Having previously shown24 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.
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
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.
After stating elsewhere27, 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.
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.
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 precipitated30 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.
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.
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 boiled33 and allowed to cool, were similarly inoculated with a drop34 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.
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
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.”
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 12° F.41 The two other flasks similarly prepared were kept at a temperature of 118 12°–127 12° 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 12°–127 12° 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 12°–18° F.
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 endowments42. 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.
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 shown43 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.
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 vessels45 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.
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
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.
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
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.
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
No. VII.—Urine remained quite bright and clear during the fifteen days in which it was kept under observation in the water-bath.48
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.)
No. IX.—Turnip Infusion remained for four days without undergoing any apparent change. Its neck was then accidentally broken at the fourth joint—a certain amount of fluid still filling the third joint. In this condition the flask was allowed to remain in the water-bath, and the fluid continued quite unchanged in appearance for five days. It was then boiled50 for three minutes, and the neck of the flask was hermetically sealed whilst the fluid was boiling. The flask being re-immersed in water-bath, the fluid continued quite clear for thirteen days. Its neck was then carefully heated in the spirit-lamp flame till, when red-hot, the rapid inbending of the glass showed that the vacuum was still preserved. This being ascertained, the flask was, after a few minutes, replaced in the bath. The next day the temperature of the bath was allowed to go up to 100° F. for three or four hours, and in the evening the fluid was observed to be very slightly turbid. In two days more (i.e., after sixteen days in vacuo) the turbidity was well marked, and when the fluid was examined microscopically it was found to contain an abundance of very languid Bacteria and Vibriones. On opening the flask there was an outrush of very fœtid gas, and the reaction of the fluid was acid.51
No. X.—Urine remained quite bright and clear during the fifteen days in which it was kept under observation in the water-bath.52
No. XI.—Hay Infusion showed a very slight amount of sediment after forty-four hours, which seemed to increase somewhat during the next three days. The fluid afterwards appeared to undergo no further change, though it remained in the warm water-bath for fifteen days.52
No. XII.—Turnip Infusion in four days showed a well-marked turbidity, and also very many flakes of a broken pellicle.52
No. XIII.—Urine in forty-four hours showed a very slight amount of sediment. During the next two days the sediment very slightly increased, but was still small in amount. At the expiration of fifteen days, no further increase in the turbidity having taken place, the fluid was examined. The vacuum was still partially preserved, as evidenced by the rapid inbending of a portion of the neck of the flask after it had been carefully made red-hot. When opened, the odour of the fluid was stale, but not fœtid, and its reaction was still faintly acid. On microscopical examination Bacteria and Torulæ were found in tolerable abundance.
No. XIV.—Hay Infusion in forty-four hours showed a very slight amount of turbidity. In sixty-eight hours the turbidity was most marked, and there was also a small amount of sediment. In another twenty-four hours it was noticed that the colour of the fluid had become much lighter, whilst the turbidity and sediment had increased. It subsequently continued in much the same state, and the flask was opened on the sixteenth day. The vacuum was found to be almost wholly impaired, whilst the odour of the fluid was sour, and not at all hay-like. On microscopical examination Bacteria, Vibriones, Leptothrix, and Torulæ, were found in abundance, and the former were very active.
No. XV.—Turnip Infusion after forty-eight hours showed a well-marked turbidity. In seventy-two hours the turbidity was more marked, and there was a slight amount of sediment. The turbidity also increased during the next twenty-four hours; though, after that, the infusion seemed to undergo no further change. The flask remained in the warm bath for fifteen days, when the fluid was examined. Its odour was not fœtid, but was somewhat like that of baked turnip. Bacteria and Vibriones existed in abundance, though their movements were extremely languid.
No. XVI.—Simple Turnip Infusion in twenty-four hours had undergone no apparent change. In thirty-six hours there was slight turbidity, and in forty-eight hours this was most marked and uniform. When the flask was opened, after seventy-two hours, there was an outrush of very fœtid gas; the reaction of the fluid was acid, and, when examined microscopically, it was found to contain multitudes of very languid Bacteria.
No. XVII.—Neutralized Infusion of Turnip + 12 gr. of Cheese,53 in thirty-six hours showed a well-marked pellicle.54 When the flask was opened, after seventy-two hours, there was a violent outrush of gas, though the fluid was still neutral. Portions of the thick pellicle were found, on microscopical examination, to be made up of Bacteria, Vibriones, and an abundance of long, interlaced Leptothrix filaments. Bacteria also existed abundantly in the fluid, though their movements were very languid.
