discovered a surprising connection between the prior researches of
chemistry and crystallographic physics and the new and entirely
unexpected results of physiological chemistry. This connection, like
the thread of Ariadne, conducted him to his recent great discoveries
in medical biology. M. Chevreul was right when, some years ago, at the
Academy of Sciences, he expressed himself thus:--
'It is by first examining in their chronological order the researches
of M. Pasteur, and then considering them as a whole, that we are
enabled to appreciate the rigour of judgment of that learned man in
forming his conclusions, and the perspicacity of a mind which, strong
in the truths which it has already discovered, is carried forward to
the establishment of new ones.'
III.
Pasteur had thus established that bodies endowed with internal
dissymmetry carried this property, in varying degrees, into their
compounds or their derivatives. When two of these bodies whose nature
has been revealed by the discovery of right-handed and left-handed
tartaric acid, where all is chemically identical--and which are only
to be distinguished from each other by their inverse crystallographic
form, and by their action on polarised light--enter into combination
with a substance which is optically and crystallographically inert, the
chemical identity ought, under these new conditions, to be preserved.
Everything remains optically and crystallographically comparable.
The inert element adds nothing to, and takes away nothing from, the
dissymmetric faculties of the active one.
To these curious studies Pasteur soon added a new chapter. He reasoned
thus:--If into these compounds I introduce a substance possessing in
itself the specific properties of dissymmetry, it is evident that this
substance, while entering into these combinations, must preserve its
own properties. The active substance would, from the moment of its
combination, add something to the properties of the molecular group
which acts like itself, and subtract something from the properties
of the group which acts in the opposite manner. The resultant effect
of these actions, sometimes concordant, sometimes antagonistic, would
cease to be alike in absolute quantity. And if this be the necessary
condition of similitude as to molecular arrangement, this similitude
would cease to exist, and with its disappearance would appear all the
differences of chemical and physical properties which constitute its
outward manifestations.
The facts were found to harmonise with these logical deductions. After
having made dissymmetry intervene as a modifier of chemical affinity,
he had a strange and manifest proof of the influence of dissymmetry in
the phenomena of life.
It had been long known, through the observations of a manufacturer
of chemical products in Germany, that the impure tartrate of lime of
commerce, if contaminated with organic matters and permitted to remain
under water in summer, would ferment and yield various products.
Pasteur caused the ordinary right-handed tartrate of ammonia to
ferment in the following manner:--He took some very pure crystalline
salt and dissolved it, adding at the same time to the liquid some
albuminoid matter, about one gramme to 100 grammes of the tartrate.
The liquid placed in a warm chamber fermented. During the process of
fermentation the liquid mass, previously limpid, became gradually
turbid, in consequence of the appearance of a small organism which
played the part of ferment. Pasteur applied this mode of fermentation
to the paratartrate of ammonia. He saw that this salt also fermented,
depositing the same organism. All appeared as if the course of things
was the same as in the case of the right-handed tartrate. But Pasteur,
having had the idea of following the course of the operation with the
aid of the polariscope, soon detected a profound difference between
the two fermentations. In the case of the paratartrate, the liquid,
at first inert, gradually assumed a sensible power of deviation to
the left, which augmented by degrees and attained a maximum. The
fermentation was then suspended; there was no longer any of the
right-handed acid in the liquid, which, when evaporated and mixed
with its own volume of alcohol, immediately furnished a beautiful
crystallisation of left-handed tartrate of ammonia.
From that moment a great new fact was established--namely, that the
molecular dissymmetry proper to organic matters intervened in a
phenomenon of the physiological order, and did so as a modifier of
chemical affinity. The kind of dissymmetry proper to the molecular
arrangement of the left-handed tartaric acid was, no doubt, the sole
cause of the difference between this acid and the right-handed acid, in
regard to the fermentation produced by a microscopic fungus. We shall
see later on that organised ferments are almost always microscopic
vegetables, which embrace in their constitution cellulose, albumen,
&c., identical with these same substances taken from the higher class
of vegetables and equally dissymmetric. We can thus understand, that
for the nutrition of the ferment and the formation of its principles
the chemical changes are more easy with one of the two tartaric acids
than with the other.
The opposition of the properties of the two tartaric acids, right and
left, at the moment when the conditions of life and nutrition of an
organised being intervened, showed themselves still more strikingly
in a very curious experiment made by Pasteur. He was the first to
prove that mildew could live and multiply on a purely mineral soil,
composed, for example, of the phosphates of potash, of magnesia, and
an ammoniacal salt of an organic acid. For such a development of
vegetable life he employed the seed of _penicillium glaucum_, which
is to be found everywhere as common mould, and to which he offered,
as its only carbon aliment, paratartaric acid. At the end of a little
time the left-handed tartaric acid appeared. Now this left-handed
acid could only show itself on the condition that a rigorously equal
quantity of the right-handed acid had been decomposed. The carbon of
the tartaric acid evidently supplied to the little plant the carbon
that was necessary for the formation of its constituents and all
their organic accessories. If the microscopic seed of penicillium
sown upon this soil was not formed of dissymmetric elements, as is
the case with all other vegetable substances, its development, its
life, its fructification would accommodate themselves equally well
with the left-handed tartaric acid as with the right. The fact that
the left-handed tartaric acid is less assimilable than its opposite
is due solely and evidently to the dissymmetry of one or other of the
primordial substances of the little plant.
