CHAPTER VI

OF THE REAL CHEMICAL, ANATOMICAL AND PHYSIOLOGICAL MEANING OF THE COAGULATION OF THE SHED BLOOD; COAGULATION OF THE BLOOD; THE BLOOD OF THE HORSE; THE SERUM OF THE BLOOD; COAGULATION OF BLOOD DILUTED WITH WATER; SECOND PHASE OF THE SPONTANEOUS ALTERATION OF THE BLOOD; THE BLOOD IN CALCINED AIR; EXPERIMENT PROVING OXYGEN HAS NO SHARE IN THE DESTRUCTION OF THE GLOBULES IN THE DEFIBRINATED BLOOD; SPONTANEOUS ALTERATION OF FLESH; SPONTANEOUS ALTERATION OF MILK; COAGULATION OF MILK; FERMENTATION OF THE EGG; SPONTANEOUS DESTRUCTION OF THE CELLULE OF YEAST; SPONTANEOUS DESTRUCTION OF TISSUES; SPONTANEOUS ALTERATION OF THE BLOOD.

Coagulation of blood diffused in water.
Second phase of the spontaneous alteration of the blood.
The following experiment demonstrates that oxygen has no influence in the phenomenon of the destruction of the globules in defibrinated blood.

The blood is a flowing tissue; Bordeu had already remarked that it was flowing flesh. This chemically, histologically and physiologically is far from being true;¹ the only thing certain is that the blood like the flesh is a tissue, and that both of them are spontaneously alterable, as are all tissues, when the natural conditions of existence of their anatomical elements are no longer realized. For instance, in the case of the muscular tissue, cadaveric rigidity follows death very quickly, and, in the case of the blood, the formation of the clot follows closely upon its issue from the vessels.

1. It was, I think, in 1742, in his thesis, entitled "Chylificationis historia," maintained, at Montpellier, at the age of 20 years, that Bordeu, among the original ideas which makes him to be regarded as one of the precursors of Bichat, put forth the idea that the blood is flowing flesh. In the seventeenth century, Amyot had already said that "the blood is engendered by the transmutaiton of some flesh which becomes a flowing liquid." (Diet, of Littre.) If an original sketch, later recognised as correct, is sufficient for the author to be historically regarded as the discoverer, assuredly Bordeu would deserve to be regarded as having discovered that the blood, like muscle flesh, is a tissue. But, as observed by Babinet, "if the ancients have said everything, they have demonstrated nothing." Bichat also inserted the blood tissue among his twenty-one elementary tissues, next to his muscular tissues.
But since Bichat other savants have so done. In my time at Montpellier the Professor of Physiology, M. Rouget, taught that the blood, because of its globules, is a tissue; and I replied that, according to the ideas then accepted, the blood is no more a liquid or a tissue than was sweetened water holding in suspension globules of yeast.
To-day, M. Ranvier also says that the blood is a tissue because it contains figured elements like the lymph. Doubtless the chief condition necessary for its being regarded as a tissue is for a product of an organism to contain some figured element, but that is not enough; according to the doctrine of Bichat it is also necessary to show that this element is living; and still that is insufficient; otherwise milk, the saliva, and even the urine and certain pathological serosities, spontaneously coagulable, would like the lymph and the blood be tissues. I will consider this further in the last chapter.

It is not disputed that the phenomenon of the coagulation of the blood is spontaneous; the standard facts concerning this phenomenon are as follows: The defibrinated blood obtained by whipping does not coagulate spontaneously, and the globules remain intact in the liquor which has lost its peculiar viscosity.

The blood of oxen and of sheep (I leave for the present the blood of the horse), received into a glass or metal vessel, seems to coagulate throughout its mass, uniformly from the periphery to the center, forming a single solid clot which follows the shape of the vessel into which it has been received. This clot contracts by degrees, up to a certain limit, expelling from it in so doing the serum of a lemon color, which thereafter becomes red-colored, getting gradually deeper, so that the contracted clot (withdrawn from the edges) floats in the serum which has been expelled from its primitive mass. As Haller has already said, the clot is formed by the network of fibres of the fibrin which imprisons the globules in its meshes.

It remains to explain these phenomena invoking only the chemical, physiological and anatomical facts studied in the preceding chapters. The necessary condition for the tissue to remain flowing is that the properties of the anatomical elements and their independence remain unchanged; that their relations with the intercellular liquor remain constant, not only in the vessel, but also after the venesection.

We know the distribution of the globules in the blood, and how they pass, one by one, into certain capillaries; the distribution of the microzymian molecular granulations is such that if the globules should disappear they will occupy all the space which the globules occupied; that is to say, that the former exist in such a manner in the blood that the globule; move in it in displacing the former unceasingly, but in immediately reoccupying the abandoned space; in short they realize the conception of Dumas, when he said of the fibrin that it exists in a flowing condition in the blood; only that this flowing condition is molecular, attached, as we have seen, as to each molecular granulation to a microzyma for nucleus, forming a limited atmosphere around each, which albuminoid atmosphere is absolutely insoluble in the blood serum.

To understand that the number of microzymas of the blood is sufficiently large in order that, surrounded by the atmosphere which constitutes them microzymian molecular granulations, they may occupy every point of the blood mass, even that of the globules which were driven away, it is sufficient to know that they exist there in innumerable quantity. This is proved in the following manner: The fibrinous microzymas, that is to say, the blood microzymas, are with the pancreatic microzymas, the smallest I have observed. They assume, in their extreme minuteness, the spherical form. The diameter of these microzymas probably does not attain 0.0005mm (mm?) in the humid state. This enables us to calculate that in the volume of 1 mm. cube there are at least 15 milliards 250 millions. Now a litre of sheep's blood furnishes 5.25 grammes of dried molecular granulations, which nearly represent the weight of the fibrin that the same-blood furnishes by whipping. But the fibrin, supposed dried, contains 1/193 of its weight of dried microzymas; then 5.25 grammes of molecular granulations, likewise dried, contains 5.25/193 = 0.0272 grammes; that is to say, 27 milligrammes of dried microzymas per litre of blood, which represents a very much greater weight of humid physiological microzymas; but in taking this figure for the weight of the microzymas in the physiological condition of humidity, and 15 milliards per milligrammes or cubic millemetre, it is seen that one litre of blood contains more than 27 times 15 milliards of microzymas. But their weight is in reality much less than this, for, humid, these microzymas can retain 80% of water; in the blood, enveloped with an albuminoid atmosphere saturated with the intercellular liquor, they certainly retain less, but in a manner to render legitimate the approximate calculations above given.

