Chapter III: Preface (2)

Seeing however, that we are able, with the aid of the microscope alone, to demonstrate that _Bacteria_ and _Torulæ_ can develop in situations where no visible germs had previously existed, it is useless, as I have said before--so far as the question of their mode of origin is concerned--to search the atmosphere to ascertain what visible germs it may contain. If some _Bacteria_ and _Torulæ_ arise from germs at all, it must be from germs which are invisible to us. The finding of visible germs in the atmosphere can, therefore, only have an indirect bearing upon the solution of the problem. Since it _can_ be shown that some visible spores and ova exist in the atmosphere, this affords a certain amount of warrant for the supposition that invisible, living, reproductive particles may also exist--more especially if the existence of an amount of organic matter, which is ordinarily invisible, can be revealed in the air, by the agency of the electric beam, or by any other means.

Nothing can be more illegitimate, however, in the way of inference, than the assumption at once indulged in by Prof. Tyndall and others (who might have been expected, by their previous scientific work, to have learned more caution) that this impalpable organic dust was largely composed of impalpable germs. Yet, without a shadow of proof, without even an attempt to prove it, the air was for a time represented to be a mere stirabout, thick with invisible germs. The briefest reflection, however, upon the probabilities of the case, should have sufficed to suggest a totally different interpretation. The surface of the earth is clothed with living things of all kinds, animal and vegetal, which are not only continually throwing off organic particles and fragments during their life, but are constantly undergoing processes of decay and molecular disintegration after their death. The actual reproductive elements of these living things are extremely small in bulk, when compared with the other parts which are not reproductive, and although _Bacteria_ and _Torulæ_ do exist abundantly, and do materially help to bring about some of the decay in question, yet their bulk, also, is extremely small in comparison with the amount of organic matter itself that is continually undergoing disintegration of a dry kind, in which _Bacteria_ and _Torulæ_ take no part. When, moreover, it is considered that in the neighbourhood of populous cities (the air of which alone exhibits this very large quantity of impalpable, mixed with palpable, organic dust), there is constantly going on a wear and tear of the textile fabrics and of the organic products of various kinds which are daily subservient to the wants of man; and that the chimneys of manufactories and dwelling-houses are also continually emitting clouds of smoke thick with imperfectly consumed organic particles, some idea may be gained of the manifold sources whence the organic particles and fragments found in the atmosphere may emanate, and also as to what proportion of them is likely to be composed of living or dead reproductive elements, or “germs.”

Thus, then, so far as the two rival doctrines of fermentation are concerned, the investigation of the nature of the solid particles contained in the atmosphere has revealed facts which are thoroughly in harmony with all the requirements of Liebig’s physical theory, though it has almost utterly failed to give anything like a scientific basis to the vital theory of Pasteur. So far from being able to show that living _Bacteria_ (which are the first and oftentimes the only organisms concerned in many processes of fermentation and putrefaction) are universally diffused through the air, Pasteur admits that these cannot be detected, and that their “germs” are not recognizable.

If, therefore, M. Pasteur still maintains the truth of his theory, it should be distinctly understood that it rests originally, not upon established facts, but upon a mere hypothesis--the hypothesis that the air teems with multitudes of invisible _Bacteria_ germs. He is driven to such a doctrine, not only by his own confessions concerning _Bacteria_, but also by the microscopical evidence to which I have referred.

So that in explaining the results of any experiments made with the view of throwing light upon the _cause_ of fermentation or putrefaction, it is especially necessary to bear in mind two considerations:--

I. That dust filtered from the atmosphere cannot be proved to include living _Bacteria_; though it is known to contain a multitude of organic particles which may be capable in the presence of water, in accordance with Liebig’s hypothesis, of acting as ferments.

II. It must also be recollected that, in the opinion of many, Life represents a higher function which is displayed by certain kinds of organic matter; and that this higher function may be deteriorated or rendered non-existent by an amount of heat which might not be adequate to decompose the organic matter itself.

It is all the more necessary to call attention to these two considerations, because M. Pasteur invariably speaks as though it had been established that the air contains multitudes of living _Bacteria_, when, really, he had only proved that the air contains a number of corpuscles resembling spores of fungi, &c. And, as I have already intimated, the existence of spores of fungi in the atmosphere, however well established, is of little or no importance as an explanation of the cause of a very large number of fermentations. Their presence is even of still less importance, owing to the fact of the co-existence with these fungus-spores, of multitudes of organic fragments, which--in accordance with the views of Liebig, Gerhardt, and other chemists--are capable of acting as ferments. To this latter consideration M. Pasteur never even alludes when he speaks (_loc. cit._ p. 40) of his “ensemencements,” and of other experiments which are equally, or even more, capable of being interpreted in accordance with Liebig’s views than with his own.

Bearing these considerations in mind, we shall be in a better position to enquire into the real interpretation that may be given to many of M. Pasteur’s results, and into the question as to how far the facts which he records are favourable to his own, or to the adverse doctrine concerning the causes of fermentation.

* * * * *

In the memoir so often alluded to on “The Organized Corpuscles which exist in the Atmosphere,” M. Pasteur adduced various kinds of evidence, tending, as he thought, to show that the first _Bacteria_ which make their appearance in putrefying or fermenting solutions, have been derived from living _Bacteria_ or their “germs,” which pre-existed in the atmosphere.

