession of particles of dust associated
with the mercury, or floating about our laboratory. We then placed our
cylindrical jar in an oven, at a temperature of 25° C. (77° F.); but
though the experiment was continued for several days following, no
fermentation manifested itself. At last, to assure ourselves that the
pulp and liquid were, notwithstanding this, well adapted to
fermentation, we introduced into the test-flask an almost imperceptible
quantity of yeast. This readily developed, and promptly produced
fermentation.[91]
It seems possible, therefore, that the germs of ferment may not exist on
bunches of sound grapes during winter, and that the well-known
experiment of Gay-Lussac on the influence of air on the fermentation of
the must of crushed grapes cannot succeed at all times.
The following observations will afford more than sufficient proof of
this statement, being, after all, but an easy method of carrying out
Gay-Lussac’s experiment, without having recourse to the use of mercury.
It may already be inferred from the preceding facts that there must be,
in the course of the year, between the end of winter and autumn, a
period when the vegetation of the cellules from which yeast proceeds
undergoes a revival. When does this period occur? In other words, how
long after winter does sterility of the plant continue, until it is
again capable of yielding ferment? To ascertain this, we conducted
numerous experiments during the summer and autumn of 1875 and the winter
of 1876. Having to conduct them in a vine-growing country—in the
vineyards of Arbois, Franche-Comté—at a distance from our laboratory, we
were compelled to adopt a simple form of apparatus for our experiments,
which, besides being very convenient, was at the same time sufficiently
exact for the object we had in view.
[Illustration: Fig. 32.]
Into common test-tubes we poured some preserved must; we then boiled it,
with the object of destroying all the germs that it might contain, and
then, having passed the flame of a spirit lamp over the upper sides of
the tubes, we closed them with corks which had been held in the flame
until they began to carbonize (Fig. 32). Having provided ourselves with
a series of tubes prepared in this manner, we carried them to a vine,
and there dropped into some of them grapes, into others bunches, from
which we had taken all the grapes, by cutting their peduncles; into
others, fragments of leaves or the wood of the branches. The corks were
again passed through the flame and replaced successively in each tube.
Some of the grapes we dropped in whole, some we crushed at the bottom of
the tubes with an iron rod that had previously been passed through the
flame; others, again, at the same moment that we introduced them into
the tubes, were cut open with scissors, likewise passed previously
through the flame, so that a portion of their interior juice might mix
with the must in the tube.
Our experiments gave the following results:—As long as the grapes were
green, about the end of July and during the first fortnight of August,
we obtained no fermentation in our must. Between the 20th and 25th of
August a few tubes underwent fermentation, by the action of the little
apiculated ferment; and in the course of September the number of tubes
that fermented increased progressively. In each series of tubes,
however, we always found a few in which there was a complete absence of
fermentation.
Here are a few actual examples. In the beginning of September we placed
grapes in thirteen tubes, into some whole, into others crushed ones,
taken from bunches of the variety known as the _ploussard_, the fruit
being already sufficiently ripe to be very pleasant to the taste. All
the tubes of this series failing to give us any trace of fermentation,
or anything besides ordinary moulds—which indeed appeared in all our
experiments, whether there was or was not fermentation—we began a new
series of experiments, under similar conditions, on September 28th, as
follows:—
Nos. 1, 2, 3 and 4 tubes containing one uncrushed grape.
No. 5 tube containing two uncrushed grapes.
No. 6 tube containing two crushed grapes.
No. 7 tube containing two crushed grapes, in 2 c.c. of water previously
boiled.
No. 8 tube with a fragment of a bunch from which grapes had been cut,
and occupying the entire depth of liquid.
No. 9 tube with a fragment of wood from a branch.
Nos. 10, 11, and 12 tubes with a fragment of leaf.
On September 29th and 30th there was no appearance of fermentation in
any of the flasks, but all contained flakes of fungoid mycelium. On the
1st of October fermentation more or less marked and active occurred in
2, 3, 4, and 5, in which uncrushed grapes were, accompanied by a general
turbidity of the liquid, and a suspension of the development of the
fungoid growths. It was still absent in 1, 6, and 7, of which the first
contained an entire, the latter crushed grapes. No. 8, containing the
woody part of the bunch, was in active fermentation. Nos. 9, 10, 11, and
12, with fragments of branch or leaves, showed no signs of fermentation.
The following day No. 1 was fermenting; but from October 5th onwards
there was no alteration in the number of fermenting tubes.
In this series we determined the presence of the small apiculated form
of yeast (_S. apiculatus_) in the tubes that fermented, only once
finding it associated with _saccharomyces pastorianus_.
We need hardly say that the grapes which we employed were perfectly
ripe, the vintage having already commenced in some of the Jura cantons.
This experiment shows that, even when the grapes are perfectly matured,
it by no means follows that each individual grape must carry germs of
ferment, and that some grapes may be crushed, in some instances several
together may be crushed, without being able to set up a fermentation. In
the presence of these novel facts, those who support the hypothesis of
the transformation of the albuminous matter contained in the juice of
grapes into yeast will no doubt admit the untenability of their
opinions, since their hypothesis requires that every grape or number of
grapes, when crushed, should ferment, in contact with air.
On the same day we prepared another series of tubes, using grapes of a
variety called the _trousseau_.
Nos. 1, 2, 3, and 4 tubes containing one whole grape.
Nos. 5 and 6 tubes containing some of the wood of a branch.
No. 7 tube containing some of the wood of a branch from which the grapes
had been detached.
In the course of the following days fermentation took place in 4, 5, and
7.
In this case three out of four of the uncrushed grapes did not cause the
must in which they were placed to ferment; whilst the same must
fermented in one of the two tubes containing wood of the branch, and in
the other remained unchanged; and, lastly, the tube containing the woody
peduncles of the bunch fermented.
We have already remarked that it was more particularly the wood of the
bunch that was charged with germs of ferment. The truth of this
assertion was proved by the following series of experiments.
On October 2nd, 1875, we charged at the vineyard twenty-four tubes, all
of which were about a third filled with pure must that had been
previously boiled.