It will be interesting to learn the thickness of the albuminoid atmosphere which surrounds each microzyma to constitute the microzymian molecular granulation, such as it exists in the blood at the moment of venesection. An approximate idea of this can be obtained by considering that the volume of the spherical molecular granulations with a condensed atmosphere of the deposit formed in the blood which has had added to it twice its volume of alcohol at 35-40 degrees is about 50 cubic centimetres per 1000 c.c. of blood; making allowance for the space occupied by the globules, we may consider that the volume of the molecular granulations, before the condensation of their atmosphere, was about twenty times greater to occupy the entire space of the 1,000 c.c. of blood; it will be presently proven that they do in reality occupy it all. The albuminoid atmosphere being thus swollen and saturated with the intercellular liquor, it can be understood that the great number of milliards of these molecular granulations arc sufficient to occupy the entire space presented by the blood, provided that their density be very little greater, if not equal, to that of the intercellular liquor which isolates them from one another. This state of the microzymian molecular granulations explains the sort of viscosity which belongs to the blood, and how the globules, whose density is greater, move about in it without being deposited and are only deposited very slowly in the ox's or sheep's blood when at rest; and we shall see how the exception presented by the blood of the horse confirms these considerations.

We have now to inquire whether, after the shedding the blood, the conditions, which I have mentioned as necessary for the blood tissue to remain flowing, can still be realized.

And first it is evident that this tissue, bearing in mind that we are considering a mixture outside of the vessels, longer in its natural physiological situation.

In this new situation the intercellular liquor, in which are united all the soluble organic and mineral products of the denutrition of the anatomical elements of the containers and of the contents, immediately changes its composition; for the disassimilated products, which have become non-usable, are no longer eliminated, and the usable can no longer be utilized or renewed; further, the anatomical elements of the flowing tissue, which have imperative need of oxygen to function properly, are more and more deprived of it; for, after having consumed all that was held in reset the flowing tissue and which the uneliminated products, thus accumulating in it, had been able to absorb, the oxygen thus consumed is not renewed by respiration. The first change then which happens in the shed-blood is that which the intercellular liquor necessarily undergoes in its composition.

The microzymian molecular granulations are first anatomical elements to be affected by this change of medium and of conditions of existence, and, we have seen, this impression is so intense and at the same time so rapid that it manifests itself in a few seconds by the profound change which occurs in the albuminoid substance of their atmosphere which, from being as it was immediately soluble in very dilute hydrochloric acid, becomes insoluble in it, dissolving in it, only as a function of time and temperature, while being transformed. It follows that this influence has the effect of coagulating this substance relatively will dilute hydrochloric acid.

The coagulation being complete, by degrees the clot divides itself spontaneously into two parts. That which, in the clot, encloses the globules; that is to say, the network of fibrin formed by the soldering of the microzymian molecular granulations, contracts then more and more, up to a certain limit, preserving the shape of the vessel in which the clot is moulded, and while the retraction takes place a part of the intercellular liquor is expelled, constituting what is called the serum, in which the retracted tissue is now immersed.

And the first serum thus expelled is transparent and lemon colored, but by degrees the oxygen which the intercellular liquor holds dissolved, as well as that which the globules contain, is consumed; then is manifested the phenomenon observed and explained by J. B. Dumas in globules deprived of oxygen; they change, and their changed coloring matter is diffused in the circumambient serum which becomes more and more of a deep red color. This is what the plasmatic hypothesis cannot explain, if one regards the plasma as a liquid in which all the components are in perfect solution. Let us now give a more direct demonstration of this fact.

All other things being equal, the rapidity of the coagulation of the blood may vary notably from one species to another. The blood of the horse, under the same conditions as that of the ox and of the sheep, is well fitted to verify the truth of the role ascribed to the third anatomical element of the flowing tissue. It is known that the (shed) blood of the horse is divided by rest into two layers: the lower, called cruor, is formed by the globules; the upper, called liquor, contains the microzymian molecular granulations. The upper layer, transparent but not limpid, flowing, possessing the peculiar viscosity, can even be decanted after the globules are precipitated and very quickly forms a clot in all its mass, in such wise, that the containing vessel maybe turned up-side down without one drop of the liquor escaping; after which the retraction, with loss of transparency, is produced and the serum commences to be progressively expressed from it, as in the case of the bloods whose globules do not separate. Now this retraction would not be produced if dealing with a substance really dissolved which, in coagulating, should become insoluble in the same medium.¹

1. The coagulation of the blood has been compared to the gelatinization of a solution of gelatine (Frey. Traite d'histologie et d'histochimie. p. 141). but a solution of pure gelatine heated in distilled water and sufficiently concentrated can be obtained absolutely limpid by careful filtration. On cooling this solution forms a jelly, more or less consistent, perfectly limpid, not undergoing any other contraction than that produced by the lowering of the temperature. Nevertheless, in fact the gelatine has coagulated, for in the gelatinized solution it has become insoluble in cold water as it was before. But the comparison made by Dumas with the state of fecula in starch is more correct. In fact in the transparent starch the fecula is not dissolved, it cannot be filtered. By cooling, after a long time, the starch undergoes a change in its appearance; it becomes more opaque and a retraction accompanied by expulsion of liquid can be observed. This happens because the fecula was not dissolved, but simply enormously distended.

The peculiarity presented by the blood of the horse may be due to the greater difference between the density of its globules and that of the intercellular liquor, and at the same time to a greater softness of the albuminoid atmosphere of the microzymian molecular granulations, which will be more swollen, and, consequently, their mass surrounded by the serum more readily traversed by the globules. I have, therefore, compared, as being the only thing accessible for experimentation, the serums of blood of the ox and of the sheep, with that of the blood of the horse, with regard to their general composition.

The rotatory power of the organic materials, taken altogether, of the serum of blood of the ox is: (a) j = — 52°.8.

In 100 c.c. of this serum there were:
Grammes.

