We should be glad to learn that the employment of a drop of ammonia would enable us to p.r.o.nounce an opinion with this degree of confidence on the nature of the lowest microscopic beings; but is M. Robin absolutely correct in his a.s.sumptions? That gentleman himself remarks that spermatozoa, which belong to animal organisms, are insoluble in ammonia, the effect of which is merely to make them paler. If a difference of action in certain reagents, in ammonia, for example, were sufficient to determine the limits of the animal and vegetable kingdoms, might we not argue that there must be a very great and natural difference between moulds and bacteria, inasmuch as the presence of a small quant.i.ty of acid in the nutritive medium facilitates the growth and propagation of the former, whilst it is able to prevent the life of bacteria and vibrios? Although as is well known, movement is not an exclusive characteristic of animals, yet we have always been inclined to regard vibrios as animals, on account of the peculiar character of their movements. How greatly they differ in this respect from the diatomacae, for example! When the vibrio encounters an obstacle it turns, or after a.s.suring itself by some visual effort or other that it cannot overcome it, it retraces its steps. The colpoda--undoubted infusoria--behave in an exactly similar manner. It is true one may argue that the zoospores of certain cryptogamia exhibit similar movements; but do not these zoospores possess as much of an animal nature as do the spermatozoa? As far as bacteria are concerned, when, as already remarked, we see them crowd round a bubble of air in a liquid to prolong their life, oxygen having failed them everywhere else, how can we avoid believing that they are animated by an instinct for life, of the same kind that we find in animals? M. Robin seems to us to be wrong in supposing that it is possible to draw any absolute line of separation between the animal and vegetable kingdoms. The settlement of this line however, we repeat again, no matter what it may be, has no serious bearing upon the questions that have been the subject of our researches.

In like manner the difficulty which M. Robin has raised in objecting to the employment of the word GERM, when we cannot specify whether the nature of that germ is animal or vegetable, is in many respects an unnecessary one. In all the questions which we have discussed, whether we were speaking of fermentation or spontaneous generation, the word GERM has been used in the sense of ORIGIN OF LIVING ORGANISM. If Liebig, for example, said of an alb.u.minous substance that it gave birth to ferment, could we contradict him more plainly than by replying "No; ferment is an organized being, the germ of which is always present, and the alb.u.minous substance merely serves by its occurrence to nourish the germ and its successive generations"?

In our Memoir of 1862, on so-called SPONTANEOUS generations, would it not have been an entire mistake to have attempted to a.s.sign specific names to the microscopic organisms which we met with in the course of our observations? Not only would we have met with extreme difficulty in the attempt, arising from the state of extreme confusion which even in the present day exists in the cla.s.sification and nomenclature of these microscopic organisms, but we should have been forced to sacrifice clearness in our work besides; at all events, we should have wandered from our princ.i.p.al object, which was the determination of the presence or absence of life in general, and had nothing to do with the manifestation of a particular kind of life in this or that species, animal or vegetable. Thus we have systematically employed the vaguest nomenclature, such as mucors, torulae, bacteria, and vibrios. There was nothing arbitrary in our doing this, whereas there is much that is arbitrary in adopting a definite system of nomenclature, and applying it to organisms but imperfectly known, the differences or resemblances between which are only recognizable through certain characteristics, the true signification of which is obscure. Take, for example, the extensive array of widely different systems which have been invented during the last few years for the species of the genera bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier, and Billroth. The confusion which prevails here is very great, although we do not of course by any means place these different works on the same footing as regards their respective merits.

M. Robin is, however, right in recognizing the impossibility of maintaining in the present day, as he formerly did, "That fermentation is an exterior phenomenon, going on outside cryptogamic cells, a phenomenon of contact. It is probably," he adds, "an interior and molecular action at work in the innermost recesses of the substance of each cell." From the day when we first proved that it is possible for all organized ferments, properly so called, to spring up and multiply from their respective germs, sown, whether consciously or by accident, in a mineral medium free from organic and nitrogenous matters other than ammonia, in which medium the fermentable matter alone is adapted to provide the ferment with whatever carbon enters into its composition, from that time forward the theories of Liebig, as well of Berzelius, which M. Robin formerly defended, have had to give place to others more in harmony with facts. We trust that the day will come when M. Robin will likewise acknowledge that he has been in error on the subject of the doctrine of spontaneous generation, which he continues to affirm, without adducing any direct proofs in support of it, at the end of the article to which we have been here replying.