Nos. 1, 2, 3, 4, 5, and 6 tubes containing one crushed grape.
No. 7 tube containing two crushed grapes.
No. 8 tube containing one crushed grape.
Nos. 9, 10, 11, and 12 tubes containing some wood of a branch of the
vine.
Nos. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 tubes containing
a fragment of the wood of a bunch from which the grapes had all been
detached and removed.
In the course of the following days some of these tubes began to
ferment; but in others only fungoid mycelia were visible.
On October 7th the following tubes were fermenting: the second of the
first eight containing whole or crushed grapes; not one of the four that
contained wood of the branches; whilst, on the other hand, of the tubes
containing wood of the bunches, 15, 17, 20, 21, 22, 23, and 24 were all
in full fermentation. In short, fermentation, and therefore germs of
yeast, were present in one single tube out of eight containing grapes;
in none of the four tubes containing wood of the vine branch; but in
seven out of the dozen containing wood of the bunches. There was no
subsequent change in the number of tubes that fermented.
The same day that we arranged this series of tubes we prepared
twenty-four other similar ones, using the _wood of bunches preserved
from the vintage of the preceding year_. Not one of these twenty-four
tubes showed the least sign of fermentation, although they contained
grape juice in presence of the wood of the bunches; this was a further
confirmation of the sterility of the germs of ferment in the case of
bunches of grapes preserved for a sufficient time.
The next question to be considered was, what length of time after the
vintage do the germs on the surface of the woody part of bunches of
grapes preserve the faculty of producing yeast? The following
experiments were undertaken to determine this point.
We have just seen that on October 2 fragments of the woody peduncles
introduced with must, on the spot, caused that must to ferment in seven
cases out of twelve. In order that we might test the wood of these same
bunches during winter we took care to wrap some fragments up in paper
previously passed through the flame. We afterwards took occasion to test
portions of these fragments as follows:—
On December 21st, 1875, we conducted an experiment with twelve. On the
following days all began to show flakes of mycelium, or numerous
multiplying cells of _mycoderms_, _torulæ_, and _dematium_; and only
four subsequently produced yeast and alcoholic fermentation. From this
we may conclude that, three months after the vintage, a large number of
the germs of yeast spread over the woody part of the bunches lose their
vitality, through desiccation by the surrounding air, since two-thirds
of the examples taken had become sterile by that time.
On January 21st we conducted a similar experiment with twelve other
tubes. At a temperature between 20° and 25° C. (68° to 77° F.)
fermentation occurred in only two of them. On March 2nd we undertook
another experiment, again using twelve tubes, and again fermentation
occurred in two tubes.
By the beginning of April the sterility was absolute. At that period of
the year (April and May) we made numerous experiments of this kind,
using the woody parts of fresh bunches of grapes and white grapes
preserved from the last vintage, plenty of which were still to be had in
a state of freshness at the provision store. We also operated on some
wood obtained from a vineyard at Meudon. In a great number of cases no
fermentation occurred; it even happened that a whole bunch of fresh
black grapes, very ripe, which were bought at Chevet’s on April 16th,
and which had been grown in a hot-house, after having been crushed, did
not ferment at all.
Up to March not one of the tubes containing the wood of dry bunches
brought from the Jura, which had fermented, showed any signs of
_apiculatus_ or of _pastorianus_, or anything besides the ordinary low
yeast of wine, _saccharomyces ellipsoideus_.[93]
It would be a study of much interest to determine if yeast exists on
other species of plants besides the vine. During the winter we could
discover it on no others. Once during the winter, experimenting on box,
we obtained fermentation in one of our tubes which contained must. In a
great number of other experiments we obtained nothing besides moulds and
growths of _dematium_, _alternaria_, and _torulaceæ_.
Our observations in Chap. III § 6, taken in connection with those which
we have just made, prove that the yeasts of fermentation, after being
dried, preserve the faculty of germination longer than the germ-cells
which are scattered over the dead wood of the vine.
As might be expected, a microscopical examination of the particles of
dust scattered over the surface of the fruit and woody peduncles of
grapes, reveals great differences in the number of these fertile
particles at different periods of the vegetation of the grapes. As long
as the grapes are green and the vine in full activity we find scarcely
any, or, at all events, very few spores which seem to belong to ordinary
fungoid growths. Towards autumn, however, when the grape is ripening and
the leaves becoming yellow, fungoid growths and numerous productions of
great fertility accumulate on the vine, on the leaves, the branches, and
the bunches. At this period we find the water in which the grapes and
the woody parts of the bunches are washed swarming with different kinds
of organized corpuscles; it is at this period, too, that the
ferment-yielding moulds attain that phase of their vegetation in which,
when mixed with the juice of grapes, they produce fermentation.
In the Jura district a peculiar kind of wine, called straw wine (_vin de
paille_), is manufactured, which seems to contradict what we have said
as to the advent of sterility towards the end of winter in the
yeast-germs formed on the surface of preserved bunches of grapes. This
straw wine is made of grapes preserved for long after the vintage on
straw. From what we have said it might be supposed that fermentation
could not occur under these circumstances. We have, in fact, no doubt
that it is often really produced by quite different yeast-germs from
those which cause fermentation in the vintage gathered in autumn.
Fermentation as effected in the manufacture of straw wine is probably
due to yeast-dust spread over the utensils of the vine-grower, and
derived from the preceding vintage. We have seen (Chap. III. § 6) that
yeast may be dried and reduced to powder, and yet preserve its faculty
of germination for several months. It would be useful, however, to
submit this surmise to the test of experiment, and it would be easy to
do so provided we took care to crush the grapes so preserved in very
clean vessels, previously heated to a temperature of 100° C. (212° F.),
having first rejected every bunch containing injured grapes, which might
have fermented or given occasion to the development of yeast.
Fermentation, we believe, would not then take place.
[Illustration: Plate 10. One of the Ferments of Acid Fruits at the
Commencement of Fermentation in its Natural Medium.]