Amount of fixed organic matters ............................................. . 8.72
Amount of fixed mineral matters, ashes .................................. 0.68
Sum of fixed matters ..................................................... 9.40

Proportions in hundredths of organic and mineral matters:
Amount of fixed organic matters ............................................... 92.76
Amount of fixed mineral matters................................................ 7.24
100.00

The rotatory power of the organic matters, taken altogether, of the blood of the horse is: (a) j = — 53°.

In 100 c.c. of the serum of the blood of the horse there were:
Grammes

Amount of fixed organic matters ................................................................. 6.70
Amount of fixed mineral matters, ashes ................................................... 0.06
Sum of fixed matters ................................................................................. 6.76

Proportions in hundredths of organic and mineral matters:
    Amount of fixed organic matters ................................................................. 99.1
    Amount of fixed mineral matters ................................................................... 0.9
            Sum of fixed matters ......................................................................... 100.0

Rotatory power of the serum of sheep's blood was: (a) j = — 64°.

And the proportions in hundredths of the fixed mineral and organic manors, this serum, were:
    Amount of fixed organic matters ................................................................ 91.4
    Amount of fixed mineral matters, ashes........................................................ 8.6
                                                                                                 100.C

The most striking thing, above all, is not alone that the serum of the blood of the horse contains less fixed materials, organic and mineral, with a rotatory power very nearly equal to that of the blood of the ox and much lower than that of sheep's blood, but especially that it contains between seven and eight times less mineral matters than either of the other two.

The serum of the blood of the horse differs then prodigiously from the two serums to which I had compared it; this amply explains at once the softness of the albuminoid atmosphere of the granulations and the rapid deposit of the globules.

Experiment thus verifies the fact that the molecular granulations occupy in the blood not only all the space occupied by the globules, but also all the space left free by their precipitation.

According to Charles Robin¹ agreeing with many authors, the blood of man, and that of the dog and of the ox, behave like that of the horse, when they are cooled down a little below freezing; they remain liquid a sufficiently long time to enable the globules to be deposited, the leukocytes, according to Donne, forming a grayish layer upon the top of the blood globules; the supernatant liquid then forms the clot, losing its transparency when the temperature reaches 12° to 14° C.(=53°.6 to57°.2,F.), and it should be added that Ch. Robin, having observed the transparency of the supernatant layer separate from the globules under these circumstances, and which he called plasma, stated that it could not be filtered.

1. Ch. Robin, Lecons surles Humeurs. p. 59 (1871).

I regret that I had not had the time to verify these experiments, but the facts may well be regarded as true, being certified by Ch. Robin. They support the theory which I lay down; the lowering of the temperature below zero (32° F.) having the effect of singularly retarding the functions of nutrition of the anatomical elements (as it retarded those of beeryeast) ought to retard the coagulation of the albuminoid atmosphere of the microzymian molecular granulations.

The formation of the classical fibrin by whipping remains to be explained, and this is now very easy. It is the result of a simultaneous mechanical and chemical action. By the mechanical action the layer of intercellular liquor which separates the molecular granulations is broken, while the granulations forcibly set free are united by their mucous albuminoid atmosphere, at the same time that the changes in the conditions of existence determine the allotropic transformation of the albuminoid substance which, coagulated as we have seen, contracts at the same time, still enveloping the microzymas; in consequence of which that which was diffused throughout all the volume of the blood is reduced to relatively small volume occupied by the classical fibrin. And by the small volume of the fibrin obtained by whipping a judgment may be formed of the enormous volume formed by the albuminoid atmosphere which surrounded the granulations of the flowing tissue at the moment of the venesection, as we have observed also in relation to the molecular granulations when separated from the alcoholized blood.

In the separation of fibrin by whipping the globules remain entire, and I have explained that if the weight of the fibrin thus produced is less than that of fibrin obtained by washing the contracted clot, it is because to the free molecular granulations of the blood are added those obtained by the destruction of the globules with their envelopes.

Such were the facts upon which rested the experimental physiological theory of the spontaneous coagulation of the blood, when, in 1895, I communicated them to the Bordeaux Congress of the French Association for the advancement of science.

If there remained any doubt as to the value of the theory concerning the spontaneous coagulation of the blood as a flowing tissue, here is what must remove them. The new experiments to which they relate are the fruit of the following considerations:

If the formation of the clot is actually the result of the spontaneous soldering of the mucous albuminoid atmospheres of the microzymian molecular granulations; if in the presence of alcohol, diluted to a suitable degree, these atmospheres are condensed, the molecular granulations remaining independent of one another, what would happen if instead of alcohol the blood should be received into water? The following is the experimental answer:

Coagulation of blood diffused in water. At the moment of venesection the blood is received into increasing volumes of distilled water up to half of its volume. The clot is formed in all cases; with small quantities the globules do not appear to be altered and the first serum has its ordinary appearance, but in proportion as the quantity of water is increased there arrives a time when the serum becomes colored. Encouraged by these trials, one day in November, at Paris, the blood of the general venesection of a Russian sheep was received, as to one part into two volumes of alcohol at 36°, carbolated with two drops per hundred c.c., and as to the other part into two volumes of distilled water carbolized in the same proportion.

The alcoholized mixture yielded the ordinary deposit of microzymian molecular granulations with the properties with which we are now familiar and naturally there was no trace of a clot.

The aqueous mixture furnished something very different from a precipitate. Like the alcoholic mixture, the aqueous mixture had been made at 9 o'clock in the morning; naturally, like the other, it was of a deep red color, since under such conditions all the haemoglobin of the blood globules had become dissolved. But at 3 p.m. the aqueous mixture was coagulated, the clot occupying the entire volume of the mixture, a volume three times greater than the blood clot without the addition of water. A little deep red liquid was already expelled; the next day the blood clot was not more contracted.

The experiment repeated with ox blood gave the same results; the blood clot was formed in the entire mass, floating in a little deep red liquid, etc.

The trembling and nearly transparent clots of the two experiments were placed to drain on a moist cloth of close texture. At a given moment the cloths were found covered with a mucous substance which, a prolonged washing, with water slightly carbolized (1 drop per 100 c.c.), then with alcohol at 25%, and again with water, did not completely decolor; at the end there remained a red false membrane which could be removed in a single piece from the damp cloth; such was the appearance and state of the fibrin of the clot formed under these conditions; its quantity is perceptibly that which is isolated by the washing of the ordinary clot. In this condition this fibrin is not dissolved immediately in hydrochloric acid at 2/1000, but like ordinary fibrin is a function of time and of temperature, and without first taking on the condition of a jelly like the fibrin from whipping, etc.

