Friday, October 20, 2006

CHAPTER 1.: The Blood and the Third Anatomical Element by Antoine Bechamp


Gay-Lussac and Thenard analyzed fibrin as they had analyzed albumen, caseine and gelatine. Thenard said that fibrin was an isolated animal matter; Chevreul said that it was an animal proximate principle and was greatly surprised, after he had discovered oxygenated water, to find that fibrin decomposed it and disengaged the oxygen, as did organic tissues; as, for example, the liver, etc. He even thought that fibrin was the only proximate principle of its kind endowed with this property.1
This fact in the history of fibrin is important; first, because it is the pivot on which turns the demonstration that this substance, reputed a proximate principle, is of the same order as the substance of the bodies which Chevreul called organic bodies; secondly, because, although neglected by physiologists and chemists, it enabled me to place beyond doubt the existence of a third anatomical element of the blood.
I did not set out with the idea of proving that fibrin is a substance of the same order as the organic tissues. Like everybody else, I regarded it as a proximate principle; I had even maintained its specificity against the chemists who contended that it was only coagulated albumen.2
Preliminary steps to the discovery of the real nature of fibrin and of the third anatomical element of the blood. The ancients regarded it as a positive fact that all animal or vege­table matter was spontaneously alterable while putrefying or
1. Thenard, "Traite elementaire de chimie," Vol. I., p. 528. 6th Ed. 1834.2. See Memoir, Essay sur les substances albuminoid. Theses de la Facuhe de Medecir.e de Strasbourg. 1856.
fermenting. In the last century (i.e., the 18th) the chemist, Macquer, established the conditions for these changes; the presence of water, the contact of air and a certain amount of heat. Long after, when in 1837 Cagniard de la Tour regarded beer-yeast as being organized and living, and fermentation as an effect of vegetation, Schwann, generalizing the new conception, endeavoured to show that no organic matter was spontaneously alterable; that the alteration had for cause the presence of organized living things, microscopic cryptogams, vibrioniens; that is to say, ferments, the origin of which, reviving the old hypothesis of Spallanzanil, he ascribed to the germs of the air.
But, notwithstanding many important verifications, the opinion of Schwann did not prevail; the presence of living products in matter undergoing change was conceded, but while some maintained that the alteration preceded the appearance of the organized products, whatever might be their origin, others, admitting the theory of Cagniard, insisted that the living things, the ferments, were the fruit of spontaneous generation.
Schwann's point of view and the hypothesis of germs of the air were so completely abandoned that in 1854 it was admitted as a fact that even cane sugar in watery solution altered spontaneously at the ordinary temperature of the air, becoming what was called invert sugar, grape sugar. Was that true? The inversion of cane sugar, the result of a chemical reaction of reduction by hydration which was produced, as Biot had observed, under the influence of strong acids could it be effected by water only, at ordinary temperature with the aid of lime alone? I wanted to know what to believe, and I instituted experiments which commenced in 1854 and were continued until 1857. Several consequences of the greatest importance resulted and, among others, the first experimental verification of the hypothesis of germs of the air which Schwann, following Spallanzanil had invoked against spontaneous generation. In short I demonstrated:
1st. That a watery solution of cane sugar remains unaltered indefinitely, at ordinary temperature, under the two following conditions: (A) Absolutely protected from access of air and (B) in contact with a limited volume of air, to which have been added certain salts or a suitable (small) quantity of creosote; as, for instance, one to two drops per 100 c.c.
2nd. That the same solution, pure, or with the addition of certain other salts in contact with the same volume of air, permitted the appearance of cryptogamic products, moulds, etc., at the same time that the inversion of the sugar was effected.
3rd. That the moulds are actually the agents, the ferments of the inversion, by secreting the necessary zymas or soluble ferment.
4th. That creosote, which hinders the birth of the moulds, etc., does not prevent developed moulds from effecting the inversion.
And as it is evident that the water and sugar of the solution cannot of themselves give birth to those cryptogamic productions which invert the cane sugar, nor to anything whatever organized and living, the conclusion is inevitable that these experiments verified the hypothesis of the existence of germs in the air.1
1. Annales de Chimie at de physique, 3d S. op. Vol. L1V. p. 28. (1858.)
Cane sugar being a proximate principle, the experiment constituted also the first demonstration that organic matters exist which are unalterable under the conditions specified by Macquer.
To be applicable generally, it was necessary to prove that what was true for cane sugar was also true for any proximate principle, even for albumin, supposed to be so readily alterable that Colin believed it could spontaneously become an alcoholic ferment.1 But there are solutions of proximate principles, even of their mixtures, which contain some albuminoid substance like the solutions of cane sugar; these solutions, with a very small quantity of creosote added, are preserved, although in contact with a limited quantity of air, so that nothing organized makes its appearance, no ferment­ation, no putrefaction takes place. But if among the materials of the mixture there are some which are directly oxydizable by the oxygen of the air, creosote will not hinder the oxydation.
1. In 1858, M. Pasteur believed so little in the existence of germs of ferments in the air, that he asserted that the lactic ferment and beer-yeast were born spontaneously of the albuminoid matter of the fermentable media.