We have devoted the greater part of this chapter to the establishing with all possible exactness the extremely important physiological fact of life without air, and its correlation to the phenomena of fermentations properly so called--that is to say, of those which are due to the presence of microscopic cellular organisms. This is the chief basis of the new theory that we propose for the explanation of these phenomena. The details into which we have entered were indispensable on account of the novelty of the subject no less than on account of the necessity we were under of combating the criticisms of the two German naturalists, Drs. Oscar Brefeld and Traube, whose works had cast some doubts on the correctness of the facts upon which we had based the preceding propositions. We have much pleasure in adding that at the very moment we were revising the proofs of this chapter, we received from M. Brefeld an essay, dated Berlin, January, 1876, in which, after describing his later experimental researches, he owns with praiseworthy frankness that Dr. Traube and he were both of them mistaken. Life without air is now a proposition which he accepts as perfectly demonstrated. He has witnessed it in the case of Mucor racemosus and has also verified it in the case of yeast. "If," he says, "after the results of my previous researches, which I conducted with all possible exactness, I was inclined to consider Pasteur's a.s.sertion as inaccurate and to attack them, I have no hesitation now in recognizing them as true, and in proclaiming the service which Pasteur has rendered to science in being the first to indicate the exact relation of things in the phenomenon of fermentation."

In his later researches, Dr. Brefeld has adopted the method which we have long employed for demonstrating the life and multiplication of butyric vibrios in the entire absence of air, as well as the method of conducting growths in mineral media a.s.sociated with fermentable substance. We need not pause to consider certain other secondary criticisms of Dr. Brefeld. A perusal of the present work will, we trust, convince him that they are based on no surer foundation than were his former criticisms.

To bring one's self to believe in a truth that has just dawned upon one is the first step towards progress; to persuade others is the second. There is a third step, less useful perhaps, but highly gratifying nevertheless, which is, to convince one's opponents.

We therefore, have experienced great satisfaction in learning that we have won over to our ideas an observer of singular ability, on a subject which is of the utmost importance to the physiology of cells.

VI. REPLY TO THE CRITICAL OBSERVATIONS OF LIEBIG, PUBLISHED IN 1870.

[Footnote: LIEBIG, Sur la fermentation et la source de la force musculaire (Annales de Chimie et de Physique, 4th series, t.

xxiii., p. 5, 1870).]

In the Memoir which we published, in 1860, on alcoholic fermentation, and in several subsequent works, we were led to a different conclusion on the causes of this very remarkable phenomenon from that which Liebig had adopted. The opinions of Mitscherlich and Berzelius had ceased to be tenable in the presence of the new facts which we had brought to light. From that time we felt sure that the celebrated chemist of Munich had adopted our conclusions, from the fact that he remained silent on this question for a long time, although it had been until then the constant subject of his study, as is shown by all his works.

Suddenly there appeared in the Annales de Chimie et de Physique a long essay, reproduced from a lecture delivered by him before the Academy of Bavaria in 1868 and 1869. In this Liebig again maintained, not, however, without certain modifications, the views which he had expressed in his former publications, and disputed the correctness of the princ.i.p.al facts enunciated in our Memoir of 1860, on which were based the arguments against his theory.