If then the albuminoid atmosphere of the hematic microzymian molecular granulations is condensed under the influence of alcohol of proper strength, the same atmosphere is much more distended, three times more, in water, and the blood still coagulates in all its mass, the globules being destroyed.

The conclusion which results from this new series of experiments is that the physiological theory of the spontaneous coagulation of the blood, founded on the existence of a third anatomical element, the microzymian molecular granulations of the flowing tissue, sufficiently explains the facts.

It is time then to erase from the language of science the words plasma, plasmine, fibrinogen and of fibrinoplastic, with which it has been encumbered.^a There must also be erased for the explanation of the phenomenon the pretended influence of the globules, of calcareous or other salts, catalytic actions of contact, etc., not to speak of various occult

[a. Term microbe must also be erased; unless it be desired to retain it to denote mankind and other short-lived animals! Trans.]

influences. An exact knowledge of the anatomy of the blood and of the conditions of existence of the anatomical elements will suffice.¹ But it is also necessary to understand in another way than has heretofore been done the meaning of what is called the coagulation of the blood. In truth, the blood does not coagulate. The experiment proves it; it is the substance of the atmosphere of the third anatomical element of the flowing tissue which, in undergoing the allotropic change of coagulation, gives to the aggregate of the phenomenon the appearance of a total coagulation; but, as we have seen, it is only an illusion.

1. It is because the conditions of existence of the anatomical elements are longer realized when the blood is preserved between two ligatures in the vessel which contains it that the coagulation of the flowing tissue is often long defer red. This explains the success of certain experiments of authors and, the most recent, those of M. Fr. Glenard (1875). (Thesis above quoted.)

The supposed spontaneous coagulation of the blood is at bottom, only the end of the first phase of the spontaneous alteration of the flowing tissue, as the cadaveric rigidity, marks the first phase of the spontaneous alteration of the muscular tissue.

But, what is it that changes in a tissue? And what is the second phase of the spontaneous alteration of the blood and at what moment does it begin?

It is of importance to the plan of this chapter to give a precise answer to these questions.

Second phase of the spontaneous alteration of the blood.

The first begins by the chemical alteration of coagulation of the albuminoid atmosphere of the microzymian molecular granulation, whence results the formation of the clot, the retraction of the latter and the expulsion of the lemon-colored serum. The globules have nothing to do with this phenomenon, as is incontestably proven by the experiment with the blood of the horse.

The second phase begins at the moment when the serum becomes colored with red, which shows that the change of the blood globules has commenced, their haemaglobin, more or less changed, being diffused in the serum. The following experiment of M. Pasteur has shown what becomes of the globules in this change. This savant made it in 1863,¹ five years after my verification of the hypothesis of germs in the air, when he had given up belief in the spontaneous generation of ferments, with the object of demonstrating that in the absence of germs the blood would not putrify because nothing living would appear in it. To understand this it is to be remembered that M. Pasteur was a protoplasmist seeing in an organism only proximate principles, admitting in it nothing figured, autonomically living, comparable to the figured ferments.

I take the recital of the experiment from a book of the author, published long after the microzymas had been discovered and the microzymian theory of the organization completed.

He commences as follows: "Let us examine into the interior of living beings, in good health, such or such of the materials which may be found there, and examine them in the state in which life has formed them, in contact with pure air."²

1. Pasteur. "Recherches sur la putrefaction." C. R.. Vol. LV1 (1863).
2. L Pasteur. "Etudes sur la beere." p. 46 (1876).

In fact, with the assistance of Cl. Barnard, he poured the blood of a dog directly into a vessel, the air in which had been calcined. The receiver, sealed with the blow-pipe, contained thus one of the materials to be examined, obtained from the interior of the animal, and thus protected from the germs of the air. I now quote textually as follows what M. Fasten thought he observed:

1. The blood does not putrify, even at the highest temperature of the atmosphere; its odor remains that of fresh blood, or takes on that of lye.

2. After an exposure of the flasks to 25°-30° C. (= 77° to 86° F.), during several weeks, nothing can be observed but an absorption of 2% to 3% of oxygen, which is replaced by a perceptibly equal volume of carbonic acid.

3. Under the circumstances in which the blood of the dog, exposed to the contact of pure air, does not putrify at all, blood crystals are formed with remarkable readiness.

4. In the first days of its being placed in the oven, slowly, at the ordinary temperature, the serum became gradully colored a deep brown.

5. In proportion as this effect is produced the blood globules disappear and the serum and the clot become filled with crystals of a brown or red color. After some weeks not a single blood globule remains either in the serum or in the clot. After a longer interval the whole of the fibrin may become collected in a single hyaline mass.¹

^{1. Ibid, p.49.}

Such was the experiment from which M. Pasteur concluded that, if protected from germs of the air, blood did not putrify at all; that is to say, was not altered by the action of any figured ferment, which in his opinion only the germs of the air could produce. I have elsewhere shown that, supposing the technique of the experiment to have been accurately carried out, the observations made thereon were incomplete, and the interpretation of the results vicious in the extreme. I will revert to this hereafter; for the present I will only show that the facts of the experiment corroborate my own.

It is evident that, taken altogether, the experiment of M. Pasteur has confirmed that which the microzymian theory never fails to prove, namely, that every tissue, every humor, withdrawn from a living healthy animal, absolutely protected from the germs of the air, necessarily alters and, consequently, alters spontaneously.

It demonstrates further that there are two distinct phases in the changes of the blood.

It is true that M. Pasteur did not stop an instant to consider the phenomenon of coagulation, but he observed that the serum, at first lemon-colored, became by degrees red, then deep brown, without insisting on the mechanism of the contraction of the clot and the expulsion and tardy coloration of the serum which marks the commencement of the second phase and which follows so exactly the consumption of oxygen which the blood contained, that the author himself testified to the absorption of a small quantity of the oxygen in his flasks with a corresponding production of carbonic acid. During the second phase, in which the haemoglobin alters more and more, blood crystals are formed, at last the globules are destroyed and disappear while the fibrin which imprisoned them in the network formed by it contracted more and more.