Let us bear in mind this capital fact which has been experimentally verified in every imaginable case: the solutions of isolated proximate principles, or mixtures of them, even albuminoid ones, first creosoted with a suitable (small) dose, exposed to the contact of a limited quantity of ordinary air, allow nothing living to appear, and remain unaltered except in cases where the mixture contains some directly oxydizable principle. In these kinds of experiments the creosote acts either by rendering the medium sterile for the germs or directly upon them, so as to prevent their development.
Organic matters reduced to proximate principles are unalterable under the conditions specified by Macquer, when the influence of the germs of the air is prevented by creosote; then they are so naturally. But Macquer did not take into account proximate principles, of which he had no idea. He really referred only to natural vegetable and animal matters, that which Thenard calls organic tissues and Chevreul organic bodies.
But among the animal matters on which Macquer experimented was milk, which he regarded as an animal emulsion, and held to be alterable of itself. Much later, Donne (an expert micrographist) and most chemists regarded milk as a solution of milk sugar, caseine and of mineral salts, holding an emulsion of butter in solution. Everybody then thought that milk was a pure mixture of proximate principles.a Such a mixture properly creosoted and in contact with a limited volume of air ought to remain unaltered indefinitely. But it was found to be otherwise. Cow's milk sufficiently creosoted at the time of milking, preserved from contact with air or in contact with only a limited amount of air, neither sours nor clots in the ordinary way. The creosote only delays the souring and the consecutive formation of the clot. But it was found that at the moment the milk became clotted, even when the phenomenon takes place in full contact with air, and with or without the addition of creosote, none of the cryptogamic productions could be found which Schwann's experiments led one to look for in it.
[aEvan Landois in his Physiology (Eng.; trans, by Stirling, 1889), makes this mistake. He makes no reference to its ferment.— Trans.]
But the souring and the clot, I do not speak of the coagulation of the milk, is only the first phase in the phenomenon of alteration. The second phase, in spite of the addition of creosote, was characterized by the constant appearance of vibrios or of bacteria. Milk then does not act as would a simple mixture of proximate principles.
These experiments and observations, which date from before 1858, were not published until 1873.1 They had greatly surprised me. Milk then was not what it had been supposed to be. There exist in it organic matters alterable, without the aid of germs of the air, and Macquer was justified in declaring them to be spontaneously alterable. And since, notwithstanding the creosote the milk already altered, soured and
1. C. R., Vol. LXXVI, p. 654.
clotted, permitted the appearance of vibrios in its substance, if these vibrios were not the products of spontaneous generation, to what did they owe their birth? It was experiments contemporary with those upon the calcareous rocks which will be studied in the last chapter of this work, as well as those which led to the discovery of the new category of living organized productions to which I have given the name of microzymas, because of their functions as ferments and of their extreme minuteness.1 It is then the microzymas of the milk itself which are the agents of its alteration and which subsequently becomes vibrios by evolution.
1. C. R., Vol. LXIII, p. 451 (1866).
The method which led to these results, as important as unexpected, the close relation between the geological ferments and the anatomical and physiological ferments of present living animals, and which at the same time answered in the negative the question of the spontaneous generation of organized ferments, is the same which has permitted the demonstration of the inherent unalterability of proximate principles, and to verify the old hypothesis of germs of the air which had been neglected. Thanks to it, it has been possible to explain anatomically and physiologically the phenomena of coagulation and the other spontaneous changes of the blood.
This method had its origin in experiments on the inversion of cane sugar, supposed to be spontaneous, and in those relating to the changes which occurred in milk, which made conspicuous the principle obtained by experiment, that creosote absolutely prevents the alteration of immediate principles by preventing the development of all living organized products, even in contact with a limited quantity of ordinary air, while the same doses under the same condi­tions did not prevent change in natural animal matters, tissues and humors, even permitting them to give birth to vibrios or to bacteria.
It is important to bear in mind that the new method (of experiment) enabled us to distinguish organic matters composed only of proximate principles, from natural vegetable and animal matters, that is to say, from organic bodies properly so-called; in short, to distinguish organic matter in the chemical meaning from that which, like milk,is organic matter in the anatomical and physiological meaning; the ferments which change the former, the organic matters in a chemical sense, that is to say, proximate principles, have for origin the germs of the air, while the ferments which change the second, that is to say, the natural organic matters, are the microzymas of their own substance, which are inherent in them as anatomical elements.
In fact, the phenomenon of the birth of the vibrios in the spontaneously altered milk, if indeed they were the result of the evolution of the microzymas of the milk, ought not to be an isolated fact, but a particular case of a general phenomenon, proper to all organic bodies, so that the fact of the birth of vibrioniens in an organic body, humor or tissue should be considered as evidence of the existence of microzymas in this tissue or this humor, even though the microscope has failed to reveal them.
Experiment has confirmed in every sense these consequences of the application of the new method of investigation to the study of the phenomenon of the spontaneous change of milk. The matter of all the tissues and humors, of all organic bodies, from the highest to the lowest—as, for instance, beer-yeast and the mother of vinegar—may give birth to vibrioniens in like conditions to those in which they are produced in the milk, or in which such conditions can be realized, if it be necessary to encourage otherwise the evolu­tion of their microzymas. And when the phenomenon of spontaneous alteration of such matter is recorded, the matter being protected from germs of the air, without the appearance of vibrioniens, it may be confidently affirmed that microzymas were present and were the agents of the change.