Some of the experiments by which he endeavoured to substantiate this position were of a very simple nature. Their narration attracted much attention at the time, as it was supposed that by their means M. Pasteur had--as he professed--conclusively shown the erroneousness of the views of those who believed in what was called “spontaneous generation.” These experiments were soon repeated by other observers, who, using different fluids, obtained quite opposite results. Thus it became obvious to impartial critics, that whilst the means adopted by M. Pasteur might be adequate to check the processes of fermentation or putrefaction in certain fluids, they were quite powerless to effect this when many other fluids were employed.

These particular experiments, however, still seem to exercise a very great influence on the minds of many in this country, who are either unaware of, or disbelieve in, the possibility of obtaining opposite results.

The chapter in which M. Pasteur detailed these experiments is thus entitled:--“_Another very simple method of demonstrating that all the organized products of Infusions (previously heated) owe their origin to the corpuscles which exist suspended in the Atmosphere._” Whilst claiming to have already rigorously established the validity of this conclusion by the experiments described in previous chapters, M. Pasteur adds:--“If there remained the least doubt on this subject, in the mind of the reader, it would be dissipated by the experiments of which I am now about to speak.” (p. 66.)

Sweetened yeast-water, urine, infusions of pear and of beetroot, were placed in flasks with long necks, variously drawn out and bent. The flasks were subsequently treated as follows. M. Pasteur says:--“I then raise the liquid to the boiling-point for several minutes until steam issues abundantly from the extremity of the drawn-out neck of the flask, which is permitted to remain open. I then allow the flask to cool. But, singular fact--and one well calculated to astonish every one acquainted with the delicacy of the experiments relating to what is called ‘spontaneous generation’--the liquid of this flask will remain indefinitely without alteration. The flask may be handled without any fear, it may be transported from place to place, allowed to experience all the seasonal variations of temperature, and its liquid does not undergo the slightest alteration, whilst it preserves its odour and its taste.” If, however, the neck of one of these flasks be broken off close to the flask itself, then, according to M. Pasteur, the previously unaltered fluid will, in a day or two, undergo the ordinary changes, and swarm with _Bacteria_ and _Mucedineæ_.

“The great interest of this method is,” M. Pasteur adds, “that it completes, unanswerably, the proof that the origin of life in infusions which have been raised to the boiling point, is solely due to the solid particles[23] which are suspended in the air.” He believes that any living things pre-existing in the fluid itself would be destroyed by the high temperature to which it had been raised; and that those contained in the air of the flask would also be destroyed, if not expelled, by the process of ebullition. Believing that the air is the source of germs from which Life is first developed in infusions, he thinks that what rapidly enters at first, on the cessation of ebullition, has its germs destroyed by contact with the almost boiling liquid; whilst the air which enters subsequently, and more slowly, is supposed to deposit its germs in the various flexures of the tubes, so that none are able to reach the fluid itself. Infusions, thus protected, do not undergo putrefaction, says M. Pasteur, because the access of pre-existing living things is necessary for the initiation of this change, and such access is prevented by the tortuous and bent neck of the flask.

[23] As expressed, the proposition may be an approximation to the truth. M. Pasteur, however, really endeavours to lead his readers to believe that the “solid particles” which are efficacious, are, in all cases, living “germs.”

Others say that _some_ fluids submitted to the conditions mentioned, will undergo putrefactive changes, and that, therefore, these experiments of M. Pasteur are utterly incapable of settling the general question as to the cause of fermentation and putrefaction, and also that concerning the origin of Life. Although acknowledging a certain difficulty in explaining the results which are sometimes attained by this method, some of us would rather confess this than confidently offer explanations--as M. Pasteur did--which may in a short time be stultified by the results of other experiments with different fluids.

Having previously shown[24] that living things could appear and multiply in such a flask as M. Pasteur describes--in any flask, in fact,--which had been hermetically sealed during the ebullition of a suitable fluid within; this was deemed to be a result so contradictory to the explanations of M. Pasteur, that it appeared needless to add my testimony, as I could have done, to that of M. Victor Meunier and others, as to the different results obtainable by operating, in M. Pasteur’s fashion, with different fluids. It seemed to me that if organisms were to be procured in flasks from which air had been altogether expelled, it was useless still to urge the preservative virtues of any process of filtration of air--with the object of showing that living things in infusions derived their origin from atmospheric germs. Obviously, if there were no atmosphere, there could be no atmospheric germs present; and if living things were, nevertheless, developed under these exclusive circumstances, how could M. Pasteur or his disciples still expect to convince others that the first living things in infusions always proceeded from pre-existing atmospheric germs--even although it could be shown, that in many cases, when these were filtered off by flasks with narrow and tortuous necks, no living things were developed in such fluids. Granting to the full the truth of such facts, they could do nothing to establish the doctrine of the origin of infusorial life from pre-existing atmospheric germs, so long as it could also be shown that living things might be developed in boiled solutions to which air, instead of being filtered, was never allowed to enter at all.

[24] ‘Nature,’ 1870, No. 36, p. 193.

It is not, therefore, because I think that some of the experiments which will subsequently be related afford any stronger or more direct support to my own conclusions, but because I think they may do this indirectly--by shaking the faith of many in some of the reasonings of M. Pasteur--that I am induced to give an account of them.[25]

[25] If his reasonings can be shown to be quite inconclusive, and if his results can be otherwise explained, some people may, at last, begin to recognize that their blind and mistaken faith in M. Pasteur’s work has been somewhat misplaced.