This picture shows clearly that during the two phases the alteration is at once chemical and anatomical, ending in the destruction and total disappearance of the globules.

But to what cause did M. Pasteur ascribe such prodigious effects? In 1863 he also experimented on muscle flesh, imitating my method of investigation, replacing creosote by alcohol. He wrapped up a voluminous mass of flesh in a linen, soaked in alcohol, and left it to itself.

"There will not be any putrefaction, he said, not in the interior, because the germs of the vibrios are absent, nor externally because the vapors of alcohol hinder the development of the germs on the surface." Nevertheless the author certified that the meat "became gamey in a pronounced manner." And why did it become gamey? Simply, said he, "because it is impossible at ordinary temperature to withdraw the interior of this flesh from the reaction of solids and liquids upon one another . . . There will necessarily always be what are called actions of contact, which develop in the flesh small quantities of new substances, which add to the savor of the meat their own savor."¹

Then at ordinary temperatures the same thing should happen with the blood as with the muscle tissues; there will be actions of contact, reactions of the solids upon the liquids; and that is why the blood changes without putrifying as flesh becomes gamey without putrefaction. There is some excuse for M. Pasteur and the academies with him to have been satisfied with these explanations, seeing that protoplasmism had been accepted as a dogma among savants. It was faith in this doctrine which caused the globule to be regarded as an organite, and the mass of the blood or of the flesh, for a collection of proximate principles, and, which is more serious, hindered M. Pasteur from seeing the microzymas among the results of his experiment, or, if he saw them, caused him to neglect them, as he had neglected the microzymas and even the vibrios in the gamey meat. However it maybe, it was by invoking actions of contact, and of reactions of solids and liquids, that M. Pasteur persuaded himself that the alterations of the blood and those of the flesh were not phenomena of putrefaction; that is to say, of fermentation. And this manner of regarding things on the part of the celebrated savant prevailed so generally that I was obliged to make an explanation on the subject of an assertion made by Balardin 1874.

M. Servel, Estor's assistant, had presented to the Academy² a work verifying the fact that with absolute protection from atmospheric germs the most divers tissues could

1. L. Pasteur, Recherches sur la putrefaction. C. R., Vol. LVI. p. 118-9 (1894).
2. C. R., Vol. LXXIX, p. 1270.

produce bacteria even in their interior, and cited other verifications made in Germany. Now, Balard, who presented the work, took the occasion to say that blood is preserved without putrid fermentation and without bacteria in the experiment of M. Pasteur.¹ I replied to M. Balard, saying that the blood is one of these substances, of which the egg is another, whereof the microzymas undergo with most difficulty the vibrionian evolution.² In spite of this the experiment of M. Pasteur was given as a pre-emptory demonstration that the interior medium contains nothing figured which could become bacterium by evolution in the substance of a tissue or of a humor; and it was asserted, in agreement with him, that the experiment proved at the same time that the bodies of animals were closed to germs from without.

1. Ibid., p. 1272
2. Ibid.. Vol. LXXX. p. 494. The note of M. Servel and my reply to M. Balard should be read with attention to have a clear idea of the state of the question in 1875.

Nevertheless, in a discussion at the Academy of Medicine, where, once more, I defended the microzymian theory, M. Pasteur took part in the discussion, maintaining his former conclusions, continuing even to deny the existence of the microzymas. It was then that I urged against him his own experiment upon the blood which is a demonstration against his own system. I said: "Do you affirm that the blood in which crystals are formed and the globules disappear is not altered? The globules of this blood are always destroyed and disappear: what then has destroyed them? Even the haemoglobin is transformed into crystals and we find in the liquid a swarm of microzymas . . . these microzymas which you have neither seen nor noticed."

No longer invoking actions of contact, M. Pasteur said: "But these transformations are made under the influence of the oxygen of the air." With regard to the presence of the microzymas he admitted it in leaving it to be believed that I had stated that they became bacteria in his experiment.¹

1.Bulletin de l'Academic de medecine, 2d series, Vol. XV. p. 679.

The presence then of the microzymas being acknowledged, the observation of the results of the experiment was completed, it matters little after that, that M. Pasteur continued to treat them as "creatures of the imagination," and that he explained the phenomena as the result of some influence of oxygen; I had reason to hope that this avowal would open the eyes of my academical opponents and that they would acknowledge that they had been deceived. But nothing of the kind; the acknowledgment exists nevertheless, and it only remains to prove that they are really the agents of the changes of the second phase of the phenomenon and of the destruction of the globules.

In the first chapter we have shown that fibrin left to itself covered by carbolized water, open even to contact with the air, is transformed into soluble products without the appearance of bacteria, leaving a residue of new microzymian molecular granulations without phenomena of fetid putrefaction; and we have also seen that the microzymas of these granulations were the ferments of the transformations. On the other hand we have also seen, that in fecula starch, the same fibrin liquefies this starch and makes it ferment, while its microzymas become bacteria; we have there two examples, in one of which the microzymas are active without evolution, in the other they undergo bacterian evolution.

The following experiment demonstrates that oxygen has no influence in the phenomenon of the destruction of the globules in defibrinated blood.

About 300 c.c. of the blood of the ox, having added to it 50 c.c. of a saturated aqueous solution of phenol, were immediately defibrinated and the blood carefully separated from the fibrin submitted to a current of carbonic acid, for the purpose of expelling the oxygen. The flask was closed and left to itself at the temperature of the month of June in Montpellier during one month, and afterwards in the oven at 30° to 33° C. (= 86° to 91°.4 F.); this blood did not undergo fetid putrefaction, the globules, very slightly altered in shape, remained whole during the first ten or twelve days. It was only about the 15th of June that there appeared a great quantity of very fine molecular granulations, of which only some rare examples had before appeared, without any trace of vibrios or of bacteria. The globules resisted for a long time further, and ended by disappearing. Here was an alteration without the presence of oxygen, whereof the microzymas could not be other than those of the globules.

The fibrin and the globules of defibrinated blood can then be destroyed by their own microzymas alone, without fetid putrefaction and without bacteria.