Another consequence results from the various facts that we have brought
out in relation to the origin of the wine-ferments, which is, that it
would be easy to cultivate one or more vine-stocks so that the grapes
gathered from them, and crushed to extract their juice, would be unable
to ferment spontaneously _even in autumn_. For this purpose it would be
sufficient to keep the bunches out of contact with particles of dust
during the vegetation of the bunches and the ripening of the grapes, and
then to effect the crushing in vessels thoroughly freed from germs of
alcoholic ferments. Moreover, every fruit and every vegetable might be
submitted to important investigations of this kind, the results of
which, in our opinion, could hardly be doubtful.
The following observations, which relate to the polymorphism of
_saccharomyces pastorianus_, seem to me to have an important bearing on
the history of alcoholic ferments, as presenting a close analogy between
the species of ferment and fungoid growths of a higher order, for
example, such fungi as _dematium_, which are generally found on dead
wood; and we would say that between the vine and other shrubs there is
only this difference, that amongst the _dematium_ forms of the vine
there occur one or more which are anaërobian, at a certain period of the
year, whilst, on the other hand, the _dematia_, _alternaria_, &c., of
other shrubs are more generally aërobian. There would be nothing
surprising in this result, considering that amongst the mucors, for
instance, we find both aërobian and anaërobian forms, and that there are
likewise torulæ-ferments or anaërobian forms, as well as torulæ-forms
exclusively aërobian.
When _saccharomyces pastorianus_ begins to develop from its natural
germs, such as are scattered over the surface of acid fruits, it takes
the form of elongated jointed filaments, branching, often pear-shaped,
and more or less voluminous. In proportion as the oxygen held in
solution in the liquid disappears and the buddings are repeated, the
length and diameter of the filaments and cells diminish, and such is the
transformation that we might, at last, suppose that we were dealing with
a different ferment of smaller dimensions.
Plate X. represents this ferment, at the commencement of fermentation in
cherry juice. In the course of a short time there is nothing to be seen
but cells of comparatively small size, disjointed and round or oval, and
filaments comparatively short and slender. This appearance is indicated
in our drawing by the cells _a_, _a_, _a_. As these latter forms
multiply with great rapidity, we soon have to search widely over the
microscopic field before we find any of the long forms from which they
spring. Instead of the forms given in Plate X., we have only those
represented in Plate XI. In other words, the aspect of these ferments
changes daily, from the very commencement of fermentation. Thus the
yeast would appear to grow smaller, coincidently with the progress of
fermentation passing from a condition in which it consists of large
cells and long ramified filaments, to a condition in which the cells are
small and the filaments short. These changes are principally due to an
alteration in the method of budding and in the life-processes of the
yeast, which speedily exhibits itself when the air supply is reduced,
and not through any intermixture of foreign ferments. So, at least, all
our observations up to the present time lead us to believe. As soon as
the oxygen has been absorbed the cells which form are oval or globular,
and the filaments do not lengthen or become so plump.
[Illustration: Plate 11. accharomyces Pastorianus, in course of Regular
Growth.]
This is, however, not the only cause of these changes in form and
aspect, although the presence of air, in greater or less quantity, has a
marked influence on the earlier developments of yeast; there is another
circumstance to be taken into account, difficult indeed to state
shortly, but which is demonstrated clearly by the microscope, and is
connected with the actual state of the germ cells. As a general rule the
budding of a cell is not an identical process when the cell is quite
young, and when it has become exhausted from want of nourishment.
Between these two conditions there is a difference which may be compared
with that which exists, for example, between a newly-formed grain which
would not germinate, and the same grain matured by rest, if we may use
the expression, that is, which has been kept long enough for its
germination to be possible. In other words, and as far as our subject is
concerned, we are not to expect that, by reviving our old yeast cells
and putting them to grow with abundance of air, in a saccharine,
nutritive medium, we shall obtain the appearance of the earlier
developments of the germ-cells on the surface of sweet and acid fruits.
We see this clearly in Plate VII., the right-hand half of which
represents the recruited budding of cells, such as those represented in
the left-hand half, in a medium peculiarly adapted to their vitality,
_and in the presence of much air_. As regards the length and size of
filaments and cells, there is little appreciable difference between the
two sides. The principal difference consists in the relative freshness
and the budding going on in individuals in the right-hand half.
There is a simple means of transforming the small, disjointed forms of
the yeast as it occurs in a deposit, at the end of a fermentation, back
into the long, tubular, pear-shaped forms peculiar to the germination of
the germ-cells, which exist amongst the particles of dust spread over
the surface of fruits. Plate X. illustrates the result of the process.
For this purpose we must effect as complete an exhaustion as possible of
the ferment _saccharomyces pastorianus_, by leaving it to itself for a
very long time, without aliment, in contact with pure air, in a damp
state; or, better still, in presence of sweetened water. We cultivate
some yeast in wort, in one of our two-necked flasks, and then carefully
decanting the fermented liquid through the right-hand neck, leave the
deposit of yeast on the sides of the flask. The glass stopper which
closes the india-rubber tube must be replaced, and the moist yeast be
left thus, in contact with pure air. The cells will steadily continue
their activity, and so gradually age, without meanwhile losing their
vitality. We use the word _age_, as we have already observed, because
the period of rejuvenescence in the case of such a yeast is so much the
slower the longer the plant has remained in that state.
Under these conditions the yeast rarely dies. It becomes attenuated and
shrivelled but still preserves its vitality, that is, the power of
reproducing itself after a lapse of several months or even several
years. In the end, however, it dies, a fact which is proved by the
cells, when sown in a nutritive medium, remaining inert.
To exhaust yeast, without destroying it, sweetened water is preferable.
Having decanted the beer, we substitute in its place water sweetened
with 10 per cent. of pure sugar. By effecting the substitution in the
following manner, we escape the risk of introducing germs from floating
particles of dust, which would nullify all experiments of this kind. We
prepare, then, a flask containing sweetened water, free from all foreign
germs, which we attach to the other flask [_i.e._, two of M. Pasteur’s
flasks with two necks, one straight and wide, the other bent and narrow
(Fig. 8)]. This is done by taking the india-rubber tube off the flask
containing yeast, and removing the glass stopper from the other
india-rubber tube attached to the flask containing the sweetened water;
then, introducing the right-hand neck of the yeast flask into the
india-rubber tube connected with the other, we raise the latter flask so
as to pour the sweetened water on to the yeast. At the same time an
assistant passes the flame of a spirit-lamp over the bent part of the
curved tube attached to the water flask, with the object of destroying
the vitality of the germs in the floating particles of dust, which enter
the flask in proportion as it is emptied into the other.