What has been hitherto said, also applies to the more recent statements concerning the efficacy of cotton-wool as an agent for filtering germs from the atmosphere. Prof. Huxley says he has never seen putrefaction or fermentation occur after certain organic fluids have been boiled for ten or fifteen minutes, if a good plug of cotton-wool has been inserted into the neck of the flask in which they are contained whilst ebullition is going on, and has, subsequently, been allowed to remain in the same situation. Using other or perhaps stronger fluids, however, I have found that such a method of proceeding is by no means adequate to stop the growth and development of organisms. And, also, even if it had been always efficacious--the reason adduced could not hold good, in the face of my other experiments, which had shown that a development of life might go on in cases where the air, which had been similarly driven out, was subsequently, in place of being filtered, prevented from gaining access to the fluid.

If germs derived from the air are the sole causes of putrefaction, then, surely, deprivation from air ought to be just as efficacious as any process of filtration of air--more especially when the filtration or the deprivation have a common starting point. And the mode of procedure, in both cases, is precisely the same up to a certain point. A fluid is boiled for a short time in order to kill the germs which may be within the flask, and to expel its previously contained air. At a certain stage of the ebullition, this may be arrested, if we have to do with a bent-neck flask, or one whose neck is plugged with cotton-wool, and no change, it is said, will subsequently take place in the contained fluid, because the air which enters is, by either of these means, filtered from its germs. But if, whilst ebullition continued, the neck of the flask had been hermetically sealed--so as altogether to prevent the re-ingress of air--and if the fluid, thus contained _in vacuo_, would nevertheless undergo fermentation, obviously the former explanation must be altogether shelved.

In the face of M. Pasteur’s explanations, and those of Professor Huxley, these frequent positive results with fluids contained _in vacuo_ are absolutely contradictory. There may naturally arise, therefore, a very grave doubt as to the validity of the explanation adduced by M. Pasteur, and adopted by Professor Huxley and others.

All these experiments to which I have been alluding are based upon the supposition (assented to by Pasteur and Huxley) that _Bacteria_ which pre-existed in the solution would certainly be destroyed by its being raised for a few minutes to a temperature of 212° F. This conclusion is, I believe, perfectly correct,[26] and in support thereof I will adduce the following additional information.

[26] M. Pasteur attempted to make a distinction in the case of slightly alkaline or neutral fluids (loc. cit., pp. 60–65). I have endeavoured to show the untenability of his conclusion in ‘Nature,’ 1870, No. 37, pp. 224–227.

_Limits of ‘Vital Resistance’ to Heat displayed by Bacteria and Torulæ._

After stating elsewhere[27], that _Vibriones_ are partly broken up or disintegrated by an exposure for a few minutes to a temperature of 212° F. in an infusion which is being boiled, and also that, in all probability, the life of _Bacteria_ would be destroyed by such a treatment, I made the following remarks:--“With reference to these organisms, however, one caution is necessary to be borne in mind by the experimenter. The movements of monads and Bacteria may be, and frequently are, of two kinds. The one variety does not differ in the least from the mere molecular or Brownian movement, which may be witnessed in similarly minute, not-living particles immersed in fluids. Whilst the other seems to be purely vital--that is, dependent upon their properties as living things. These vital movements are altogether different from the mere dancing oscillations which not-living particles display, as may be seen when the monad or Bacterium darts about over comparatively large areas, so as frequently to disappear from the field. After an infusion has been exposed for a second or two to the boiling temperature, these vital movements no longer occur, though almost all the monads and Bacteria may be seen to display the Brownian movement in a well-marked degree. They seem to be reduced by the shortest exposure to a temperature of 100° C. to the condition of mere not-living particles, and then they become subjected to the unimpaired influence of the physical conditions which determine these movements.” I now have various facts to add in confirmation of these conclusions, and in extension of our knowledge concerning the vital resistance to heat of _Bacteria_ and _Torulæ_.

[27] ‘Nature,’ 1870, No. 35, p. 171.

It would be a most important step if we could ascertain some means by which these primary movements of living _Bacteria_ might be distinguished from the secondary, or communicated, movements of not-living particles. In many cases, organisms that are truly living may only exhibit very languid movements, which, as movements, are quite indistinguishable from those that the same _Bacteria_ may display when they are really dead. Because the movements, therefore, are of this doubtful character, persons are apt, unfairly, to argue that the _Bacteria_ which present them, are no more living than are the minute particles of carbon obtained from the flame of a lamp, which may exhibit similar movements. This, however, is a point of view which becomes obviously misleading if too much stress is laid upon it; and it is more especially so in this case, when those _Bacteria_ which display the most characteristic sign of vitality--viz., “spontaneous” division or reproduction--do, at the time, almost always exhibit only the same languid movements. Mobility is, in fact, not an essential characteristic of living _Bacteria_, whilst _the occurrence of the act of reproduction is the most indubitable sign of their life_. It should be remembered, therefore, that any _Bacteria_ which are almost motionless, or which exhibit mere Brownian movements, _may_ be living, whilst those which spontaneously divide and reproduce, are certainly alive--whatever may be the kind of movement they present.