If in the experiment with the blood of the ox, defibrinated or not, no blood crystals were formed it was because the haemoglobin of this blood is one of those which either do not yield them at all or do so with difficulty.¹

1 As to the assertion of M. Pasteur relative to the influence of the oxygen of the air, he knew that a long lime before I had refuted this in advance (see Les Microzymas, p. 253. and following (1883),Chamalet, 60, Passage de Choiseul): it is there shown that the blood taken from the crural artery of a dog, with the addition of a little of the saturated solution of creosote, submitted to a continuous current of common air is preserved arterialized and the globules remain perfect a long lime; these last, however, end by being destroyed so that the microzymas become free, often without the appearance of bacteria, and always without felid putrefaction. When the current of air is replaced by pure oxygen the same thing happens and the crystals of blood are formed at between 24° to 26° C. of temperature. It is on the contrary, in carbonic acid, that the blood globules of the dog are destroyed most quickly and the crystals are formed most readily between 35 to 40 C, always without fetid putrefaction.
Later I showed that under :he same conditions as that of the blood of the dog, the bloods of the ox. of the pig, of the fowl, of the duck give neither crystals nor yet the soluble haemoglobin of the ox. Bulletin of the Academy of Medicine, 2d Series. Vol. XVII. p. 225 (1887).
I will add that under the prolonged action of the current of air on the blood of the dog I have found that the quantity of normal urea was increased. This statement should be verified.

Assuredly, if in spite of the changes certified to by himself, M. Pasteur came to the conclusion that muscle, flesh and blood were not liable to become putrid, it must have been because he firmly believed that ferments had for their only source the germs of the air and that the protoplasmic system of organization was founded on rigorous observation. And I venture to say that he knew he was in error and that, later, it was with design that he disputed the microzymian theory, being unwilling to confess that he had observed badly and had taken the wrong road.

Prof. Joseph Bechamp reviewed the experiment on flesh as I had done for that of the blood. He repeated the experiment of M. Pasteur without using alcohol as an antiseptic, and in the centre of the piece of meat, there where M. Pasteur said that the germs of vibrios were absent, he found the microzymas in evolution and vibrios or bacteria. At the same time he found the tissue disorganizing.¹

^{1. C.R. Vol LXXXIX, p. 573.}

When I had led M. Pasteur to acknowledge the presence of the microzymas in the altered blood, I was anxious to make him confess the presence of bacteria in the interior of the gamey mass of flesh in his other experiments. But he refused, saying: "I do not know what you mean in speaking of one of my experiments on flesh."

The excessive role ascribed to the germs of the air by this savant and his pretended demonstration of the imputrescibility of organic matters in general when protected from the germs of the air, have diverted science into a deplorable road. He thus threw doubt upon a truth long since acquired; namely, that all natural organic matters, vegetable and animal, are liable to spontaneous change by a phenomenon of fermentation under the conditions specified by Macquer.

This truth must be re-established if we would understand the real meaning of the experiment of M. Pasteur upon the blood; to do this we must connect it with the introductory matter which precedes the first chapter of this work, which led up to the discovery of the real nature of fibrin, which was 11 ic point of departure for the discovery of the real nature of the blood.

I call to mind then that I proved how a solution of sugar or of any other proximate principle, or their mixtures, were changeable on contact with the air, owing to the ferments horn of the germs of this air. M. Pasteur, who had previously asserted the spontaneous generation of ferments, repeated my experiments and was convinced. Then he generalized and asserted that it would be the same in the case of urine and of milk, resembling in this sweetened yeast broth which, being boiled, was not altered if creosoted or left to itself in calcined air.

Before the experiment on the blood or on the flesh, this celebrated savant had experimented on urine and on milk. As to fresh milk, he admitted, a priori, that it soured owing to ferments born of the germs of the air, and that it was coagulated by the lactic acid which coagulated its casein. But here we have boiled milk coagulating in calcined air without becoming sour, while vibrios appeared in it. He was surprised at this, but did not in anywise seek to fathom the mystery, maintaining that in milk the germs of the air resist heating to 100 ° C. and become vibrios, to which he ascribed the coagulation.¹

1. His Memoir should be read (Ann. de Chimie et de Physique, Vol. LXIV, pp. 58-63) to realize the efforts made by M. Pasteur to convince himself that the germs of the air are the sole origin of the vibrios.

I have narrated, in the introduction, how I applied the new method of research to milk and thence to various other tissues; similarly I studied from the point of view of their chemical and anatomical changes urine, birds' eggs, fruits which become over-ripe, sprouted barley, frozen plants after a thaw and globules of beer yeast, etc.

Now for the chemical and anatomical facts regarding milk, upon which I cannot insist too strongly. The first phase of its alteration is the separation of the milk globules in the cream; this separation corresponds in an inverse sense to the separation of the blood globules in the cruor of the blood of the horse; the second phase is the souring which precedes the formation of the clot; and this souring corresponds to a fermentation which produces alcohol, acetic acid and lactic acid, the agents whereof are solely the microzymas proper to the milk, for at the moment when the clot is formed, whether the milk has been creosoted or not, the microscope discloses only these microzymas, which have become more readily visible. The vibrios or bacteria which then appear mark the anatomical phase of the phenomenon. But for the complete understanding of the phenomenon of the alterations which occur in milk, it is necessary, as in the case of the blood, to recognize that after its issue from the gland its anatomical elements are no longer in their normal conditions of existence. Also perfectly fresh milk does not contain lactic acid-contrary to the opinion of Berzelius—but it contains alcohol and acetic acid; it hence results that the production of lactic acid after the milking indicates a functional change in the microzymas of the milk; and this formation of lactic acid taking place without the disengagement of gas, especially of hydrogen, indicates in addition that the microzymas of the milk are different from those of the blood.

And since it relates to the phenomenon of coagulation, and that the clot of milk has been compared to the clot of blood, it must also be recognized that the milk clot is not the coagulation of the casein by lactic acid. The casein, in fact, is an insoluble albuminoid proximate principle, which exists in the milk in the state of a soluble caseinate; the acids, whether it be the lactic or the acetic acid, saturate the alkali and the casein is precipitated; it results from this that that .which is called coagulation of the casein in the milk, which is spontaneously altered, is the slow precipitation of the casein by the acids which render the milk sour.