The sweetened water, which is thus brought into contact with yeast of
greater or less freshness, soon begins to ferment. Fermentation
accomplished, the vinous liquid is decanted and replaced by fresh
sweetened water, which ferments in its turn, although even at this stage
with greater difficulty than the first; this second dose is again
decanted, and again replaced by fresh sweetened water, and this process
is repeated three or four times. The yeast becomes weaker and weaker,
and eventually is unable to cause any fermentation in sweetened water
poured on it.
This exhaustion of yeast in sweetened water may be produced more quickly
by the following means:—It is sufficient to sow a mere trace of pure
yeast in a large quantity of sweetened water, say 100 c.c. (nearly four
fluid ounces), that is, instead of pouring the contents of a bottle of
sweetened water upon the whole deposit of yeast in the flask which
contained the fermented wort, we simply take a little yeast, by means of
a fine tube, from the deposit at the bottom of the flask, and introduce
it into the flask of sweetened water. This large proportion of liquid is
itself sufficient to exhaust the small quantity of yeast, quickly
checking the feeble fermentation which it had induced, so feeble indeed
as frequently not to be detected by the eye, from the fact that the
amount of liquid present is more than sufficient to dissolve any bubbles
of carbonic acid gas that might otherwise have been liberated.
It is a remarkable fact that the yeast, which during its protracted stay
in the sweetened water becomes enfeebled to such a degree that it can no
longer excite the least fermentation in that water, but will remain in
its presence for an indefinite time in a state of inert dust, does not
die. In some of my experiments the yeast has remained alive in the
sweetened water for more than two years.[94] It is almost unnecessary to
point out that these results are altogether out of keeping with the
various properties that are usually attributed to yeast.[95]
In these experiments we may use yeast-water[96] instead of water
sweetened with sugar. Into some flasks of pure yeast-water we put a
little yeast, taking all precautions to prevent the introduction of
foreign germs. No fermentation results, there being no sugar present;
the yeast, however, begins to bud, and this budding is more or less
marked according to the quantity of carbohydrate food which we introduce
along with the specimen. An interior chemical action also goes on,
causing a gradual change in the aspect of the yeast. The plasma of the
cells collects about the centres, assuming a yellowish-brown colour,
becoming granular, and forming within the cells masses more or less
irregular in shape, very rarely spherical.
We may observe here that these conditions seem to be peculiarly adapted
to show the character of the interior sporulation of the cells
discovered by Dr. Rees. Notwithstanding this we have never succeeded in
finding it distinctly, under these circumstances.
The fact which should claim all our attention, we repeat, is, that this
exhausted, shrivelled-up, aged-looking yeast preserves its faculty of
germination for several years; that, moreover, this faculty may be
aroused by placing it in aerated nutritive media, in which case it will
exhibit all the peculiarities which, under similar conditions,
characterize some of the germ-cells found on the surface of our sweet
domestic fruits. In other words, this yeast, instead of multiplying, as
it always does in the course of several growths in saccharine musts, in
the form of cells which detach themselves readily as soon as they have
nearly attained the form and size of the mother-cells, begins to shoot
out into such beautiful forms as those of _dematium pullulans_,
producing like that ferment long, well-grown, branching filaments, as
well as plump and frequently pyriform cells, as represented in Plate X.
The following figures (33 to 37) and descriptions of the observations to
which they relate will furnish fresh proofs of our assertions. In these
figures we see _saccharomyces pastorianus_, which has been exhausted in
sweetened water or in yeast-water, undergo revival in saccharine musts,
give rise to elongated, branching, pear-shaped forms, such as belong to
the original ferments of fruits, and afterwards assume the most minute
forms that we find in fermentations progressing or completed.
Let us examine Fig. 33. The history of this growth is as follows:—
Some spontaneous yeast which, after repeated cultivation, had acquired
the aspect represented in Plate XI.—which aspect the _saccharomyces
pastorianus_ generally assumes under these circumstances—was exhausted
in sweetened water, and subsequently revived in must at 10° C. to 11° C.
(51° F.). At this temperature germination was not very marked before the
end of eight days; at a temperature of 20° C. (68° F.), it only took
three days, under similar conditions. The sketch includes but one of the
long branches from which the ferment cells and the budding joints took
rise, but there were a great number more. Some of the forms represented
in the figure bear a striking resemblance, it appears to us, to some of
those of _dematium_, in Plate IX.; and even we may trace out the several
peculiarities of form which distinguish the figures in the latter plate.
[Illustration: Fig. 33.]
The next figure (34) represents the earliest forms of germination of
another specimen of _saccharomyces pastorianus_ in wort, after it had
been exhausted by four successive growths in sweetened water. We here
see the large ferment-form which appears at the commencement of
fermentation, in acid fruits, such as cherries and gooseberries (Plate
IX.), associated with smaller forms, which follow it and emanate from
it, in proportion as the process of budding is repeated. The field was
covered with this minute form, and we had to search about considerably
before we could find any of the large cells and the long, branching,
jointed filaments which we have sketched. The reason of this was, that
these large, extended filaments only appear at the beginning, when there
is still an abundance of air, giving place, after repeated budding, to
minute cells or short filaments, the ever-increasing number of which
soon hides the others from sight.
[Illustration: Fig. 34.]
[Illustration: Fig. 35.]
Fig. 35 represents _saccharomyces pastorianus_ again, as it appears
after having been exhausted by two years preservation in yeast-water, in
contact with pure air. Strange to say, it has lost its elongated
appearance, and would appear to have originated from a round ferment.
The cells are much exhausted, and most of them seem to have a double
border; their interior is very granular and of a yellowish colour. One
might readily take the specimen to be a dead old ferment, which,
however, it by no means is.