"I had admitted," he says, "that the resolution of fermentable matter into compounds of a simpler kind must be traced to some process of decomposition taking place in the ferment, and that the action of this same ferment on the fermentable matter must continue or cease according to the prolongation or cessation of the alteration produced in the ferment. The molecular change in the sugar, would, consequently, be brought about by the destruction or modification of one or more of the component parts of the ferment, and could only take place through the contact of the two substances. M. Pasteur regards fermentation in the following light: The chemical action of fermentation is essentially a phenomenon correlative with a vital action, beginning and ending with it. He believes that alcoholic fermentation can never occur without the simultaneous occurrence of organization, development, and multiplication of globules, or continuous life, carried on from globules already formed. But the idea that the decomposition of sugar during fermentation is due to the development of the cellules of the ferment, is in contradiction with the fact that the ferment is able to bring about the fermentation of a pure solution of sugar. The greater part of the ferment is composed of a substance that is rich in nitrogen and contains sulphur. It contains, moreover, an appreciable quant.i.ty of phosphates, hence it is difficult to conceive how, in the absence of these elements in a pure solution of sugar undergoing fermentation, the number of cells is capable of any increase."

Notwithstanding Liebig's belief to the contrary, the idea that the decomposition of sugar during fermentation is intimately connected with a development of the cellules of the ferment, or a prolongation of the life of cellules already formed, is in no way opposed to the fact that the ferment is capable of bringing about the fermentation of a pure solution of sugar. It is manifest to any one who has studied such fermentation with the microscope, even in those cases where the sweetened water has been absolutely pure, that ferment-cells do multiply, the reason being that the cells carry with them all the food-supplies necessary for the life of the ferment. They may be observed budding, at least many of them, and there can be no doubt that those which do not bud still continue to live; life has other ways of manifesting itself besides development and cell-proliferation.

If we refer to the figures on page 81 of our Memoir of 1860, Experiments D, E, F, H, I, we shall see that the weight of yeast, in the case of the fermentation of a pure solution of sugar, undergoes a considerable increase, even without taking into account the fact that the sugared water gains from the yeast certain soluble parts, since in the experiments just mentioned, the weights of solid yeast, washed and dried at 100 degrees C.

(212 degrees F.), are much greater than those of the raw yeast employed, dried at the same temperature.

In these experiments we employed the following weights of yeast, expressed in grammes (1 gramme=15.43 grains):

(1) 2.313

(2) 2.626

(3) 1.198

(4) 0.699

(5) 0.326

(6) 0.476

which became, after fermentation, we repeat, without taking into account the matters which the sugared water gained from the yeast:

grammes. grains.

(1) 2.486 Increase 0.173 = 2.65

(2) 2.963 Increase 0.337 = 5.16

(3) 1.700 Increase 0.502 = 7.7

(4) 0.712 Increase 0.013 = 0.2

(5) 0.325 Increase 0.009 = 0.14

(6) 0.590 Increase 0.114 = 1.75

Have we not in this marked increase in weight a proof of life, or, to adopt an expression which may be preferred, a proof of a profound chemical work of nutrition and a.s.similation?

We may cite on this subject one of our earlier experiments, which is to be found in the Comptes rendus de l'Academie for the year 1857, and which clearly shows the great influence exerted on fermentation by the soluble portion that the sugared water takes up from the globules of ferment:

"We take two equal quant.i.ties of fresh yeast that have been washed very freely. One of these we cause to ferment in water containing nothing but sugar, and, after removing from the other all its soluble particles--by boiling it in an excess of water and then filtering it to separate the globules--we add to the filtered liquid as much sugar as was used in the first case along with a mere trace of fresh yeast insufficient, as far as its weight is concerned, to affect the results of our experiment. The globules which we have sown bud, the liquid becomes turbid, a deposit of yeast gradually forms, and, side by side with these appearances, the decomposition of the sugar is effected, and in the course of a few hours manifests itself clearly. These results are such as we might have antic.i.p.ated. The following fact, however, is of importance. In effecting by these means the organization into globules of the soluble part of the yeast that we used in the second case, we find that a considerable quant.i.ty of sugar is decomposed. The following are the results of our experiment; 5 grammes of yeast caused the fermentation of 12.9 grammes of sugar in six days, at the end of which time it was exhausted. The soluble portion of a like quant.i.ty of 5 grammes of the same yeast caused the fermentation of 10 grammes of sugar in nine days, after which the yeast developed by the sowing was likewise exhausted."