In any particular case, however, can we decide whether _Bacteria_, that have been submitted to a given temperature, and which exhibit movements resembling those known as Brownian, are really dead or living? If the movements are primary, or dependent upon the inherent molecular activity of the organism itself, they ought, it might be argued, to continue when the molecules of the fluid are at rest; if, on the other hand, they are mere secondary or communicated movements, impressed upon the organisms as they would be upon any other similarly minute particles, by the molecular oscillations of the fluid in which they are contained, then the movements ought to grow less, and gradually cease, as the fluid approaches a state of molecular rest--if this be attainable. Following out this idea, some months ago, I first tested the correctness of the assumption by experimenting with fluids containing various kinds of not-living particles; such as carbon-particles from the flame of a lamp, or freshly precipitated baric sulphate. However perfect may have been the Brownian movements when portions of these fluids were first examined beneath a covering-glass, they always gradually diminished, after the specimen had been mounted by surrounding the covering-glass with some cement or varnish. Thus prepared, no evaporation could take place from the thin film of fluid, and after one, three, four, or more hours--the slide remaining undisturbed--most of the particles had subsided, and were found to have come to a state of rest. In order still further to test these views, I took an infusion of turnip, containing a multitude of _Bacteria_ whose movements were of the languid description, and divided it into two portions. One of these portions was boiled for about a minute, whilst the other was not interfered with. Then, after the boiled solution had been cooled, a drop was taken from each and placed at some little distance from one another on the same glass slip; covering-glasses half an inch in diameter were laid on, and the superfluous fluid beneath each was removed by a piece of blotting-paper. When only the thinnest film of fluid was left, the covering-glasses were surrounded by a thick, quickly-drying cement.[28] Examined with the microscope immediately afterwards, it was generally found that the _Bacteria_ which had been boiled presented a shrunken and shrivelled aspect--whilst some of them were more or less disintegrated--though, as far as movement was concerned, there was little to distinguish that which they manifested, from that of their plumper-looking relatives which had not been boiled.

[28] I always employ a solution of gum mastic and bismuth in chloroform. If a different varnish be employed, it is of course necessary to ascertain whether its application is injurious to the enclosed _Bacteria_.

If the specimens were examined again after twenty-four or more hours, there was still very little difference perceptible between them, as regards their movements. And the same was the case when the specimens were examined after a lapse of some days or weeks. One important difference does, however, soon become obvious. The _Bacteria_ which have not been boiled, undergo a most unmistakeable increase within their imprisoned habitat; whilst those which have been boiled, do not increase. The two films may be almost colourless at first (if the _Bacteria_ are not very abundant), but after a few days, that composed of unboiled fluid begins to show an obvious and increasing cloudiness, which is never manifested by the other. Microscopical examination shows that this cloudiness is due to a proportionate increase in the number of _Bacteria_.

Is the continuance of the movements of the organisms which had been boiled attributable to their extreme lightness, and to the slight difference between their specific gravity and that of the fluid in which they are immersed? I soon became convinced that this was one, if not the chief reason, when I found that _Bacteria_ which had been submitted to very much higher temperatures, behaved in precisely the same manner as those which had been merely boiled, and also that other particles which--though obviously dead--had a similar specific lightness, also continued to exhibit their Brownian movements for days and weeks. This was the case more especially with the minute fat particles in a mounted specimen of boiled milk,[29] and also with very minute particles which were gradually precipitated[30] from a hay infusion that had been heated to 302° F. for four hours. Trials with many different substances, indeed, after a time convinced me that the most rapid cessation of Brownian movements in stationary films,[31] occurred where the particles were heavy or large; and that the duration of the movement was more and more prolonged, as the particles experimented with, were lighter or more minute. So that, when we have to do with _Bacteria_, the minute oil globules of milk, or with other similarly light particles, the movements continue for an indefinite time, and are, in part, mere exponents of the molecular unrest of the fluid. They are always capable of being increased or renewed by the incidence of heat or other disturbing agencies.

[29] If an unboiled specimen of milk be mounted, a multiplication of living particles takes place here and there amongst the fat globules, just as the multiplication of _Bacteria_ occurs in a vegetable infusion; but in the boiled specimen no trace of such multiplication can ever be detected.

[30] Those particles which come to rest, in such cases, are always in contact with one or other of the contiguous surfaces of glass.

[31] The specific gravity of the fluid being constant. Where this is dense or viscid, as with glycerine, Brownian movements do not occur at all.

In respect of the movements which they may exhibit, therefore, really living, though languid, _Bacteria_, cannot always be discriminated from dead _Bacteria_. Both may only display mere Brownian movements.

It becomes obvious, then, that in doubtful cases we ought not to rely very strongly upon the character of their movements, as evidence of the death of _Bacteria_--although these may frequently be of so extensive a nature as to render it not at all doubtful whether the _Bacteria_ which display them are living. In the experiments which I am about to relate, we shall be able to pronounce that the _Bacteria_ are living or dead, by reference to the continuance or cessation of their most essentially vital characteristic. If _Bacteria_ fail to multiply in a suitable fluid, and under suitable conditions, we have the best proof that can be obtained of their death.