Fig. 36 represents the germination of this ferment, which had previously
been revived in a flask of wort, at the temperature of the air, in May,
1875. The following are the details of our observation:—
We sowed a trace of the exhausted yeast (Fig. 35) in a flask of wort on
May 16th. The sketch (Fig. 36) was made on May 19th, but on the 18th
there was a sensible revival. It will be seen how much the little
ferment had developed in the course of three days from the time when the
process commenced. If we had waited a few days longer before taking our
sample, we should probably have had difficulty in finding any cells or
filaments of the large ferment form, as there would have been so few of
them in comparison with the others.
[Illustration: Fig. 36.]
In the above figure we should remark the chain of large cells and
long-jointed processes, _a_, _b_, _c_, _d_ : _d_ is one of the cells
that we sowed; it has become transparent, and its contents, which are
slightly granular, have lost their brownish tint; _c_ is a large cell
which sprang from the preceding one; its outline is clear, and it is
full of fine, yellowish granules, which present a perfect resemblance to
the large ferment-cells of fruits, proceeding from the germ-cells on the
surface of those fruits, when it begins to appear in sweet juices; _b_
is a long filament, sprung from the preceding cell; and, last of all,
_a_ is a joint and its bud, in which the border is not yet very clearly
defined; it has scarcely any granules, and is finer than the others,
belonging, in short, to the small ferment form represented in Plate XI.
Here, then, we see the transition of the large ferment to the small, on
the same branch, after two generations from the germination of the
germ-cell _d_. This observation corroborates the opinion maintained by
us, that in Figs. 33, 34, 36, as in Plate X., we have not a mixture of
two ferments, the one consisting of large, elongated filaments, the
other of small cells, but one and the same ferment, the differences in
the form and size of which depend on particular conditions. The smallest
ferment-form very soon becomes the only one visible, and it preserves
its peculiar appearance in successive growths from inability to return
to the full, elongated, filamentous forms before undergoing a prolonged
exhaustion. The ferment of _mucor_ would probably afford similar
indications: it would be very interesting to find out.
[Illustration: Fig. 37. 1 Div. = 1/450th of millimetre (1/11250th of
in.).]
The following is one of the most curious of the forms presented by
_saccharomyces pastorianus_, occurring after exhaustion in a sweet
mineral liquid. The ferment, taken from a closed vat, in which it had
been used for beer, was sown in the mineral liquid on July 4th, 1873.
The following days the ferment developed feebly, but perceptibly, and
gradually increased in bulk. The flask was left to itself in an oven at
25° C. (77° F.) until December 3rd, when we ascertained that all the
sugar had fermented. We then sowed a trace of the deposit, which had
become abundant, in a flask of pure wort. On December 4th there was no
perceptible change. On December 5th, however, fermentation was in active
progress; a large quantity of froth covered the surface of the liquid,
and a considerable deposit of ferment had already taken place at the
bottom. We made a microscopical examination of this deposit, a sketch of
which we append (Fig. 37). The dark, double-bordered cells are those
which were sown but did not rejuvenesce. We may notice in different
places several of these same cells, recognizable by their granular
contents, which they are beginning to lose, to make room for germinating
cells and joints, often numerous. For instance, in the group at the
bottom of our figure one of the cells is in course of rejuvenescence and
germination, and has given rise to no fewer than six cells, filaments,
or groups of filaments. In different fields of our microscope we met
with a crowd of branches, more or less ramified, and chains of cells, of
greater or less length, of which we have sketched a few. In proportion
as the budding of these branches is repeated, the cells and joints
become more readily disunited, grow small, and assume the appearance of
_saccharomyces pastorianus_ in ordinary growths, almost as represented
in Plate XI. At first, when the old, exhausted cells begin to germinate,
their appearance rather resembles that of _dematium pullulans_, as seen
in the germination of many of the corpuscles on the surface of clusters
of grapes or fruits, or their woody parts, some specimens of which are
to be found in Plate IX.
We may briefly summarize the leading facts demonstrated in the above
paragraph. We have seen that there are different alcoholic ferments. In
the fermentation of natural saccharine juices, which, especially when
acid, so readily undergo a decided alcoholic fermentation, the ferments
originate in certain germ-cells, which are spread in the form of minute
spherical bodies of a yellow or brown colour, isolated or in groups,
over the exterior surface of the epidermis of the plant, and which are
gifted with an extraordinary power of budding with ease and rapidity in
fermentable liquids. The presence of atmospheric oxygen is indispensable
to the germination of these germ-cells, a fact which explains
Gay-Lussac’s observation that atmospheric oxygen is necessary for the
commencement of spontaneous fermentation in must.[97] Of these various
ferments one deserves special mention—namely, the variety termed
_saccharomyces pastorianus_. As is the case with all ferments, when we
gather it from the deposits produced in must that has been fermented by
its action, it is composed entirely of oval or spherical cells or of
short joints. When again placed in a similar must it buds, like all the
ordinary ferments, and the buds detach themselves from the joints or
mother-cells as soon as they have attained the size of these latter,
from which time in the new deposit is reproduced the original
ferment-form from which it sprung, and so on. Under certain conditions
of exhaustion, however, which may be easily obtained, and which we have
already accurately described, the cells undergo an absolute change as
regards their capabilities of budding and germinating. Each cell,
modified in its structure by the conditions we have mentioned, shows a
tendency to shoot out all around its surface, with astonishing rapidity,
into a multitude of buds, from many of which spring branching chains,
covered in parts, and more especially at the internodes, with cells and
jointed filaments, which fall off and bud in their turn, soon to present
the forms of the yeast deposit. In this way _saccharomyces pastorianus_
seems to afford a kind of bond of union between the race of ferments on
the one hand, and certain kinds of ordinary fungoid growths on the
other. Of these latter the plant which De Bary has named _dematium_, and
which is generally found on the surface of leaves or dead wood, more
especially, however, on the wood of the vine at the end of autumn, the
time of the vintage, presents a striking example.
There seems every reason to believe that at this period of the year one
or more of the varieties of _dematium_ furnish cells of yeast, or even
that the ordinary aërobian varieties of _dematium_ produce at a certain
stage of their vegetation, in addition to aërobian cells and torulæ,
other cells and torulæ which are anaërobian, that is, alcoholic
ferments.