How is it possible to maintain that, in the fermentation of water containing nothing but sugar, the soluble portion of the yeast does not act, either in the production of new globules or the perfection of old ones, when we see, in the preceding experiment, that after this nitrogenous and mineral portion has been removed by boiling, it immediately serves for the production of new globules, which, under the influence of the sowing of a mere trace of globules, causes the fermentation of so much sugar?

[Footnote: It is important that we should here remark that, in the fermentation of pure solution of sugar by means of yeast, the oxygen originally dissolved in the water, as well as that appropriated by the globules of yeast in their contact with air, has a considerable effect on the activity of the fermentation. As a matter of fact, if we pa.s.s a strong current of carbonic acid through the sugared water and the water in which the yeast has been treated, the fermentation will be rendered extremely sluggish, and the few new cells of yeast which form will a.s.sume strange and abnormal aspects. Indeed this might have been expected, for we have seen that yeast, when somewhat old, is incapable of development or of causing fermentation even in a fermentable medium containing all the nutritive principles of yeast if the liquid has been deprived of air; much more should we expect this to be the case in pure sugared water, likewise deprived of air.]

In short, Liebig is not justified in saying that the solution of pure sugar, caused to ferment by means of yeast, contains none of the elements needed for the growth of yeast, neither nitrogen, sulphur nor phosphorus, and that, consequently, it should not be possible, by our theory, for the sugar to ferment. On the contrary, the solution does contain all these elements, as a consequence of the introduction and presence of the yeast.

Let us proceed without examination of Liebig's criticisms:

"To this," he goes on to say, "must be added the decomposing action which yeast exercises on a great number of substances, and which resembles that which sugar undergoes. I have shown that malate of lime ferments readily enough through the action of yeast, and that it splits up into three other calcareous salts, namely, the acetate, the carbonate and the succinate. If the action of yeast consists in its increase and multiplication, it is difficult to conceive this action in the case of malate of lime and other calcareous salts of vegetable acids."

This statement, with all due deference to the opinion of our ill.u.s.trious critic, is by no means correct. Yeast has no action on malate of lime, or on other calcareous salts formed by vegetable acids. Liebig had previously, much to his own satisfaction, brought forward urea as being capable of transformation into carbonate of ammonia during alcoholic fermentation in contact with yeast. This has been proved to be erroneous. It is an error of the same kind that Liebig again brings forward here. In the fermentation of which he speaks (that of malate of lime), certain spontaneous ferments are produced, the germs of which are a.s.sociated with the yeast, and develop in the mixture of yeast and malate. The yeast merely serves as a source of food for these new ferments without taking any direct part in the fermentations of which we are speaking. Our researches leave no doubt on this point, as is evident from the observations on the fermentation of tartrate of lime previously given.

It is true that there are circ.u.mstances under which yeast brings about modifications in different substances. Doebereiner and, Mitscherlich, more especially, have shown that yeast imparts to water a soluble material, which liquefies cane-sugar and produces inversion in it by causing it to take up the elements of water, just as diastase behaves to starch or emulsin to amygdalin.

M. Berthelot also has shown that this substance may be isolated by precipitating it with alcohol, in the same way as diastase is precipitated from its solutions. [Footnote: DOEBEREINER, Journal de Chimie de Schweigger, vol. xii., p. 129, and Journal de Pharmacie, vol. i., p. 342.

MITSCHERLICH, Monatsberichte d. Kon. Preuss. Akad. d. Wissen, eu Berlin, and Rapports annuels da Berzelius, Paris, 1843, 3rd year.

On the occasion of a communication on the inversion of cane-sugar by H. Rose, published in 1840, M. Mitscherlich observed: "The inversion of cane-sugar in alcoholic fermentation is not due to the globules of yeast, but to a soluble matter in the water with which they mix. The liquid obtained by straining off the ferment on a filter paper possesses the property of converting cane-sugar into uncrystallizable sugar."

BERTHELOT, Comptes rendus de l'Academie. Meeting of May 28th, 1860, M. Berthelot confirms the preceding experiment of Mitscherlich, and proves, moreover, that the soluble matter of which the author speaks may be precipitated with alcohol without losing its invertive power.