Having made many experiments with solutions of ammonic tartrate and sodic phosphate, I have almost invariably observed that such solutions--when exposed to the air without having been boiled--become turbid in the course of a few days owing to the presence of myriads of _Bacteria_ and _Vibriones_, with some _Torulæ_. These organisms seem to appear and multiply in such a solution almost as readily as they do in an organic infusion. On the other hand, having frequently boiled such solutions, and closed the flasks during ebullition, I have invariably found, on subsequent examination of these fluids, that whatever else may have been met with, _Bacteria_ and _Vibriones_ were always absent. The difference was most notable, and it seemed only intelligible on the supposition that any living _Bacteria_ or dead ferments which may have pre-existed in the solution, were deprived of their virtues by the preliminary boiling. These experiments also seemed to show that such solutions, after having been boiled, and shut up in hermetically-sealed flasks, from which all air had been expelled, were quite incapable of giving birth to _Bacteria_. The unboiled fluid, exposed to the air, might have become turbid, because it was able to nourish any living _Bacteria_ which it may have contained, or because it was capable of evolving these _de novo_, under the influence of dead ferments whose activity had not been destroyed by heat. Hence we have a fluid which is eminently suitable for testing the vital resistance of _Bacteria_,--one which, although quite capable of nourishing and favouring their reproduction, does not appear capable of evolving them, when, after previous ebullition, it is enclosed in a hermetically sealed flask from which all air has been expelled. Three flasks were half-filled with this solution.[32] The neck of the first (_a_) was allowed to remain open, and no addition was made to the fluid. To the second (_b_), after it had been boiled and had become cool, was added half a minim of a similar saline solution, which had been previously exposed to the air, and which was quite turbid with _Bacteria_, _Vibriones_ and _Torulæ_. From this flask--after its inoculation with the living organisms--the air was exhausted by means of an air-pump, and its neck was hermetically sealed during the ebullition of the fluid, without the flask and its contents having been exposed to a heat of more than 90° F. The third flask (_c_) was similarly inoculated with living _Bacteria_, although its contents were boiled for ten minutes (at 212° F.), and its neck was hermetically sealed during ebullition. The results were as follows:--the solution in the first flask (_a_), became turbid in four or five days; the solution in the second (_b_), became turbid after thirty-six hours; whilst that in the third flask (_c_), remained perfectly clear. This latter flask was opened on the twelfth day, whilst its contents were still clear, and on microscopical examination of the fluid no living _Bacteria_ were to be found. This particular experiment was repeated three times with similarly negative results, although on two occasions the fluid was only boiled for one minute instead of ten.

[32] In the proportion of ten grains of neutral ammonic tartrate, with three grains of neutral sodic phosphate, to an ounce of distilled water.

It seemed, moreover, that by having recourse to experiments of the same kind, the exact degree of heat, which is fatal to _Bacteria_ and _Torulæ_ might be ascertained. I accordingly endeavoured to determine this point. Portions of the same saline solution, after having been boiled[33] and allowed to cool, were similarly inoculated with a drop[34] of very turbid fluid, containing hundreds of living _Bacteria_, _Vibriones_, and _Torulæ_. A drying apparatus was fixed to an air-pump, and the flask containing the inoculated fluid was securely connected with the former by means of a piece of tight india-rubber tubing,[35] after its neck had been drawn out and narrowed, at about two inches from the extremity. The flask containing the inoculated fluid was then allowed to dip into a beaker holding water at 122° F., in which a thermometer was immersed. The temperature of the fluid was maintained at this point for fifteen minutes,[36] by means of a spirit lamp beneath the beaker. The air was then exhausted from the flask by means of the pump, till the fluid began to boil; ebullition was allowed to continue for a minute or two, so as to expel as much air as possible from the flask, and then, during its continuance, the narrowed neck of the flask was hermetically sealed by means of a spirit-lamp flame and a blowpipe. Other flasks were similarly prepared, except that they were exposed to successively higher degrees of heat--the fluid being boiled off, in different cases, at temperatures of 131°, 140°, 149°, 158°, and 167° F. All the flasks being similarly inoculated with living _Bacteria_, _Vibriones_, and _Torulæ_, and similarly sealed during ebullition, they differed from one another only in respect to the degree of heat to which they had been submitted. Their bulbs were subsequently placed in a water bath, which during both day and night was maintained at a temperature of from 85° to 95° F. The results have been as follows:--The flasks whose contents had been heated to 122° and 131° F. respectively, began to exhibit a bluish tinge in the contained fluid after the first or second day; and after two or three more days, the fluid in each became quite turbid and opaque, owing to the presence and multiplication of myriads of _Bacteria_, _Vibriones_ and _Torulæ_; the fluids in the flasks, however, which had been exposed to the higher temperature of 140°, 149°, 158°, and 167° F., showed not the slightest trace of turbidity, and no diminution in the clearness of the fluid while they were kept under observation--that is, for a period of twelve or fourteen days. One kind of conclusion only is to be drawn from these experiments, the conditions of which were in every way similar, except as regards the degree of heat to which the inoculated fluids were subjected--seeing that the organisms were contained in a fluid, which had been proved to be eminently suitable for their growth and multiplication.[37] If inoculated fluids which have been raised to 122° and 131° F. for ten minutes, are found in the course of a few days to become turbid, then, obviously, the organisms cannot have been killed by such exposure; whilst, if similar fluids, similarly inoculated, which have been raised to temperatures of 140°, 149°, 158°, and 167° F. remain sterile, such sterility can only be explained by the supposition that the organisms have been killed by exposure to these temperatures.

[33] It was necessary to boil the solution first, in order to destroy any living things or dead ferments which it might contain. It must contain one or the other, because an unboiled solution of this kind, in a corked bottle about half full, will always become turbid; whilst, after it has been boiled, it may be kept indefinitely under similar conditions without becoming turbid.

[34] The proportion was one drop of the fluid, opaque with organisms, to an ounce of the clear solution.

[35] Into which a piece of glass tube had been slipped to prevent collapse.

[36] Allowing even five minutes for the temperature of the 1 oz. of fluid to become equal to that of the bath, it would then have remained exposed to this amount of heat for about ten minutes.

[37] Fluids which had remained sterile would always, in the course of thirty-six or forty-eight hours after inoculation with living _Bacteria_, become more or less turbid.