In this manner we arrive at the confirmation of an idea entertained by
most authors who have studied yeast closely—namely, that it must be an
organ detached from some more complex vegetable form. We may also add
that in the case of _saccharomyces_ the chains of filaments, both
tubular and fusiform, and septate cells more or less pyriform
originating in them, when attentively observed, remind us forcibly of
the filamentous chains and spore-balls, or conidia of _mucor racemosus_
when submerged, so that one might suppose that the spore-ferment of our
_dematium_ is itself an organ detached from some still more complex
vegetable form, in the same way that conidia-ferment of _mucor
racemosus_ belongs to that more complex fungoid growth.
In the following passage De Bary uses, for the first time, the words
_dematium pullulans_ (Hofmeister, vol. ii. p. 182, 1866). The German
naturalist begins by citing the opinions of Bail, Berkeley, and H.
Hoffmann, the first of whom maintains that _mucor mucedo_ becomes
transformed into the yeast of beer, the second that yeast is a peculiar
state of _penicillium_, and the third that it may be generated by fungi
of very different nature, and especially by _penicillium glaucum_ and
_mucor mucedo_. He goes on to say: “I have taken great pains to repeat
the experiments of Bail, Berkeley, and H. Hoffmann, but I have never
been able to confirm the results which they have stated, either in the
case of growths in microscopic cells or in experiments performed in
test-tubes with the purest possible substances—specially prepared
solutions or must of wine and spores of penicillium, _mucor mucedo_,
_botrytis cinerea_, &c.” On this point M. De Bary arrives at exactly the
same results which we communicated to the _Société Philomathique_ and
the _Société Chimique_ of Paris, as already given in Chap. IV. § 4, p.
128, note.
M. De Bary goes on to say: “In researches of this kind it is difficult
to eliminate two sources of error. On the one hand, it is beyond doubt
that cells of ferment are actually scattered over everything, and that,
consequently, they may easily get into the experimental liquid along
with the spores that we sow, and so occasion mistakes. On the other
hand, there are a great many fungi which develop budding processes
similar to yeast, but incapable of producing fermentation, which yet in
some cases spring directly from spores as well as from mycelium,
especially we may instance _exoascus_. This last observation is
especially applicable to the extraordinarily numerous variety of fungi
which rank under the Dematiei and Sphaeriacei, and which I shall term,
for convenience of naming, _dematium pullulans_.”[98]
We shall conclude this paragraph with a remark that has doubtless
presented itself to the minds of our readers, which is, that it would be
impossible to carry out the experiments we have described if we could
not make sure of dealing with pure ferments, or, at least, with mixtures
the components of which are sufficiently well known for us to assign to
each the effect produced by it in the total phenomena observed. It would
be extremely difficult to continue growths of yeast-deposit in sweetened
water or in a moist atmosphere if the little plant were mixed with
spores of other fungoid growths, a variety of ferment-forms, and germs
of bacteria, vibrios, or infusoria in general. All these foreign
organisms would tend to develop just in proportion as the conditions of
the media were more or less favourable to their growth, and, in a very
few days, our flasks would be filled with swarms of beings which, in
most cases, would entirely conceal the facts relating to those forms,
the separate study of which it was our object to follow out. We shall
have occasion, therefore, to examine, in a subsequent paragraph, the
preparation of ferments in a state of purity. At present we may state
that yeast, which in its ordinary condition is a mass of cells so liable
to change that its preservation in a moist state is impossible,
manifesting in the course of a few days during the winter, and in
twenty-four hours during the heat of summer, all the signs of incipient
putrefaction, thereby losing its distinctive characteristics, is
nevertheless capable, when pure, of enduring the highest atmospheric
temperatures for whole years without showing the least signs of putrid
change or contamination with any other microscopic organisms, and
without the cells losing their power of reproduction. In the presence of
facts like these, the theory of spontaneous generation must seem
chimerical. The hypothesis of the possible transformation of yeast into
_penicillium glaucum_, bacteria, and vibrios, or conversely, which the
theories of Turpin, H. Hoffmann, Berkeley, Trécul, Hallier, and Béchamp
involve, is equally refuted by these facts.
§ II.—On “Spontaneous” Ferment.
The expression _spontaneous ferment_ may be applied to any ferment that
appears in a fermentable liquid without having been purposely sown in
it. In this respect the ferments mentioned in the preceding paragraph,
those of all saccharine juices of fruit which ferment when left to
themselves—the ferments of wine, for example—are spontaneous. The term,
however, is not altogether appropriate, because, after all, the process
is the same as if an actual sowing had been made, since, as we have
shown, it is absolutely necessary for the juice to come into contact
with the surfaces of the fruit, so that the ferment may be mixed with
it, and so produce subsequent fermentation. Therefore, although we may
apply the term _spontaneous ferment_ to the ferments of fruits, we
intend that expression to apply in this paragraph solely to those
ferments that are generated in a saccharine liquid, in which, by
previous boiling, we have destroyed all ferment germs, and which,
nevertheless, enters into fermentation after being exposed in free
contact with air. In such a case it is entirely from the particles of
dust floating in the air that the ferment germs that appear in the
liquid are derived. Such are typical _spontaneous_ fermentations, and it
is of the ferment so obtained that we are about to speak.