M. Bechamp has applied Mitscherlich's observation, concerning the soluble fermentative part of yeast, to fungoid growths, and has made the interesting discovery that fungoid growths, like yeast, yield to water a substance that inverts sugar. When the production of fungoid growths is prevented by means of an antiseptic, the inversion of sugar does not take place.

We may here say a few words respecting M. Bechamp's claim to priority of discovery. It is a well-known fact that we were the first to demonstrate that living ferments might be completely developed if their germs were placed in pure water together with sugar, ammonia, and phosphates. Relying on this established fact, that moulds are capable of development in sweetened water in which, according to M. Bechamp, they invert the sugar, our author a.s.serts that he has proved that "living organized ferments may originate in media which contain no alb.u.minous substances." (See Comptes rendus, vol. ixxv., p. 1519.) To be logical, M. Bechamp might say that he has proved that certain moulds originate in pure sweetened water without nitrogen or phosphates or other mineral elements, for such a deduction might very well be drawn from his work, in which we do not find the least expression of astonishment at the possibility of moulds developing in pure water containing nothing but sugar without other mineral or organic principles.

M. Bechamp's first note on the inversion of sugar was published in 1855. In it we find nothing relating to the influence of moulds. His second, in which that influence is noticed, was published in January, 1858, that is, subsequently to our work on lactic fermentation, which appeared in November, 1857. In that work we established for the first time that the lactic ferment is a living, organized being, that alb.u.minous substances have no share in the production of fermentation, and that they only serve as the food of the ferment. M. Bechamp's note was even subsequent to our first work on alcoholic fermentation, which appeared on December 21st, 1857. It is since the appearance of these two works of ours that the preponderating influence of the life of microscopic organism in the phenomena of fermentation has been better understood. Immediately after their appearance M. Bechamp, who from 1855 had made no observation on the action of fungoid growths on sugar, although he had remarked their presence, modified his former conclusions. (Comptes rendus, January 4th, 1858.)] These are remarkable facts, which are, however, at present but vaguely connected with the alcoholic fermentation of sugar by means of yeast. The researches in which we have proved the existence of special forms of living ferments in many fermentations, which one might have supposed to have been produced by simple contact action, had established beyond doubt the existence of profound differences between those fermentations, which we have distinguished as fermentations proper, and the phenomena connected with soluble substances. The more we advance, the more clearly we are able to detect these differences. M. Dumas has insisted on the fact that the ferments of fermentation proper multiply and reproduce themselves in the process whilst the others are destroyed. [Footnote: "There are two cla.s.ses of ferments; the first, of which the yeast of beer may be taken as the type, perpetuate and renew themselves if they can find in the liquid in which they produce fermentation food enough for their wants; the second, of which diastase is the type, always sacrifice themselves in the exercise of their activity." (DUMAS, Comptes rendus de l'Academie, t. lxxv., p.

277, 1872.)] Still more recently M. Muntz has shown that chloroform prevents fermentations proper, but does not interfere with the action of diastase (Comptes rendus, 1875). M. Bouchardat had already established the fact that hydrocyanic acid, salts of mercury, ether, alcohol, creosote, and the oils of turpentine, lemon, cloves, and mustard destroy or check alcoholic fermentations, whilst in no way interfering with the glucoside fermentations (Annales de Chimie et de Physique. 3rd series, t.

xiv., 1845). We may add in praise of M. Bouchardat's sagacity, that that skilful observer has always considered these results as a proof that alcoholic fermentation is dependent on the life of the yeast-cell, and that a distinction should be made between the two orders of fermentation.

M. Paul Bert, in his remarkable studies on the influence of barometric pressure on the phenomena of life, has recognized the fact that compressed oxygen is fatal to certain ferments, whilst under similar conditions it does not interfere with the action of those substances cla.s.sed under the name of SOLUBLE FERMENTS, such as diastase (the ferment which inverts cane sugar) emulsin and others. During their stay in compressed air, ferments proper ceased their activity, nor did they resume it, even after exposure to air at ordinary pressures, provided the access of germs was prevented.

We now come to Liebig's princ.i.p.al objection, with which he concludes his ingenious argument, and to which no less than eight or nine pages of the Annales are devoted.