Some of these experiments have been repeated several times with the same results. On three occasions, I have found the fluid speedily become turbid, which had only been exposed to 131° F. for ten minutes, whilst on three other occasions I have found the inoculated fluid remain clear, after it had been exposed to a heat of 140° F. for ten minutes.[38]

[38] There is, however, another point of extreme interest in connection with these experiments, bearing upon the supposed universal distribution of “germs” of _Bacteria_ and other organisms, which I will now mention. One of the flasks, which had been exposed to 140° F., and which had been hermetically sealed at this temperature, had its neck cracked (accidentally) about half an hour afterwards. Thinking it would be as well, notwithstanding this, to keep it and observe the result, its bulb was immersed in the same water-bath with the other flasks which had been prepared at the same time. Whilst the fluid in one of these which had been exposed to a heat of 131° F., became turbid in the course of a few days, this, which had been exposed to a heat of 140° F. and whose neck was also extensively cracked, remained quite clear for seven days, although to such an extent exposed to the access of germs. Its eminent suitability for nourishing the germs of such organisms was also shown, because, on the seventh day, the fluid being still clear, the blade of a penknife was dipped into it, after having been previously immersed in a solution containing living _Bacteria_ and _Torulæ_, and in thirty-six hours after this inoculation, the fluid had become turbid, owing to the presence of myriads of these organisms. So that even where obvious cracks occur, and the vacuum is altogether impaired by the consequent inrush of air, such air does not necessarily carry with it germs of _Bacteria_--which have been supposed to be universally diffused, and capable of passing through cracks so minute as to be invisible. These results, important as they are, have not at all surprised me, because one may frequently find a previously boiled solution of the kind under consideration, remaining free from turbidity for two weeks or more, although the neck of the flask has been merely covered by a loose paper-cap (see p. 30).

In experimenting upon rather higher organisms, with which there is little difficulty in ascertaining, by microscopical examination, whether they are living or dead, I have found that an exposure even to the lower temperature of 131° F. for five minutes, always suffices to destroy all signs of life in Vibrios, Amœbæ, Monads, Chlamydomonads, Euglenæ, Desmids, Vorticellæ, and all other Ciliated Infusoria which were observed, as well as in free Nematoids, Rotifers, and other organisms contained in the fluids which had been heated.

These results are quite in harmony with the observations and experiments of M. Pouchet and of Professor Wyman, as to the capability of resisting heat displayed by _Vibriones_ and all kinds of ciliated infusoria. According to the former,[39] the majority of ciliated infusoria are killed at, or even below, the temperature of 122° F., whilst large _Vibriones_ are all killed at a temperature of 131° F.[40] According to the observations of Professor Wyman, the motions of all ciliated infusoria are stopped at less than 130° F., whilst _Vibriones_, taken from the most various sources, also seemed to be killed at temperatures between 130°–136.4° F. Similarly, we find Baron Liebig quite recently making the following remarks concerning a species of _Torula_:--“A temperature of 60° C. [140° F.] kills the yeast cells; after exposure to this temperature in water, they no longer undergo fermentation, and do not cause fermentation in a sugar solution.... In like manner, active fermentation in a saccharine liquid is stopped when the liquid is heated to 60° C., and it does not recommence again on cooling the liquid.”

[39] ‘Nouvelles Expériences,’ etc., 1864, p. 38.

[40] ‘American Journal of Science and Arts,’ Oct. 1867.

That the organisms in question--being minute naked portions of living matter--should be killed by exposure to the influence of a fluid at these temperatures will perhaps not seem very improbable to those who have attempted to keep their fingers for any length of time in water heated to a similar extent. With watch in hand I immersed my fingers in one of the experimental beakers containing water at 131° F., and found that, in spite of my desires, they were hastily withdrawn, after an exposure of less than _five-and-twenty seconds_.

Wishing to ascertain what difference there would be if the inoculated fluids were exposed for a very long time, instead of for ten minutes only, to certain temperatures, I prepared three flasks in the same manner--each containing some of the previously boiled solution, which, when cold, had been inoculated with living _Bacteria_, _Vibriones_, and _Torulæ_. These flasks and their contents were then submitted to the influence of the following conditions:--One of them was heated for a few minutes in a beaker containing water at 113° F., and then by means of the air-pump a partial vacuum was procured, till the fluid began to boil. After the remainder of the air had been expelled by the ebullition of the fluid, the neck of the flask was hermetically sealed, and the flask itself was subsequently immersed in the water of the beaker, which was kept for four hours at a temperature between 113° and 118-1/2° F.[41] The two other flasks similarly prepared were kept at a temperature of 118-1/2° - 127-1/2° F. for four hours. In two days, the fluid in the first flask became slightly turbid, whilst in two days more the turbidity was most marked. The fluid in the two other flasks which had been exposed to the temperature of 118-1/2° - 127-1/2° F. for four hours, remained quite clear and unaltered during the twelve days in which they were kept in the warm bath under observation. These experiments seem to show, therefore, that the prolongation of the period of exposure to four hours, suffices to lower the vital resistance to heat of _Bacteria_ and _Torulæ_ by 14-1/2° - 18° F.

[41] During nearly the whole of the time the temperature was kept at 113° F. It only rose to the higher temperature for about ten minutes.