In the course of the researches which we undertook in order to ascertain
whether _mycoderma vini_, or vinous efflorescence, became transformed,
in the case of beer, into actual alcoholic ferment—researches which were
the more protracted and varied in consequence of their leading to the
condemnation as erroneous, on the faith of new and more precise
experiments, such as those given in Chap. IV. § 2, of that
transformation, in which we had for long believed—we had occasion to
observe several spontaneous fermentations of this kind in various
saccharine liquids. We then proceeded to describe our method of
conducting the experiments. Having brought about the development of a
film of _mycoderma vini_ or _cerevisiæ_ on the surface of a liquid,
fermented or not, we submerged that film in wort, which we afterwards
put into long-necked flasks, in which alcoholic fermentation generally
took place in the course of a few days. This fermentation in no way
resulted from the transformation of the cells constituting the
efflorescence into ferment. The mycodermic film merely acted as a
receptacle of true ferment germs, wafted thither with the particles of
dust floating in the air of the laboratory, which germs developed in the
liquid into actual alcoholic ferments amongst the cells of the submerged
mycoderma. By conducting experiments in this manner we brought about
several spontaneous fermentations, the germs of which could have been
introduced by nothing but the particles of dust in the air. These
fermentations, which we were obliged to follow very carefully with the
microscope from the time when they first manifested themselves, on
account of the transformation that we were seeking, which transformation
we thought might possibly be that of the cells of _mycoderma vini_ into
cells of ferment, generally gave us during the first days of
fermentation the large, elongated, branchy ferment represented in Plate
X., which was succeeded by the small ferment represented in Plate
XI.[99]
[Illustration: Plate 12. Ferment-cells from a Spontaneous Fermentation
just starting.]
Here let me describe one of these experiments. In the beginning of
March, 1872, we grew some _mycoderma vini_, obtained from wine, on some
wort contained in a shallow basin. On March 6th we submerged the
efflorescence and put it all together, liquid and film, into a
long-necked flask. On March 9th we detected incipient fermentation, and
on March 12th we took a sketch of the yeast of the deposit, as given in
Plate XII. This is the large and long branching, more or less
pear-shaped form, which occurs at the beginning of fermentation in the
sweet and acid musts of our domestic fruits. On March 16th we made
another sketch of the deposit, in which the proportion of cells, in the
form of elongated segments and filaments, reminded us, in some measure,
of the filamentous mycelium of typical fungoid growths much diminished.
In this case, however, the majority of cells were oval, round, and in
short segments. On this day, March 16th, we added some fresh wort to
that which had fermented, with the object of prolonging the duration of
fermentation and increasing the proportion of yeast. On March 19th we
made a fresh sketch, which it is not necessary either to reproduce;
suffice it to say, that the yeast was now considerably more regular and
uniform in appearance.
[Illustration: Fig. 38.]
Spontaneous ferment, therefore, very often occurs in this large
ferment-form, which, by repeated developments in the act of
fermentation, becomes reduced by degrees after successive generations to
the ferment which, following Dr. Rees, we have named _saccharomyces
pastorianus_, a polymorphous ferment which must be studied closely that
it may not be confounded with others, inasmuch as it is so universally
diffused that we very seldom fail to find it in any ferment which has
been exposed in contact with ordinary air, at least, we may repeat, in a
laboratory devoted to researches on fermentation. We have found the same
thing occur in a brewery, being there mixed with the ferments used in
brewing.
There are, no doubt, several varieties of this _saccharomyces_. We
sometimes find amongst the spontaneous ferments which repeated growths
have brought to a more or less uniform state, the forms represented in
Plate XI., but the cells and segments much smaller. Amongst others, Dr.
Rees has distinguished a _saccharomyces exiguus_.
Fig. 38 represents another spontaneous ferment, which appeared in a
boiled saccharine wort, which entered into fermentation after being
exposed to the air of the laboratory.
The sketch was made directly after the fermentation had commenced.
Probably this is simply one of the earlier forms assumed by the
_saccharomyces_, or by one of its varieties. It will be seen that the
alcoholic ferment is associated with another little filiform ferment,
probably the lactic. The spontaneous ferments are almost always impure,
a circumstance that may be readily understood if we bear in mind the
results described in Chapters III. and IV.
§ III.—On “High” and “Low” Ferments.
The ferments mentioned in the preceding paragraphs do not belong,
properly speaking, to industrial products; that is to say, in actual
practice there are no operations in which the ferments of fruits and
spontaneous ferments are employed for the purpose. It is quite true that
these ferments are the cause of the fermentations from which wine,
cider, gin, rum, gentiana, mead, &c., are derived, but these
fermentations are spontaneous, they take place without the intervention
of man, and without man’s directing their production, or taking any
notice of the agent which starts them.
In the manufacture of beer, on the other hand, the practice is quite
different. We may say that the wort is never left to ferment
spontaneously, the fermentation being invariably produced by the
addition of yeast formed on the spot in a preceding operation, or
procured from some other working brewery, which, again, had at some time
been supplied from a third brewery, which itself had derived it from
another, and so on, as far back as the oldest brewery that can be
imagined. A brewer never prepares his own yeast. We have already had
occasion to remark that the interchange of yeasts amongst breweries is a
time-honoured custom, which has been observed in all countries at all
periods, as far back as we can trace the history of brewing. The yeasts
which in the present day produce beer in the brewery of Tourtel, near
Nancy, in that of Grüber, at Strasburg, that of Dreher, at Vienna, and
others, came originally from breweries, where and when it would be hard
to say. In the case of the first working brewery, the yeast was, no
doubt, derived from some spontaneous fermentation, which took place in
an infusion of barley that had been left to itself, or, from some
natural spontaneous ferment, and nothing could be easier than to realize
this fact again. In the brewing industry there are two distinct modes of
fermentation:—“high” fermentation and “low” fermentation, some of the
distinctive characteristics of which we have pointed out in Chapter I.
It may be questioned whether the spontaneous yeast employed in the first
brewery, or that which a wort left to itself in the present day would
yield, would be of the “high” or “low” type. It may be concluded from
what we have said on the subject of spontaneous fermentations in wort,
that wort, left to itself, would furnish ferments more or less
resembling those of wine. We have never obtained in spontaneous
fermentations of wort either a distinctly “high” ferment, or a
distinctly “low” one, properly so called; nor, further, have we ever
obtained either one of these distinct kinds, with its industrial
characteristics, in experiments on the ferments of fruits. What, then,
was the origin of the “high” and “low” ferments now used by brewers?
What was the nature of their original germs? These are questions which
we are unable to answer, but we are very much inclined to think that we
have here another example of the modifications which plants as well as
races of animals undergo, and which become hereditary in the course of
prolonged domestication. We know nothing of corn in its wild state, we
cannot tell what its first grain was like. We know nothing of the
silk-worm in its original state, and we are ignorant of the characters
of the race that furnished the first egg.