* * * * *
Thus for the first time was introduced into physiological studies and
considerations the fact of the influence of the molecular dissymmetry
of natural organic products.
Pasteur always speaks with enthusiasm of the grand future reserved for
researches which have this influence for their object; for molecular
dissymmetry is the only sharp line of demarcation which exists between
the chemistry of inorganic and that of organic nature.
FOOTNOTE:
[7] [M. Pasteur appears to use the word _devenir_ as a substantive in a
sense equivalent to the German _Werdende_.]
_FERMENTATION._
Arrived at this unexpected turn in the road which he had hitherto
pursued, Pasteur paused for an instant. Should he commit himself to the
course which abruptly opened before him? His scientific instincts urged
him to do so, but the prudence and reserve which show themselves to be
the basis of his character, whenever he finds himself called upon to
make a choice of which the necessity is not absolutely demonstrated,
held him back. Was it not wiser to continue in the domain of molecular
physics and chemistry? M. Biot counselled his doing so; the route had
been made plain, success awaited him at each step, but an incident
connected with the University triumphed over his hesitations.
He had just been nominated, at thirty-two years of age, Dean of the
Faculté des Sciences at Lille. One of the principal industries of the
Département du Nord is the fabrication of alcohol from beetroot and
from corn. Pasteur resolved to devote a portion of his lectures to the
study of fermentation. He felt that if he could make himself directly
useful to his hearers he would thereby excite general sympathy with,
and direct attention to the new Faculté. The young man congratulated
himself on this idea, and the man of science rejoiced in it still more.
He was filled by the reflections suggested to him by the strangeness of
the phenomena which he had just encountered in regard to the molecular
dissymmetry of the two tartaric acids, in connection with the life
of a microscopic organism. He saw new light thrown upon the obscure
problem of fermentation. The part so active performed by an infinitely
small organism could not, he thought, be an isolated fact. Behind this
phenomenon must lie some great general law.
I.
All that has lived must die, and all that is dead must be
disintegrated, dissolved or gasified; the elements which are the
substratum of life must enter into new cycles of life. If things were
otherwise, the matter of organised beings would encumber the surface of
the earth, and the law of the perpetuity of life would be compromised
by the gradual exhaustion of its materials. One grand phenomenon
presides over this vast work, the phenomenon of fermentation. But
this is only a word, and it suggests to the mind simply the internal
movements which all organised matter manifests spontaneously after
death, without the intervention of the hand of man. What is, then,
the cause of the processes of fermentation, of putrefaction, and of
slow combustion? How is the disappearance of the dead body or of the
fallen plant to be accounted for? What is the explanation of the
foaming of the must in the vintage cask? of dough, which, abandoned to
itself, rises and becomes sour? of milk, which curdles? of blood, which
putrefies? of the heap of straw, which becomes manure? of dead leaves
and plants embedded in the earth, which transform themselves into soil?
Many different attempts were made to account for this mystery before
science was in a condition to approach it. In our age, and at the time
when Pasteur was led to the study of the question, one theory held
almost undisputed sway. It was a very ancient theory, to which Liebig,
in reviving it, had given the weight of his name. 'The ferments,' said
Liebig, 'are all nitrogenous substances--albumen, fibrine, caseine;
or the liquids which embrace them, milk, blood, urine--in a state of
alteration which they undergo in contact with the air.'
The oxygen of the air was, according to this system, the first cause of
the molecular breaking up of the nitrogenous substances. The molecular
motions are gradually communicated from particle to particle in the
interior of the fermentable matter, which is thus resolved into new
products.
These theoretic ideas regarding the part played in fermentation
by the oxygen of the air were based upon experiments made in the
beginning of the century by Gay-Lussac. In examining the process of
Appert for the preservation of animal and vegetable substances--a
process which consisted in inclosing these substances in hermetically
sealed vessels and heating them afterwards to a sufficiently high
temperature--Gay-Lussac had seen, for example, the must of the grape,
which had been preserved without alteration during a whole year,
caused to enter into a state of fermentation by the simple fact of its
transference to another vessel--that is to say, by having been brought
for an instant into contact with the oxygen of the air. The oxygen of
the air appeared, then, to be the _primum movens_ of fermentation.