The following is the application of the method to fibrin, regarded as an organic body:
Demonstration that fibrin is not a proximate principle, but a false membrane of microzymas. Birth of bacteria in the fibrin.
The fibrin is produced mechanically from the blood by whipping the latter; being regarded as an organic body, it ought like milk to contain microzymas capable of undergoing vibrionian evolution.
To demonstrate this, M. Estor and I employed a modification of the method which had been used to demonstrate the microzymas of the chalk and of muscle flesh. The modification consisted in preparing a starch of the fecula of potatoes, to boil it for a long time, to creosote it while boiling and to introduce into it the solid substance to be studied at the moment it was extracted from the creosoted water, into which it had been immersed to protect it from the influence of germs of the air. The experiment was as follows: Fibrin was obtained by whipping under the following conditions; at the moment of the venesection creosote was added to the blood, and at the same time it was whipped with a bundle of metallic wires which had just been washed in boiling creo­soted water; then the fibrin was washed in creosoted water. Into 100 grammes of creosoted starch 15 grammes of freshly prepared humid fibrin were introduced and the flask sealed and placed in the oven heated to from 30° to 40° C. (= 86° —104° F.). The starch, exactly as happened with muscle tissue, became liquefied by degrees, and after a time the presence of bacteria in the mixture was evident; but it was observed that the liquifaction of the starch generally preceded the appearance of the bacteria.
The foregoing is a general view of the phenomena, but differences were observed in its manifestations, according to the species and age of the animal as well as the source of the blood. Generally the fibrin of young animals disintegrates in fluid starch, while the bacteria develop. The duration of the liquifaction of the starch is also variable.
It is known that boiled milk clots, which means that the microzymas are not killed at the temperature of boiling; on the other hand, to prevent the chalk from liquifying starch,a I was obliged to heat it (moist) to more than 200° C. (=392° F.). The microzymas of the fibrin resist up to 100° C. (= 212° F.). The fibrin was boiled for several minutes in distilled water before it was placed in the starch. In this case the liquifaction is still further delayed and even ceases to be produced if the boiling of the fibrin is too prolonged; but the bacteria appear none the less; and these bacteria present always the same morphological characters.
[aFor explanation of the action of the microzymas of the calcareous rocks see "Role de la craie dans les fermentations," Bull. Soc. Ch., Vol. VI, p. 484 (1866); also "Les Microzymas," third conference; also post-chapter VIII of this work.—Trans.)
To complete the demonstration it should be added, and M. Estor was witness of the fact, that fibrin, exactly as was the case with the mother of vinegar (a sort of vegetable membrane of visible microzymas), and under like conditions can produce lactic and butyric fermentation, a fact which will be further considered hereafter.
Such was the experiment and its complement, whence it was concluded that fibrin, like milk, like flesh, like the tissue of liver, etc., contains microzymas, since, like them, it gives birth to bacteria without the aid of germs of the air.
Under the conditions of the experiment these micro­zymas could only have come from the blood. Efforts were then made to find them in the blood itself at the moment of the venesection. This was a delicate investigation and will be reverted to hereafter, as it is allied to the whole subject of this work.
Fibrin, whether obtained by whipping or by washing the clot (we will see presently wherein these two preparations differ), is not a proximate principle, but constitutes a membrane or fibre composed of microzymas. In short, it is not organic matter in the chemical meaning, but an organic body in the anatomical and physiological meaning. Nevertheless this demonstration that fibrin contains microzymas is indirect; and one might still contend that the bacteria has been born spontaneously, in the mixture of fibrin with starch. In any case it left unsettled the nature of the substance which, in the false membrane, is like an intermicrozymian gangue, as also the quantitative relation between the microzymas and this substance. It was therefore very desir­able to obtain these microzymas isolated, as Estor and I had isolated those of the liver.
The fibrinous microzymas and their properties compared with those of the fibrin.
The question of the solubility of fibrin in dilute hydrochloric acid had been long discussed and it occurred to me that I might there find a means whereby the microzymas might be isolated, as they ought to be insoluble in it, as were those of the chalk. On going over the history of experiments on the fibrin, I found many experiments and observations relating to fibrin deserving attention which had been neglected.
Thenard had already described the action of dilute hydrochloric acid upon the fibrin of the blood and the formation of hydrochlorates of this substance, one of which, the gelatinous, was soluble in tepid water.1 Long afterwards, Bouchardat called attention to the fact that Chevreul had
1. Thenard, Traiee elementaire de chimie, Vol. III, p. 430 (1815).
demonstrated that fibrin always contained fat and he asked himself if even deprived of fatty bodies it would be a pure proximate principle? To demonstrate that that which had been generally admitted was not well founded, he made the following experiment:
He heated fresh, moist fibrin with ten times its weight of very dilute hydrochloric acid (1 to 2000) and observed that it swelled up, and by a prolonged maceration was at last dissolved, but that there always remained manifest a portion of a product which was not attacked by an excess of this very dilute acid employed as a solvent. Bouchardat called the undissolved part epidermose, and albuminose that which was dissolved.1 It was from this experiment that Bouchardat justly concluded that the fibrin is decidedly not a proximate principle.