With regard to the coagulation of boiled milk, where there is no souring, that is a phenomenon of another kind in which the caseinates, the albuminates and the zymas of the milk modified by the heat take part; it is a zymastic action, which may be likened to coagulation by rennet, the zymas whereof has its origin in some functional modification by heat of the microzymas of the milk. And this functional modification of these microzymas is so certain that, if there be added to the milk quantities of creosote or of carbolic acid sufficiently great to prevent the vibrionian evolution of the microzymas, there will be no souring or coagulation of the milk; the albuminoid matters undergo other transformations and at last, if the action continues for a long time, at 30° to 35 ° C., the milk globules are destroyed, the fatty bodies which they contain being set free.

The preceding facts regard especially the milks of the cow and of the goat, which are casein milk.

The milks of the ass and of women do not contain casein, but they sour spontaneously without coagulating and yield no clot on the addition of rennet.¹

Normal human urine, creosoted, ferments without disengaging gas, producing alcohol, acetic acid and benzoic acid proceeding from hyppuric acid, while the epithelial cellules are destroyed and the microzymas evolute.²

1. On the histological constitution and the comparative chemical composition of the milks of the cow, of the goat, of the ass and of woman"; "On the spontaneous alterations of milk and on the changes which heating produces in it." (M. Chamalet. Passage de Choisel, 60. Paris.)
2. C. R., Vol. LXI. p. 374, and Les Microzymas, etc.. p. 713.

The liver, plunged in carbolized water, produces, with the disengagement of carbonic acid, hydrogen and sulphuretted hydrogen, alcohol, acetic acid, lactic acid, while its cellules are destroyed and its microzymas evolve and become bacteria.¹

But the changes of eggs and of beer yeast are specially conclusive. The egg of the bird is an organism whose function is to produce a bird. Donne, by vigorous jolting, destroyed this organism, mixing, in the shell, the yolk with the white and thus produced a kind of alteration which I studied. The egg of an ostrich thus treated, at a temperature of 30°-35° C. (86°-95° F.), fermented and produced so much gas that the internal pressure became sufficiently strong to throw out a small part of the contents, on a hole being made in the shell. The gases set free were carbonic acid, hydrogen and a trace of sulphuretted hydrogen. When the gaseous disengagement ceased there was no longer any sulphuretted hydrogen. All the vitellin globules had disappeared and the microzymas were preserved with their form, without any trace of vibrios or other organized production. All the glucose of the egg had disappeared while the albuminoid matter had been preserved, the soluble being coagulable by heat. The products of fermentation were alcohol, acetic acid and butyric acid, showing that they had been produced from the lactate. Here then was a fermentation strictly defined, wherein the microzymas, like those of the blood, did not undergo vibrionian evolution.² In order that the vitellin microzymas may evolve other conditions are necessary.

1. C. R, Vol. LXXV. p. 1830.
2. C. R., Vol. LXVII, p. 523.

The case of beer yeast is still more interesting, for it has to do with a living being reduced to a cellule, whose alteration and total destruction will throw a strong light upon those of the blood globule. Suppose an alcoholic fermentation of cane sugar for which a little more yeast had been employed than was needed for the complete fermentation of the sugar. The fermentation being accomplished the yeast will be deposited in the fermented liquor and be preserved there unaltered indefinitely, as if in lethargy, with its form and its properties. This determined, let us take some fresh yeast, as it comes from the brewery, washed in distilled water to purify it from what it has brought away from the vat, and steep it in from three to four times its weight of creosoted distilled water to destroy the influence of germs of the air. In this situation, so different from its normal condition of existence, at the temperature of about 30° C. (86° F.) and without any trace of air, it will for a long time disengage pure carbonic acid, producing at the same time a relatively great amount of alcohol, acetic acid and other products, preserving its form all the time. Evidently it has only been able to produce all these these things at the expense of its own substance, of its contents, since its tegument at first remains whole. And if the process of alteration is allowed to continue, this tegument itself will disappear, its microzymas will become free and vibrios appear.¹

The following is the method by which the mechanism of the spontaneous destruction of the cellule of beer yeast can be most easily studied. It is well known that yeast does not cause fecula to ferment. But what is not known is, that it liquefies the starch of fecula and is completely destroyed in producing the liquifaction, leaving of its organism nothing but its microzymas, the soluble part of their content being left in the circumambient medium. The phenomenon lasts a greater or less time according to the quantity of creosote employed to destroy the influence of the germs of the air. If the quantity of creosote is small the microzymas undergo vibrionian evolution, if it is sufficient the microzymas do not evolve.² But that is not all. Thus studied, the phenomenon of the spontaneous destruction of the cellule of beer yeast has enabled me to confirm the generality of the fact which I had long before observed in studying the microzymian origin of the vibrioniens.

1. For details and developments see C. R,. Vol. LVIII. p. 601; "Sur les fermentations par les ferments organises" (1864).
2. Ann. de chimie el de physique. 4th Series. Vol. XXIII. p. 443. and Sur la nature el l'origine des ferments (1871).

While the globule of yeast is being destroyed and its microzymas set free and begin to undergo vibrionian evolution, several phases of this evolution are to be observed, which Estor and I have described from the commencement of our researches upon the liver, etc.,¹ namely, at first the microzymas are scarcely altered in their size and form; then microzymas coupled in the form of the figure 8, then chaplets of microzymas of from 3 to 10 and 20 grains, all of the same size; then vibrios properly so called; then bacteria often very large, motile or not; also the amylobacters of Trecul, free or fastened end to end. When the phenomenon is not checked by an addition of creosote or carbolic acid, all these productions maybe seen at the same time in the field of the microscope. Now if without changing any of the conditions of the experiment the observation of it is continued, it will be seen that all the forms other than the single microzymas disappear successively; first the amylobacters disappear; new forms of smaller dimensions appear and disappear in turn, so that in the end there remain only swarms of motile forms scarcely differing from the original microzymas which had evoluted.

1. C. R. Vol. LXVI. p. 421, and p. 859 (1868).

Speaking then in the language of anatomy, we may say that the microzymas become vibrioniens by evolution; the vibrios, the bacteria, the vibrioniens in general, return to the microzymian form by an inverse phenomenon of evolution, the ultimate forms differing in little or nothing from the microzymas, the anatomical element of the cellule.

It is thus directly demonstrated that a yeast globule, a cellule in general, in being destroyed sets its own microzymas free; that these, if the necessary conditions are realized, become vibrioniens by evolution, which, in the same apparent conditions, by an inverse phenomenon reproduce the microzymas.