The illustrious chemists Berzelius and Mitscherlich explained the
phenomena of fermentation otherwise. They placed these phenomena in the
obscure class known as _phenomena of contact_. The ferment, in their
view, took nothing from, and added nothing to, the fermentable matter.
It was an albuminoid substance, endowed with a force to which the name
_catalytic_ was given. The ferment in fact acted by its mere presence.
A very curious observation, however, had been made in France by
Cagniard-Latour and in Germany by Schwann. Cagniard-Latour, however,
was the first to publish this observation, which was destined to
become so fruitful. One of the ferments most in use, and known as
early as the leavening of dough or the turning of milk, is the deposit
formed in beer barrels, which is commonly called _yeast_. Repeating an
observation of the naturalist Leuwenhoeck, Cagniard-Latour saw this
yeast, which was composed of cells, multiplying itself by budding, and
he proposed to himself the question whether the fermentation of sugar
was not connected with this act of cellular vegetation. But as in
other fermentations the existence of an organism had not been observed
even by the most careful search, the hypothesis of Cagniard-Latour of
a possible relation between the organisation of the ferment and the
property of being a ferment was abandoned, though not without regret
by some physiologists. M. Dumas, for example, recognised that in the
budding of the yeast globules there must be some clue to the phenomenon
of fermentation. I, however, repeat that as nothing of the kind had
been found elsewhere, and as all other fermentations presented the
common character of requiring, to put them in train, organic matter in
a state of decomposition, the hypothesis of Cagniard-Latour remained a
simple incident, instead of having the value of a scientific principle.
Liebig, moreover, carrying general opinion along with him, contended
that it is not because of its being organised that yeast is active,
but because of its being in contact with air. It is the dead
portion of the yeast--that which has lived and is in the course of
alteration--which acts upon the sugar.
The new memoirs published on the subject agreed in rejecting the
hypothesis of any influence whatever of organisation or of life in the
process of fermentation. Books, memoirs, dogmatic teaching, all were
favourable to the theoretic ideas of Liebig. If a few rare observers
indicated the presence in certain fermentations of living organisms,
this presence was, in their opinion, a purely accidental fact, which,
instead of favouring the phenomenon of fermentation, was injurious to
it.
From his first investigation on lactic fermentation Pasteur was led to
take an entirely different view of the matter. In this fermentation he
recognised the presence and the action of a living organism, which was
the ferment, just as yeast was the ferment of alcoholic fermentation.
The lactic ferment was formed of cells, or rather of little rods nipped
at their centres, extremely small, being hardly the thousandth part
of a millimeter in diameter.[8] It reproduced itself by fission--that
is to say, the little rod divided itself at its middle and formed two
shorter rods, which became elongated, nipped, in their turn, at their
centres, each giving rise, as before, to two rods. Each of these,
again, soon divided itself into two, and so on. Why had not this
been observed prior to Pasteur? For the simple reason that chemists
had never observed the production of lactic fermentation except in
complex substances. They mixed chalk with their milk for the purpose
of preserving the neutrality of the fermenting medium. They employed
substances such as caseine, gluten, animal membranes, all of which,
when examined by the microscope, exhibited a multitude of mineral or
organic granules, with which the lactic ferment was confounded. Thus
the first care of Pasteur, with the view of proving the presence of
the ferment and its life, was to replace the cheesy matter and all its
congeners by a soluble, nitrogenous body, which would permit of the
microscopic examination of all the living cellular products.
In a memoir presented to the Academy of Sciences in 1857 Pasteur
stated that there were 'cases where it is possible to recognise in
lactic fermentation, as practised by chemists and manufacturers, above
the deposit of chalk and the nitrogenous matter, a grey substance
which forms a zone on the surface of the deposit. Its examination
by the microscope hardly permits of its being distinguished from
the disintegrated caseum or gluten which has served to start the
fermentation. So that nothing indicates that it is a special kind
of matter which had its birth during the fermentation. It is this,
nevertheless, which plays the principal part.'
To isolate this substance and to prepare it in a state of purity,
Pasteur boiled a little yeast with from fifteen to twenty times its
weight of water. He then carefully filtered the liquid, dissolved in it
about fifty grammes of sugar to the litre, and added to it some chalk.
Taking then, by means of a drawn-out tube, from a good ordinary lactic
fermentation a trace of the grey matter of which we have just spoken,
he placed it as the seed of the ferment in the limpid saccharine
solution. By the next day a lively and regular fermentation had set
in, the liquid becoming turbid and the chalk disappearing, and one
could distinguish a deposit which progressed continually as the chalk
dissolved. This deposit was the lactic ferment.