1. C.R., Vol. XIV, p. 962 (1842).
The part of the fibrin that Bouchardat regarded under the name of epidermose, as one of the two proximate principles constituting fibrin, was precisely the microzymas which I proposed to isolate. In consequence of the viscosity of the hydrochloric solution, essentially capable of change as Bouchardat had said, and of the slowness of filtration, he had not determined the quantity of the epidermose. By employing a less diluted hydrochloric acid (1 to 3 c.c. of fuming acid per 1000 c.c.) the viscosity was diminished, and on adding to it 2-3 drops of phenol per 100 c.c. the suspected alteration was absolutely prevented, and the deposition of the insoluble part could be awaited. Even then it requires from ten to twelve days to filter a litre of the liquid. For quantitative experiments it must be left at rest and the deposit must not be turned out upon the filter until the filtration of the supernatant liquid is completed.
The grayish brown mass retained on the filter was resolved under the microscope into exceedingly fine molecular granulations, which are the microzymas, and some shapeless fragments proceeding doubtless from the blood globules destroyed during the preparation of the fibrin. To procure these molecular granulations as free as possible from foreign fragments the mass when removed from the filter is steeped in hydrochloric acid (1 in 1000), and the liquor, creosoted or carbolized, is passed through a fine linen mesh and left to deposit. The deposit is collected on a filter of very fine mesh, is successively washed with water to remove every trace of acid, and finally with ether, slightly alcoholized, to remove the fat. The matter when removed from the filter is then dried in a dry vacuum, is agglomerated, and brownish in color.
The moist fibrinous microzymas, completely drained, are composed (in hundredths) as follows:
Organic matter chiefly albuminoid........... 13.553 Mineral matter........................................ 0.384 Water (by difference).............................. 86.063 100.000
Like all organized beings, they contain mineral matter and much fixed water. Their organic matter is chiefly albuminoid; in fact, dry, it dissolves in fuming hydrochloric acid, developing when hot a violet color; and if to the hydrochloric acid water is added a white precipitate of albuminoid matter is obtained.
The minuteness of these humid microzymas, swollen with water, is extreme. Under the microscope they appear to be spherical in form, animated with the brownian movements, the diameter whereof hardly attains 0.0005 (half a thousandth of a millimeter).
Their quantity is very small. From some determinations, unavoidably somewhat uncertain, I estimated that the humid, drained fibrin of the general blood of an ox yields about one thousandth of its weight of microzymas dried at 100°. Taking this figure of 1/1000 as the best approximation, and considering that 1,000 grammes of drained fibrin contain 193 grammes of fibrin dried at 100°, it is evident that the weight of the dried microzymas is 1/193 of the dry fibrin; in short 100 parts of fibrin dried at 100° contain 0.518 parts of microzymas dried at the same temperature. This quantity appears to be very small, and one might think that in the blood it might be neglected, and that consequently the microzymas take no part in the phenomena studied. It is not so, for we shall see that they are anatomical elements and physiological agents of rare energy; and that if it was inter­esting to weigh them, it is still more so to count them.
Let us first show that in the fibrin they are at the same time that which liquifies starch and from which bacteria are derived, that which decomposes oxygenated water, and that which determines its apparent solution in dilute hydrochloric acid.
1. The fibrinous microzymas liquify starch and then become bacteria.
The microzymas of 60 grammes of fibrin obtained from the blood of an ox or of a dog, fresh, still humid, well washed so as to remove every trace of acid, are sufficient to liquify 50 grammes of potato starch at 45° to 50° C. (= 113°-122° F.). The liquifaction is completed in 16 hours; if the reaction is prolonged, Fehling's reagent is reduced. Other things equal, the liquifaction is more rapid with the microzymas of the fibrin of a dog. Finally bacteria appear, while another fermentation begins and the liquid becomes acid.
To estimate the influence of the concentration of the acid in the extraction of the microzymas the fibrin in another operation was treated with hydrochloric acid, 3 to 1,000. The microzymas did not lose any of their activity.
II. The microzymas of fibrin decompose oxygenated water.
The humid microzymas, crude, or with the fat removed by ether, as well as such as have been dried in a dry vacuum, decompose oxygenated water, setting the oxygen free, but with much greater energy than the fibrin from which they had been obtained; showing therein an energy hardly less than bioxide of manganese. I ascertained that the microzymas of the fibrin of the blood of all the animals examined by me acted in like manner.
Later the theory of these facts will be explained, but to anticipate the objection regarding the germs of the air I call attention to the four facts following:
1. Fibrinous microzymas which have liquified starch are still able to decompose oxygenated water;
2. Fibrinous microzymas which have exhausted their decomposing action upon oxygenated water can no longer liquify starch and do not develop into bacteria.
3. Fibrinous microzymas which have been subjected to boiling at 100° C. (= 212° F.) do not liquify starch and do not decompose oxygenated water;
4. Fibrinous microzymas lose the property of decomposing oxygenated water with lapse of time.
But fibrinous microzymas washed in ether, so as to remove their fat, dried in vacuo and protected from contact with the air in a closed tube, preserve for a long time the property of decomposing oxygenated water, but lose by degrees their energy; after ten years they had lost it altogether, without having appreciably lost weight.
Here was another essential property of the microzymas which I formulated!