Such experiments would seem to be most important and crucial in their nature. They may be considered to settle the question as to the vital resistance of these particular _Bacteria_, whilst other evidence points conclusively in the direction that all _Bacteria_, whencesoever they have been derived, possess essentially similar vital endowments[42]. Seeing also that the solutions have been inoculated with a drop of a fluid in which _Bacteria_, _Vibriones_, and _Torulæ_ are multiplying rapidly, we must suppose that they are multiplying in their accustomed manner, as much by the known method of fission, as by any unknown and assumed method of reproduction. In such a fluid, at all events, there would be all the kinds of reproductive elements common to _Bacteria_, whether visible or invisible, and these would have been alike subjected to the influence of the same temperature. These experiments seem to show, therefore, that even if _Bacteria_ do multiply by means of invisible gemmules as well as by the known process of fission, such invisible particles possess no higher power of resisting the destructive influence of heat than the parent _Bacteria_ themselves possess. This result is, moreover, as I venture to think, in accordance with what might have been anticipated _à priori_. _Bacteria_ seem to be composed of homogeneous living matter, and any gemmule, however minute, could only be a portion of such living matter, endowed with similar properties.

[42] The _Bacteria_ and _Vibriones_ with which Professor Wyman experimented were derived from different sources; and so far as I also have been able to ascertain, the _Bacteria_ of different fluids are similarly affected by exposure to similar degrees of heat. Thus, if on the same slip, though under different covering glasses, specimens of a hay infusion, turbid with _Bacteria_, are mounted, (_a_) without being heated, (_b_) after the fluid has been raised to 122° F. for ten minutes, and (_c_) after the fluid has been heated to 140° F. for ten minutes, it will be found that, in the course of a few days, the _Bacteria_ under _a_ and _b_ have notably increased in quantity, whilst those under _c_ do not become more numerous, however long the slide is kept. Facts of the same kind are observable if a turnip infusion, containing living _Bacteria_, is experimented with; and the phenomena are in no way different if a solution of ammonic tartrate and sodic phosphate (containing _Bacteria_) be employed instead of one of these vegetable infusions. The multiplication of the _Bacteria_ beneath the covering-glass, when it occurs, is soon rendered obvious, even to the naked eye, by the increasing cloudiness of the film.

_Extent to which boiled Fermentable Fluids may be preserved in Vessels with Bent Necks, or in those whose Necks are guarded by a Plug of Cotton-Wool._

Having thus satisfied ourselves as to the truth of the conclusion that _Bacteria_ are killed when the fluid containing them is boiled (at 212° F.), we are in a position to proceed with the inquiry as to the evidence which exists in respect to the statements made by M. Pasteur, Professor Huxley, and others, that fermentable fluids which have been boiled, will not undergo fermentation, either in vessels whose necks have been many times bent, or in those into whose necks a plug of cotton-wool has been inserted during the ebullition of their contained fluid. Organisms are not found in such cases, they say, because the “germs” from which the low organisms of infusions are usually produced, are arrested either in the flexures of the tube or in the cotton-wool. As I have before stated, however, it is obvious that if this explanation be the correct one, the preservation should be equally well marked in all cases--quite irrespectively of the amount of albumenoid or other nitrogenous material which may be contained in the fluid. Any exceptions to the rule should at once suggest doubts as to the validity of the explanation.

It was shown[43] in 1865 by M. Victor Meunier that some fluids were preserved after having been boiled in a vessel of this kind, whilst others, submitted to the same treatment, speedily became turbid from the presence of _Bacteria_ and other organisms.[44] By these experiments he ascertained that strong infusions did frequently change, whilst weak ones might be preserved; and that even a strong infusion might be prevented from undergoing change if the period of ebullition were sufficiently prolonged.

[43] ‘Compt. Rend.,’ t. lxi. p. 1060.

[44] When boiled solutions, containing mannite, with a little nitrate and phosphate of ammonia, were employed, they always remained sterile. Similar negative results followed the employment of ox-gall. Of three decoctions of beef with which M. Meunier experimented, the two stronger of them were found to contain swarms of _Bacteria_ in about twelve days. Of three other flasks containing boiled urine, two also proved fertile.

The fluids most frequently employed by M. Pasteur were yeast-water, the same sweetened by sugar, urine, infusion of beetroot, and infusion of pear.

Taking urine as a fair example of such a fluid, I have found that the statements of M. Pasteur and of Professor Lister are perfectly correct. This fluid may generally remain for an indefinite period in such vessels[45] without becoming turbid, or undergoing any apparent change. The same is generally found to be the case with an infusion of turnip, and occasionally an infusion of hay may be similarly prevented from undergoing fermentation. On the other hand, if the turnip-solution be neutralized by the addition of a little ammonic carbonate, or liquor potassæ; or, better still, if even half a grain of new cheese be added to the infusion before it is boiled, then I have found that the fluid speedily becomes turbid, owing to the appearance of multitudes of _Bacteria_. In an infusion to which a fragment of cheese had been added, I have seen a pellicle form in three days, which, on microscopical examination, proved to be composed of an aggregation of _Bacteria_, _Vibriones_, and _Leptothrix_ filaments. A mixture of albuminous urine and turnip-infusion has also rapidly become turbid in a vessel of this kind owing to the appearance of multitudes of _Bacteria_, and so has a mixture containing one-third of healthy urine with two-thirds of infusion of turnip.

[45] I have employed flasks of about 1-1/2 oz. in capacity, provided with necks two feet in length. In each case, after the flask has been half filled with the fluid, the neck has been bent eight times at an acute angle.

Other infusions have been boiled for ten minutes in a vessel with a horizontal neck two feet long, into which, during ebullition, a good plug of cotton-wool had been carefully pushed down for a depth of twelve or fourteen inches, and cautiously increased in quantity during the continuance of the ebullition; whilst immediately after the withdrawal of the heat, the plug was pressed closer, and all the outer unoccupied portion of the tube was rapidly filled up in the same manner.