These reflections may seem to favour the supposition that there is a
real difference between “high” yeast and “low” yeast, and that both of
these differ from spontaneous ferments and the ferments of domestic
fruits. These are propositions demanding most careful consideration, for
it is generally admitted that these ferments become intermixed, that
their morphological differences are merely a question of medium, and
that the transition of one to the other is a simple matter. The
following facts seem to contradict such statements.
[Illustration: Fig. 39.]
“High” Ferment.—Fig. 39 represents some “high” yeast taken from a
deposit after fermentation, and Fig. 40 the same yeast in course of
propagation in some aerated wort. In comparing “high” yeast with other
alcoholic ferments at the same stage of development, there are three
points which are especially striking: the diameter of its cells is
relatively large, their general aspect is rounder, and when they are
undergoing propagation their mode of budding produces a markedly
ramified appearance, so that the cells always occur in clusters and
branches. Fig. 40 gives a very exact idea of these characters. To
investigate satisfactorily the branching habit of growth peculiar to
this ferment we should examine it during the first few hours of its
propagation, when, under the influence of the oxygen dissolved in the
fermentable liquid, its vital activity is greatest. Later on, often on
the day following the sowing, the groups become disconnected, and at the
end of the fermentation the cells have quite separated from each other,
not more than 2 or 3 per cent. remaining united, and even these in
groups of not more than two cells together. This is represented in Fig.
39.
To give an idea of the rapidity with which this ferment multiplies, we
may state that our sketch (Fig. 40) was made under the following
conditions:—On April 28th, 1874, we caused a flask of wort to ferment by
means of a trace of “high” yeast. On the morning of the next day, that
is fourteen hours afterwards, an appreciable deposit of yeast had
formed, and some frothy patches appeared on the surface of the liquid,
showing that fermentation had set in. On May 1st we decanted the beer,
substituting for it water sweetened with 10 per cent. of sugar. On May
2nd we decanted the sweetened water, and substituted a fresh quantity
containing the same percentage of sugar. On May 3rd, at mid-day, we took
some of the fermenting liquid from this flask and put it into a flask of
wort; five hours after the introduction of the ferment we made the
sketch in question. The field is covered with branching clusters, the
groups being sketched exactly as they occurred in the field. Their
activity was due to the condition of the ferment, and to the perfect
fitness of the nutritive medium for its vegetation. In sweetened water
the budding of the cells was considerably less active; no branching
groups of cells are to be found. Budding, nevertheless, occurs to a
considerable extent, but it is limited to one bud, or two at the most,
to each cell. Fermentation in pure sweetened water is mostly correlative
with the duration of vital activity in the globules already formed.
[Illustration: Fig. 40.]
Let us next suffer our yeast to exhaust itself by keeping it in a great
excess of sweetened water for a very long time; we shall then be able to
observe its process of revival, and see if we can find any facts
analogous to those presented by _saccharomyces pastorianus_ (Chapter V.
§ 1).
With this object in view, on May 6th, 1874, we impregnated two fresh
flasks of sweetened water with some of the contents of the
before-mentioned flask, which we had refilled with sweetened water on
May 2nd. On May 13th we decanted the liquid, which was still very sweet,
from one of these two fresh flasks, which could hardly be said to have
fermented at all—the quantity of yeast in them being so small—and
replaced it with some wort. Strange to say, on the morning of the 14th
we found an appreciable growth of yeast, and a froth of carbonic acid
gas on the surface of the liquid. The yeast therefore was not dead,
although its fermentative powers had been exhausted. There was, however,
no remarkable feature in connection with its revival, nor did we find
the slightest trace of any of the elongated ferment-form. What we got
was simply the ramified groups of “high” yeast again, in round cells,
but nothing more.
Fearing that our yeast might not have remained for a sufficient time in
the sweetened water for exhaustion, we set aside, for a whole year, the
other flask which we had prepared on May 6th. On May 16th, 1875, we
decanted the sweetened liquid and replaced it with wort. This time,
however, there was no revival of the yeast; it had perished.
Fortunately, we had also saved the flask of yeast and sweetened water
which was prepared on May 2nd, 1874, as already mentioned, and in this
case, as will be seen, the vitality of the yeast had not been
extinguished, doubtless, in consequence of the formation of what we
shall presently designate by the name of _aërobian ferment_. On May
16th, 1875, we decanted the liquid from this last flask, and replaced it
with wort. On the next day the surface of the wort was covered with a
thin froth, indicating the commencement of fermentation. The microscope
revealed nothing extraordinary, or indicative of the fermentation of any
special ferment. To assure ourselves that our ferment had remained
“high,” we sowed some of it in a fresh flask of wort on May 19th, and
then, seven hours after impregnation, submitting it to examination, we
could find nothing but ramified groups in fine condition, without a
single elongated cell, indeed, it would have been impossible to find a
more beautiful specimen of “high” yeast, or one of a more decided
character.
It would seem, therefore, that “high” yeast cannot, under any
circumstances, assume the form and character of the ferment
_saccharomyces pastorianus_, or of other known ferments. We are
justified, therefore, in regarding it as a distinct species of ferment,
an opinion which is supported by other circumstances.
1. In equal quantities of saccharine wort a considerably greater growth
of “high” yeast is obtained than of other yeasts. We need no very
rigorous proofs to convince ourselves of this fact: for by simply
causing equal volumes of the same wort to ferment, the one being pitched
with _saccharomyces pastorianus_, for example, the other with “high”
yeast, we shall obtain a perceptibly greater volume of “high” yeast than
of the other, in certain cases even five or six times as much.
2. “High” yeast is of a tougher texture than the others, separating,
when the fermented liquor and its deposit is shaken up, into lumps which
refuse to disappear; whereas _saccharomyces pastorianus_ diffuses
through the whole liquid with the greatest ease.
3. “High” yeast produces a special beer, with a peculiar flavour, well
known to consumers, but little esteemed at the present day. Hence the
gradual displacement of breweries worked on the old “high” fermentation
system by others in which “low” yeast (of which more anon) alone is
employed.