III. Fibrin owes to its living microzymas the faculty of being dissolved in very dilute hydrochloric acid.
Bouchardat, following Thenard, observed that before dissolving in dilute acid fibrin swelled up in a translucent, colorless gelatinous mass1 and that solution was effected only after prolonged maceration. The progress of solution is so slow that Liebig, for a long time, held that fibrin was insoluble in dilute hydrochloric acid; and we shall see that it was upon this remark that he founded the distinction between muscular fibrin (masculine or syntonine) and blood fibrin. Dumas, on the other hand, verified the fact of solubility and showed that at the temperature of 40° C. (= 104° F.) solution was more rapid. According to Dumas the phenomenon is a function of time and temperature. I shall prove that it is at the same time especially a function of the activity of the microzymas.
1. It was this gelatinous mass thai Thenard correctly regarded as a hydrochlorate of organic matter.
First let us remember that creosote or phenic acid delays the souring and coagulation of milk as well as the vibrionian evolution of its microzymas. Phenol similarly retards the supposed solution of fibrin in very dilute hydrochloric acid. The following will demonstrate the fact:
A mass of 600 grammes of fresh and humid fibrin of ox's blood is divided into four equal parts—A, B, C, D, each of 150 grammes, which are treated in flasks of like capacity in the following manner:} A, 2,000 c.c. of hydrochloric acid, 2 to 1,000; B, the same volume of acid and 40 drops of phenol; C, the same volume of acid and 60 drops of phenol; D, 2,000 c.c. of boiling distilled water. The ebulition is maintained at 100° C. for two minutes. Left to cool and 4 c.c. of fuming hydrochloric acid are added, so that it was also diluted to 2 to 1,000.
The four flasks were covered and placed in the same enclosure; temperature kept at 24° to 28° C. (= 75.2° to 82.4° F.).
In A, B, C the fibrin swelled into a gelatinous mass. In d the fibrin remained a dull white, without becoming gelatinous. In A, the gelatinous mass was dissolved in three days. In B, the solution was effected in four days. In C, the solution was effected in six days. In D, the unswollen fibre remained a dull white; there was no change at the end of a fortnight, though with free access of air.
The phenomenon at the same temperature is then a function of time; it must also be so of the microzymas, since phenic acid retards it the more, the greater the dose, even as it delayed the coagulation of milk, and finally boiling for a sufficient time prevented it entirely, as it had prevented the fibrin and the fibrinous microzymas from liquifying starch and from decomposing oxygenated water. The function ascribed to the microzymas will be made still more clear when it is shown that that which is called the dissolving of fibrin is really the result of a reaction of a profound trans­formation undergone by that part of the fibrin which is in solution. The theory of the phenomenon will also be ex­plained presently; at present we will confine ourselves to saying that in the order of the ideas of these experiments the supposed solution in very dilute hydrochloric acid is, at bottom, only a mode of spontaneous alteration of the fibrin under special conditions. We have now to consider the normal method of its spontaneous alteration.
IV. Fibrin changes spontaneously without undergoing fetid putrefaction.
Gay-Lussac had observed that fresh fibrin, in an open flask, in contact with water which was renewed from time to time, putrefied and disappeared almost wholly, leaving only an insignificant insoluble residue. At the time this observation was made, it was believed that albuminoid proximate principles, as well as others, were spontaneously alterable. This was before the experiments of Schwann regarding the influence of the germs of the air. On the study of this change being again taken up to determine its products, among those which are dissolved, there was observed an albuminoid matter coagulable by heat, which was taken for albumin, also leucine, valeric acid, butyric acid, hydro-sulphate of ammonia, etc. In reality, in the experiment of Gay-Lussac, the alteration was a complex phenomenon, in which the ferments born of the germs of the air take part, and which are the agents of the fetid putrefaction. If the influence of these germs be annulled, the result is different. A mass of fresh fibrin, prepared with the usual care, was immediately im­mersed in distilled water (first carbolized by 3-4 drops per 100 c.c.), so that it was covered with a bed of liquid. Under these conditions, after five to six weeks, at a temperature ranging from 15° to 25° C. (= 59°-77° F.) the fibrin had disappeared; in its place were a clear transparent liquid and a considerable precipitate. No odor except that of the carbolic acid; no vibrios either in the liquor or in the precipitate. The alteration then had taken place without any fetid putrefaction. What was its nature? Later in chapter 11 the nature of these dissolved bodies will be compared with those of the change of fibrin in dilute hydrochloric acid. Let us see of what the precipitate consisted.
The molecular granulations of the change without fetid putrefaction of the fibrin:
In the precipitate, which is greater than the precipitate of microzymas after the disappearance of the fibrin in dilute dydrochloric acid, the microscope shows us a very great number of very small spherical molecular granulations, much more bulky than the fibrinous microzymas, and some shapeless remains, probably of fibrin or of the envelopes of blood globules. To procure these molecular granulations pure the precipitate, which is thick, is steeped in water slightly carbolized, then passed through a close-meshed silk cloth, purified again by levigation, collected on a filter, to be there again washed with water and finally with ether slightly charged with alcohol to remove the fat, and then again with water.