So that as Estor and I have demonstrated in the development of the embryonic cellules of the fowl, and as I have demonstrated in the case of beer yeast and in the case of cellules which may develop in the mother of vinegar, the microzymas which are the commencement of all cellular and tissue organization are also their end, being, as we have seen, the end even of the bacteria.

Now that which is true of the microzymas of beer yeast is true also of the microzymas of all cellules, of all tissues, both of animals and of plants. And this fact has been confirmed unwittingly even by those who deny the microzymas and who, to avoid naming them, have called them punctiform ferments; a microzyma or microzymas-producers at the beginning, microzymas at the end, such are the beginnings and the ends of a bacterium and of a cellule.

Thus all natural animal and vegetable matters, that is to say, organized as Bichat conceived them and defined their organization, the anatomical elements morphologically definite, are the only things living in them; yes, all these matters, from the highest in organization down to beeryeast, are spontaneously alterable from the moment that they are no longer in the situation of their natural conditions of existence, chemically and anatomically.

In insisting upon their chemical alterations, especially upon the production of alcohol, of acetic acid, of lactic acid and of benzoic acid, with or without the disengagement of carbonic acid gas, etc., I wished to show that these alterations belong to the class of the best known fermentations, which assume a living figured ferment. But even in the spontaneous alteration of beeryeast, alcohol and acetic acid are not the only products formed; I described others in 1864; on further studying these latter I have found succinic acid, a special gummy substance, a ternary, furnishing mucic acid, leucin and tyrosin, nitrogenous compounds whose formation bears witness that the albuminoids of the yeast contribute to the changes; later others have been found equally nitrogenous, etc. In extending these researches upon yeast to the spontaneous alterations of the flesh of the horse and that of fish, these researches have been verified by isolating similar or analogous products.

But since these spontaneous chemical alterations belong to the class of fermentations which presuppose the presence of a figured ferment, what is this ferment? For if the beer yeast which causes sugar to ferment puts in it a part of itself, of its transformed content which is recovered among the products of normal fermentation, it is not destroyed; it remains whole, its tegument preserving to it its form, with its own anatomical element-microzymas. On the other hand, when it produces alcohol spontaneously, without sugar, it alters, and is destroyed, as are destroyed the cellules and the organization of the blood, of flesh, of the liver, etc. It is not then these cellules and these tissues which are the ferments of the spontaneous fermentations. M. Pasteur sought in the altered blood the vibrio born of the germs of the air and, not finding it, concluded that there was neither fermentation nor even chemical alteration in it; there are nevertheless fermentations without vibrios and without cellules in which are produced alcohol and acetic acid; in the first phase of the alteration of milk, for instance, and in that of eggs jolted up within the shell. These ferments are precisely the microzymas, often the vibrioniens resulting from their evolution, and microzymas which are the result of the destruction of the latter, for at a given moment, either at the commencement or at the end of the phenomenon, there is in the medium which is altering or of which the alteration is completed no production morphologically defined other than the microzymas of origin or the microzymas resulting from their destruction.

And this is not a gratuitous assertion, for I have experimentally proved that the microzymas of animal origin and those of the yeast are actually the figured ferments which produce, with sugar or fecula, alcohol, acetic acid, lactic acid, and by fermentation of the lactate of chalk, butyric acid. And it is precisely the microzymas of the microzymian molecular granulations of the blood or those of the blood globules which belong to that class.

From all these experiments it results incontestably that the microzymas of living organisms in general, and those of the blood and of the blood globules in particular, are anatomical elements and are themselves figured ferments; that is to say, that they are living and organized in the same manner as it is admitted that yeast is so; as are also the vibrioniens which these microzymas may become by evolution, out of the same organized substance. But the microzymas are living beings of an entirely special order without analogy, on which I have insisted for a long time and again insist upon as crowning the demonstration that the blood is veritably a tissue.

And now what happens when this or any tissue whatever alters? First it is no longer preserved in the state in which it exists and functions in the organism, in coordination, to speak as does Dr. Antoine Gros, in general coordination with the functioning of all the organs and of their tissues; it has then, as we have established for the albuminoid atmosphere of the hematic microzymian molecular granulations and for the coloring matter of the contents of the red globule, to undergo, owing to the change in the conditions of its existence, some chemical change in some of its parts; it is in short that its special anatomical elements change their form and their function to the extent of being destroyed and disappearing, leaving the microzymas as the only trace of their existence which, according to circumstances, do or do not undergo vibrionian evolution. And the anatomical change maybe so rapid, as is well known to histologists, that one is obliged to take steps to preserve the integrity of the tissues. In fact, one or two minutes may suffice, after the blood has been shed, to render it impossible to demonstrate the third anatomical element.

We must conclude then that in all the experiments, including those of M. Pasteur, the chemical and anatomical alteration in the blood is the work solely of the microzymas, which, in certain conditions, do not become bacteria. As to the question, to what order the chemical phenomenon belongs, it is now solved; since every chemical transformation of a proximate principle of organic matter, under the influence of a figured ferment, is called fermentation or putrefaction, it is evident that the spontaneous chemical alterations of the blood are the result of a fermentation or of a putrefaction without fetid products.

Assuredly, whenever the experiment on the blood shall be taken up upon a larger scale, even under the conditions of that of M. Pasteur, other products will be discovered besides those which I have pointed out, and among them I should not be surprised if alcohol should be found to be one of them.

It is now evident what is the real meaning of the phenomenon called the spontaneous coagulation of the blood; it is the following: The blood, being a tissue, is necessarily alterable itself, as is well known are all tissues, and as are all natural organic matters, animal or vegetable, that which is called coagulation being only the first phase of its more complete change, which extends to disorganization and to the disappearance of its globules. And the phenomenon in its entirety is the work of the microzymas, which, acting physiologically as ferments, effect the chemical transformation of the proximate principles, and thereby the anatomical changes, which end in the disorganization of the tissues and of the cellules.

But, as I have said, the microzymas are living beings of a special order without analogy, as I have shown in other publications, upon which I have promised to insist afresh to give to this work and to its demonstration their highest character of certainty and also to refute new errors upon which I had only touched in a casual manner in a former work.¹This will be the subject of the following chapter.

1. Microzymas et Microbes, etc. M. Chamalet. pub., Paris, 60 Passage de Choiseul, 60.