Preserved in such a vessel, a specimen of urine remained unchanged; a hay-infusion also underwent no apparent alteration; whilst a very strong infusion of turnip became turbid in five days, and ultimately showed a large quantity of deposit.[46]

[46] These are the only experiments which I have performed with the very long plugs of cotton-wool, though in other previous trials with plugs about 1-1/2 in. long, I have several times obtained positive results.

Thus the rules laid down by Pasteur and others are not universal, and therefore, as I have previously pointed out, the explanation which he adduced of the preservation of those particular fluids which remained unchanged is at once rendered doubtful. More especially is there room for doubt on this subject when, as I have found, the result of the experiment can be, within certain limits, predicated beforehand, according to the nature of the fluid employed. If all organisms proceed from pre-existing germs, and these can be filtered from the air by a certain mechanical contrivance, then, if it be alleged that it is on account of such filtration that certain boiled fluids do not change, all fluids placed under these conditions ought, on this theory, to be similarly preserved. Exceptional cases cannot be accounted for on this hypothesis. To others, however, who say that organisms are capable of arising _de novo_, and that fermentation can be initiated without the agency of living things, the above facts appear quite natural. The more complex the nitrogenous or protein materials contained in a solution, the more is it fitted to undergo fermentative changes, which may be accompanied by the _de novo_ origination of living things. Therefore the above results are just as compatible with the notions of M. Liebig and his school, as they are antagonistic to those of M. Pasteur. Certain fluids, it is found, do not undergo change; whilst other fluids, of a more complex description, will ferment under the influence of similar conditions. Prolonged ebullition also, by breaking up some of the more unstable compounds of a solution (those which most easily initiate these changes) will retard or prevent its fermentation.

The complete untenability of M. Pasteur’s explanations are, however, best revealed by having recourse to a series of comparative experiments, in which portions of the same fluid are boiled for an equal length of time in vessels of different kinds, and are then subsequently submitted, in a water-bath, to the influence of the same temperature.

I have made many experiments of this kind with different solutions, some of which I will now record. Owing to the different behaviour of the same fluids under different conditions, we are enabled to draw some most important conclusions; and owing to the different behaviour of different fluids under these respective conditions, our attention is strongly drawn to other facts which ought considerably to influence our judgment as to the relative merits of the two doctrines concerning the cause of fermentation and putrefaction.

COMPARATIVE EXPERIMENTS.

In the following experiments, each fluid (unless a statement is made to the contrary) was _boiled continuously for ten minutes_, after having been placed in its flask. Then, with the neck either open, sealed, or plugged, the bulb of the flask was immersed in a water-bath maintained at a temperature of 80°–95° F., during both day and night.[47]

[47] When infusions have been employed, these have all been made as strong as possible, and have been filtered before use. _Warm_ water has been added in quantity just sufficient to cover the substance to be infused (cut into very small pieces), and the mixture has then been kept at a temperature of from 110°–130° F. for three or four hours.

FIRST SET OF EXPERIMENTS (I.–XV.).

_a. Fluid exposed to Air in a Flask with a short Open Neck._

*No. I.--Urine* in twenty-four hours was still clear and free from deposit. In forty-four hours the fluid was very slightly turbid, and on microscopical examination _Bacteria_ and _Torulæ_ were found, though not in very great abundance. In sixty-eight hours the fluid was quite turbid.

*No. II.--Hay Infusion* in twenty-four hours was still clear. In forty-four hours the fluid was very turbid, and a drop on examination showed multitudes of _Bacteria_ of different kinds, exhibiting languid movements. In sixty-eight hours the turbidity had become much more marked, and there was also a certain amount of sediment.

*No. III.--Turnip Infusion* in twenty-four hours showed a very slight degree of turbidity. A drop examined microscopically revealed a number of very minute, but very active, _Bacteria_. In forty-four hours the turbidity had become very well marked.

_b. Fluid in contact with Ordinary Air and its Particles; Neck of Flask Sealed after the Fluid had become Cold._

*No. IV.--Urine* remained quite bright and clear during the fifteen days in which it was kept under observation in the water-bath.[48]

*No. V.--Hay Infusion* after forty-four hours showed a well-marked turbidity. In sixty-eight hours there was an increase in the amount of turbidity, and also some sediment. During the next forty-eight hours turbidity and sediment gradually increased, whilst the colour of the fluid (originally that of port wine) became several shades lighter. Except that it grew still lighter in colour, and that the amount of sediment increased, it underwent no further obvious change during the fifteen days in which it remained in the bath.[48]

*No. VI.--Turnip Infusion* underwent no change during the fifteen days in which it was kept in the bath under observation.[48]

_c. Fluid in a Flask with a Neck two feet long, and having Eight acute Flexures._

*No. VII.--Urine* remained quite bright and clear during the fifteen days in which it was kept under observation in the water-bath.[48]

[48] Flask still in my possession, unopened.

*No. VIII.--Hay Infusion* remained bright and clear for twelve days. On the thirteenth day a very slight (almost inappreciable) sediment was seen, which scarcely underwent any obvious increase during the next eight days, though on the two following days (twenty-second and twenty-third) the turbidity became most obvious: much sediment was deposited, and the fluid assumed a much lighter colour.[49] (On the twenty-second day the temperature of the bath was raised to 100° F., for two or three hours.)

[49] Flask still in my possession, unopened.