In this condition the molecular granulations preserve their form; they decompose oxygenated water, liquify starch and again decompose oxygenated water after having effected this liquifaction; in short, they possess the properties of fibrin and of its microzymas, but they are neither fibrin nor its microzymas.
In fact, these molecular granulations, the insoluble remains of the disappeared fibrin, treated with hydrochloric acid (2 in 1,000), dissolve much more rapidly than the fibrin, leaving undissolved microzymas identical with, as slender as, and endowed with the same properties as, those of fresh fibrin.
This last observation is important. It is a consequence of the fact that fibrin, under the conditions of the experiment, alters spontaneously without fetid putrefaction, without vibrios, leaving a residuum of molecular granulations which contain microzymas identical with those obtained from fibrin treated with dilute hydrochloric acid. It is explicable only in one way. As milk, treated with a sufficient dose of phenic acid, becomes changed otherwise than milk not so treated or only slightly so, without the microzymas becom­ing vibrios, so the microzymas of the fibrin have trans­formed, in a certain way, the intermicrozymian substance which is in it, as gangue, without undergoing vibrionian evolution, but remaining enveloped as in an atmosphere of albuminoid matter insoluble in water, but easily soluble in very dilute hydrochloric acid, the microzymas being set free.
The great importance of taking these molecular gran­ulations into consideration will be seen when studying in the third chapter the state of the fibrin in the blood. Meanwhile, the fact that the fibrin changes spontaneously in carbolized water, that is to say, without the aid of germs of the air, is a fresh proof that fibrin is not a proximate principle.
In the next chapter we shall see what is the nature of the albuminoid matters of the spontaneous alteration of fibrin in carbolized water, and compare it with that of the change which takes place under the influence of hydrochloric acid.
Meanwhile, such are the proofs, all agreeing, founded on the new method of investigation, to the effect that fibrin, like milk, the liver, etc., is neither a proximate principle nor a compound of such principles, but that like them it is an organic body, containing special microzymas; and further that these living microzymas are what, in the fibrin, liquify starch and can become vibrionien by evolution, decomposes oxygenated water; determines the change of this fibrin either in very dilute hydrochloric acid or in carbolized water.
To complete the history of the microzymas of the fibrin, we must try to discover by what mechanism they decompose oxygenated water and liquify starch, cither isolated or in fibrin; and how it is that they are the agents which determine the spontaneous alteration of fibrin, both in very dilute hydrochloric acid and in carbolized water.
Theory of the decomposition of oxygenated water by fibrin and by the fibrinous microzymas.
I stated at the commencement of this chapter that Thenard, having discovered that organic tissues (for example, the liver) decompose oxygenated water, thought that fibrin decomposed it through being a proximate principle and was the only substance of this order that did so. But what is really the nature of the phenomenon of this decomposition? Thenard said that fibrin and organic tissues "decompose oxygenated water in the same manner as metals (platinum, for instance) without giving up any of their principles, without absorbing the smallest quantity of oxygen, without undergoing the least visible change." In short, that oxygenated water is decomposed by fibrin owing to what has since been called "action through presence," "catalytic action of contact," such as metals or the bioxide of manganese. Such was the state of science a few years ago and is so, perhaps, today. It was necessary for a more exact knowledge of the blood and of organization in general to fix exactly the meaning of this, both as to fact and as to principle; the more so that they were advanced by Thenard himself as a possible explanation of the phenomenon of fermentation, and were the point of departure of the hypothesis called actions of presence, of catalytic contact, which have been the cause that the true theory of fermentation has been so much misunderstood.
In reality, the decomposition of oxygenated water by fibrin, with disengagement of oxygen, is the result not of an action by presence merely, as with the bioxide of manganese, but of a chemical reaction, as is evident from the following experiments:
30 grammes of fibrin of fresh ox-blood, containing 3 gr. 79 of matter dried at 100° C., have successively decomposed three times 60 c.c. of oxygenated water at 10.5 volumes of oxygen. At the second and third addition, the disengagement became gradually slower, so that at the third, after twenty-four hours, no more gas was given off, although the oxygen­ated water was not all decomposed. Altogether 1,600 c.c. of oxygen were set free from 1,890 c.c., which the 180 c.c. of oxygenated water employed, contained. It is evident that if the fibrin had given up nothing, if there had not been some reaction, the successive liquors resulting from the action of the oxygenated water ought not to contain any organic matter. But these liquors on being evaporated left a combustible residue, whose weight,—deducting the ashes,— were 0.16 grammes dried at 100°---i.e., 0.533 for 100 of humid fibrin or 2.76 per cent, of fibrin dried at 100° C.
The fibrinous microzymas also yield up somewhat of their substance in decomposing oxygenated water. Six grammes of these microzymas, fresh, humid, containing 0.84 gr. of matter dried at 100°, having exhausted their decomposing action, the evaporated liquors have left as resi­due, dried at 100°, 0 gr. 06 of organic combustible matter, deducting the ashes; that is to say, 1 for 100 of humid matter-that is, 7.5 per cent, of the weight of the dried microzymas.