Pasteur reproduced this experiment by substituting for the water of the
yeast a clear decoction of nitrogenous plastic substances. The ferment
invariably presented the same aspect and the same multiplication. These
results, however, did not yet satisfy Pasteur. He desired more rigour
in a subject of such theoretic importance. Might not the partisans of
Liebig's theory argue, if not without subtlety yet with a semblance
of justice, that the fermentation was not due to the formation and
progressive growth of this feeble nitrogenous globular deposit, but
rather to the nitrogenous matter dissolved during the decoction of the
yeast used in the composition of the liquor? Up to a certain point
it might be maintained that the dissolved matters which had been in
contact with the oxygen of the air had been thrown into molecular
motion, that this motion had been communicated to the fermentable
matter, and that the deposit of the pretended organised ferment was but
an accident--one of the physical changes or one of the precipitates
so frequently observed in the modifications of albuminoid matters. In
the observation of Cagniard-Latour and of Schwann as to the life of
the yeast, Liebig saw nothing more. 'One cannot deny,' said he, 'the
organisation of the yeast or its multiplication by budding, but these
living cells are always associated with other dead cells in process of
molecular alteration. It is these molecular motions which communicate
themselves to the molecules of the sugar, break them up, and cause them
to ferment.'
The arguments of Liebig derived great strength from the belief
which was shared by all chemists that the cells of yeast perish
during fermentation and form lactate of ammonia. On examining this
assertion, Pasteur found that not only was there no ammonia formed
during alcoholic fermentation, but that even if ammonia were added it
disappeared, entering into the formation of new yeast cells. Was not
this a proof of the potency of the organised ferment?
Tormented, however, by the idea that, notwithstanding all these facts,
the reasonings of Liebig might still find some credit, Pasteur worked
earnestly to discover new facts capable of demonstrating that Liebig's
theory was absolutely false. He made two crucial experiments, the one
relating to the yeast of beer, or of alcohol, and the other relating
to the lactic ferment. He introduced into a pure solution of sugar a
small quantity of crystallisable salt of ammonia, then some phosphates
of potash and magnesia, and he sowed in this medium an imponderable
quantity, if we may so express it, of fresh cells of yeast. The cells
thus sown multiplied, and the sugar fermented. In other words, the
phosphorus, the potassium, the magnesium of the mineral salts, united
to form the substances which compose the ferment. By this experiment,
so simple and yet so demonstrative, the power of the organisation
of the ferment was once for all established. The contact theory of
Berzelius had no longer any meaning, since it was evident that the
fermentable matter here furnished to the ferment one of its essential
elements, namely, carbon. Liebig's theory of communicated molecular
motion, originating in a nitrogenous albuminoid substance, had no
better claim, since such substances had been discarded. The whole
process took place between the sugar and a ferment germ which owed its
life and development to nutritive matters, the most important of which
was the fermentable substance. Fermentation, in short, was simply a
phenomenon of nutrition. The ferment augmented in weight, feeding upon
the sugar, and its vitality was such that it contrived to build up the
complex materials of its own organisation by means of sugar and purely
mineral elements.
In a second experiment, Pasteur demonstrated that, notwithstanding
their smallness and the possibility of confounding them with the
amorphous granules of caseine and gluten, the little particles of
lactic ferment were indeed alive, and that they, and they only, were
the cause of lactic fermentation. He mixed with some water, sweetened
with sugar, a small quantity of a salt of ammonia, some alkaline
and earthy phosphates, and some pure carbonate of lime obtained by
precipitation. At the end of twenty-four hours the liquid began to get
turbid and to give off gas. The fermentation continued for some days.
The ammonia disappeared, leaving a deposit of phosphates and calcareous
salt. Some lactate of lime was formed, and at the same time one could
notice the deposition of the little lactic ferment. The germs of the
lactic ferment had, in this case, been derived from particles of dust
adhering to the substances themselves, of which the mixtures were made,
or to the vessels used, or from the surrounding air. The chapter on
spontaneous generation will render this clear.
It suffices here to state that the results of this second experiment
were absolutely conclusive, and that the theories of contact force or
of communicated motion, which up to that time had reigned in science,
were completely overthrown.
II.
The light shed by these experiments quickly extended its sphere; and
Pasteur lost no time in discovering a new ferment, that of butyric
acid. Having shown the absolute independence which exists between the
ferment of butyric acid and the others, he found, contrary to the
general belief, that the lactic ferment is incapable of giving rise
to butyric acid, and that there exists a butyric fermentation having
its own special ferment. This ferment consists of a species of vibrio.
Little transparent cylindrical rods, rounded at their extremities,
isolated or united in chains of two or three, or sometimes even more,
form these vibrios. They move by gliding, the body straight, or bending
and undulating. They reproduce themselves by fission, and to this mode
of generation their frequent arrangement in the form of a chain is due.
Sometimes one of the little rods, with a train of others behind it,
agitates itself in a lively manner as if to detach itself from the
rest. Often, also, the little rod, after being broken off, holds on
still to its chain by a mucous transparent thread.