Fibrin and its microzymas then do not decompose oxygenated water in the same manner as do platinum or the bioxide of manganese, since they both give up part of their substance which is found transformed in solution in the oxygenated water. If Thenard thought that fibrin gave up nothing it was because, on the one hand, he took into account only the disengaged oxygen, which seemed to him the whole of that which the oxygenated water could furnish; that which had been absorbed being very minute, and, on the other hand, that the fibrin seemed to him not to have undergone any change. But the change really has been great, since that which remains has no longer any action on oxygenated water, does not liquify starch and does not yield bacteria.
These remarks apply to the microzymas which are recovered, similar morphologically, to what they had been before being treated, but do not now liquify starch nor become bacteria by evolution.
It is then a fact, decomposition with oxygen set free from oxygenated water by fibrin or by its isolated microzymas, is correlative with a chemical reaction, with a change in the property of the substance which has exhausted its decomposing activity. And on comparing, in hundredths, the quantities (of the products of the reaction) which are dis­solved, of the fibrin and of the isolated microzymas, it is found that the latter furnish much more than the former. They furnish much more, even if we consider only the quantity of microzymas contained in the fibrin used, viz., o gr. 0335 for 60 grammes of humid fibrin or 5 gr' .79 of that dried at 100° C. In fact, if the dried fibrin yields or contains 2.76, calculating that which its microzymas would give by comparison with what is given by the isolated microzymas, it is found to be 4 per cent, instead of 7 per cent., which is that given by these latter. I do not lay much stress on this differ­ence because it may be due partly to the difficulties and uncertainties attendant upon the weighing. But none the less by means of these comparisons it is clear that the microzymas, whether isolated or not, give up more than does the fibrin, which tends to show that the intermicrozymian gangue of the fibrin does not exert any decomposing action upon oxygenated water, as will be presently directly demon­strated. Anyway it is evident that some substance belonging to the organization of the microzyma—probably a proximate principle—is yielded and transformed, and that it is not the entire microzyma which is the agent of the decompos­ition, since the greatest part of its mass remains, preserving its form. But what is this substance? Without being able to define it exactly we shall see that it is essentially albuminoid. Whatever it may be, it is important to know that it only effects the decomposition on certain conditions. For instance, oxygenated water, which contains a free acid, is not decom­posed either by fibrin or by the fibrinous microzymas, and, reciprocally, the fibrin dissolved by hydrochloric acid under the same conditions as the experiment of Bouchardat, in which the acid is very dilute, and containing the microzymas, decomposes it only when it is neutral. But albuminoid mat­ters combine with several acids; it is without doubt a hydro-chlorate, a sulphate, etc., of this substance, whatever it may be, which is not changed by oxygenated water, and oxygen­ated water which is not decomposed by it. In illustration of this the following very interesting case of the influence of a special acid is given.
Liebig observed that fibrin steeped in a very dilute solution of hydrocyanic acid did not decompose oxygenated water. The observation was true but incomplete, as the influence of this acid is only temporary. In fact, if the quantity of oxygenated water is sufficient, the liberation of oxygen recommences at the end of a time, the longer the greater the quantity of hydrocyanic acid. The decomposition recommences because the oxygenated water destroys the hydrocyanic acid by a phenomenon of oxydation without liberation of oxygen.1
1. C. R.. Vol. XCV, p. 926 (1887). I have since further studied this subject. Hydrocyanic acid and oxygenated water react upon each other; at first without the liberation of gas; an oxamide is formed which crystallizes; at the same time there is a liberation of heat, which increasing, the oxydation is accomplished with production of urea and liberation of oxygen. It is then solely because hydrocyanic acid and oxygenated water react first of all, that the microzymas of fibrin are protected, and not as has been supposed, because hydrocyanic acid acts as a poison upon these microzymas. The fact that after the destruction of the hydrocyanic acid, the fibrin again decomposes oxygenated water, proves that what happens is not a phenomenon of poisoning. This will be further treated hereafter.
Theory of the liquifaction of fecula starch by fibrin and by the fibrinous microzymas.
Fibrin and its microzymas are insoluble in water; on the other hand, Payen demonstrated that fecula exists in a special condition of hydration and of swelling, in which it is similarly insoluble.
How then can these insoluble bodies act upon one another, the one, fecula, dissolving, while the other, the fib­rin or the microzyrnas, remain insoluble? The explanation is the same as before given of the inversion of cane sugar by moulds, born of the germs of the air in its aqueous solution, but which are insoluble, as are the microzymas.
I have proved directly that these moulds, born of other organized ferments, and other microzymas, produce in themselves and secrete soluble products of an albuminoid nature, which are of the same order as those called soluble ferments and which were confused in the same category with organized insoluble ferments. Having thus established the anatomical origin of soluble ferments, to mark the union of dependence between the product and the producer, I gave the name of zymas to what had been termed soluble ferment.
This established, as the microzymasa of sprouted barley produce diastase or hordeozymas, as the pancreas or its microzymas produce pancreazymas, which liquify and saccharify starch, so the fibrinous microzymas produce the zymas which effect its liquifaction.
[aFor the purpose of continuing "the conspiracy of silence" beneath which the marvellous discoveries of Bechamp have been obscured for so many years, this word and its congeners are never used in the writings of the chief conspirators, nor in those of the numerous leaders of the profession who have been duped by them.—Trans.]
And since every zymas is of the albuminoid order, as the fibrinous microzymas which have exhausted their decom­posing action upon oxygenated water do not liquify starch, we can say that the substance which in the fibrinous microzymas is given up and transformed by oxygenated water, is precisely this zymas, an albuminoid substance which liquifies the starch of fecula.
Theory of the spontaneous alteration of fibrin, whether in very dilute hydrochloric acid or in carbolized water.
The two constitutive parts of fibrin are equally insoluble in water and in very dilute dydrochloric acid. As for the liquifaction of starch by them, the same question arises: how can these two insoluble bodies act upon one another, the one remaining insoluble, the microzymas; the other, the albuminoid matter, entering into solution? The answer is the same. In the same manner that fecula is made soluble and transformed by the zymas which the microzymas secrete, so the albuminoid matter is dissolved by this zymas while being transformed.
The explanation of the phenomenon is thus very simple. Only in the case in which very dilute hydrochloric acid inter­venes does the transforming chemical action of the zymas secreted by the microzymas act upon the insoluble combination, which the albuminoid matter makes, at first with the hydrochloric acid; while in the carbolized water it acts directly on the insoluble albuminoid matter as on the amylaceous matter of the starch. But the action of the zymas being exer­cised on the one hand on the hydrochloric combination of the albuminoid matter and on the other on this matter itself, it is not to be wondered at that the soluble products of the reaction differ in some respects, as will be explained in the second chapter.
It is now easy to understand why the previous coction of the fibrin hinders alike its solution in dilute hydrochloric acid and in carbolized water. It is because heat at 100° kills the microzymas as it destroys the activity of all zymases, and doubtless because the inter-microzymian albuminoid matter has undergone the special coagulation which hinders it from effecting the gelatinous combination with hydrochloric acid before spoken of.
To sum up: Fibrin is not a proximate principle. It decomposes oxygenated water correlatively to a change in the zymas produced by its microzymas, which zymas is the agent of the liquifaction of starch and of the changes under­gone by its albuminoid matter, whether in dilute hydrochloric acid or in carbolized water, conditions in which its micro­zymas do not undergo vibrionian evolution. In short, these microzymas, whether in the fibrin or isolated, are not the agents of the decomposition of the oxygenated water after the manner of ferments—that is to say, physiologically by a phenomenon of fermentation, but only as producers of the proximate principle which the oxygenated water changes as it changes hydrocyanic acid.
To complete a knowledge of fibrin and of its micro-zymas, I recall the facts that Estor and I, in our note, described an experiment from which we concluded that "in the presence of pure calcic carbonate and so long as the microzymas of the fibrin continued to evolve they behaved, as regards fibrin, at the same time as alcoholic ferment, and as acetic, lactic and butyric ferments.1 Among these experi­ments I will describe two, because they were conducted on a sufficiently large scale the better to establish results. The proportions of the materials employed were as follows:
Fecula of potatoes, 5 parts, transformed into starch in 85 per cent, of water; pure calcic carbonate, 1 part; fibrin fresh, moist, newly prepared, 0.13 parts; temperature of the oven 35° to 40° C. (= 95°-104° F.).
1. C. R., Vol. LIX.715-716.
The two experiments were started on the 22nd of May. The next day disengagement of gas commenced, a mixture of carbonic acid and of hydrogen. From the 8th day the gas was analyzed repeatedly, and was found to be composed as follows, in hundredths:

gaseous mixture is thus seen to have varied with the complication of the reaction.
One of the experiments was stopped on the 10th September for the purpose of making the analysis. There was still a large amount of fecula not transformed; the products of the fermentation were as follows:
Absolute alcohol............................................................ 21 cent, cubes. Proprionic acid ................................................................... 12 grammes. Butyric acid .............................................................................. 150 " Chrystallized acetate of soda ................................................. 650 " Chrystallized lactate of chalk.................................................. 709 "
The second operation, upon a greater scale, was continued until the lactate formed had been transformed; the analysis of the products was made on the 10th of May of the following year. The experiment then had lasted nearly a year. There was still some fecula not transformed. There were found:
Alcohol mixed with higher alcohols........................................ 78 c.c. Proprionic acid.........................................................................80 gr. Butyric acid ............................................................................680 gr. Acids higher than the butyric up to caprylic ......................... 245 gr. Crystallized acetate of soda....................................................725 gr.

Thus, as in the classical lactic fermentations, the ferment which produced the lactic acid is that also which destroys this acid in the lactate of lime. It is only necessary to observe that the products formed by the microzymas of the fibrin differ greatly, both in proportion and in quality, from those of ordinary lactic fermentations, and especially from those by mother of vinegar. I shall, by and by, insist further on the fact that the bacteria of the microzymas which evolve in the first phase have gradually but completely disappeared in the second, so that at the end there only remained a few forms closely allied to the microzymas.

But I insist here on the fact that for the two experiments 200 grammes of fresh fibrin were employed containing at the start at most 0 gr. 2 of microzymas to effect the prodigious transformations of the fecula. The fibrinous microzymas are then figured ferments of rare energy.

Such were the preliminaries to the discovery of the third anatomical element of the blood. For a complete understanding of the fibrin and the products of its changes it is necessary to know in what light to regard the albuminose of Bouchardat, which this savant believed existed in the supposed solution of fibrin in dilute hydrochloric acid, and to do this we must have a better knowledge of the albuminoids.

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