No. XVIII.—Simple Turnip Infusion after forty-eight hours showed no change. It was kept in water-bath for twelve days, and during the whole of this time the fluid remained quite clear. The tube was then broken 1 12 inch above the bulb (which was re-immersed in the bath), leaving the fluid exposed to the air through the straight open tube. The fluid at this time was odourless, and its re-action was still faintly acid.
The infusion remained thus exposed for six days without undergoing any apparent change. On the eighth day a very slight whitish sediment was noticed, which had increased in quantity by the tenth day, though there was still no trace of general turbidity. On the eleventh day some of the sediment was examined in a drop of the fluid, and it was found to be wholly composed of rather large Torulæ cells—the largest being about 13000 in diameter, though all the smaller sizes were abundantly represented. Not a single Bacterium or Vibrio could be detected, and the fluid was still quite odourless.55
No. XIX.—Neutral Turnip Infusion + 12 gr. of Cheese, showed no perceptible change in twenty-four hours, though in thirty-six hours there was a well-marked pellicle on the surface. When the neck of the flask was broken after seventy-two hours, the fluid was found to be very fœtid, whilst its re-action had become slightly acid. Portions of the pellicle were found to be made up by aggregations of Bacteria, Vibriones, and an abundance of Leptothrix filaments. The Bacteria all exhibited very languid movements.
No. XX.—Simple Turnip Infusion in twenty-four hours showed a very slight amount of turbidity; in thirty-six hours this had increased, and in forty-eight hours there were multitudes of curdy flocculi floating in a tolerably clear fluid. The flask was opened after seventy-two hours, when there seemed to be only a very slight inrush of air. The odour of the fluid was somewhat fœtid, and its re-action was acid. There were multitudes of Bacteria and Vibriones, partly separate and partly aggregated (constituting the flocculi above mentioned). The separate Bacteria exhibited only very languid movements.
No. XXI.—Neutral Turnip Infusion + 12 gr. of Cheese, showed a well-marked pellicle on its surface in twenty-four hours. In thirty-six hours the first pellicle had, in great part, sunk to the bottom of the flask, though its place on the surface was already taken by a new, though thin, scum-like layer. After seventy-two hours, the flask was opened; there was no fœtid odour of the fluid, and its re-action was still neutral. Examined microscopically the fluid showed an abundance of Bacteria, and also of short monilated filaments. There were, however, none of the ordinary kind of Vibriones, and no Leptothrix. All the Bacteria exhibited very languid movements.
No. XXII.—Urine in twenty-four hours showed no change; though in forty-six hours the turbidity was well marked.56 Examined microscopically it was found to contain an abundance of Bacteria.
No. XXIII.—Urine in eighteen hours showed a distinct pellicle, though there was not much general turbidity. During the next few days the old pellicle fell to the bottom, and a new one formed.
No. XXIV.—Urine in forty-eight hours showed no change. After twelve days there was still no general turbidity, though there was a slight flocculent deposit of an uncertain nature. Two days afterwards the flask was broken, when the odour of the fluid was still found to resemble that of fresh urine, and its re-action was acid. The flocculi were made up of granular aggregations, in the midst of which were a few bodies closely resembling Torulæ, though they were somewhat doubtful in nature. Neither Bacteria nor Vibriones could be found. The flask, having a short open neck, was then replaced in the warm bath. In sixteen hours the whole fluid had become turbid; it was also slightly fœtid; and on microscopical examination it was found to be swarming with Bacteria, Vibriones, and Leptothrix.
No. XXV.—Turnip Infusion + 12 gr. of Cheese in forty-eight hours showed no change, though in seventy-two hours there was a well-marked pellicle, in which some bubbles of gas were engaged. After ninety-six hours the neck of the flask was broken; the fluid was found to be fœtid, and it had an acid re-action. On microscopical examination, a portion of the pellicle was seen to consist of multitudes of Bacteria, Vibriones, and jointed Leptothrix filaments.
No. XXVI.—Simple Turnip Infusion remained clear, and showed no appreciable change for seven days. On the eighth day a slight general turbidity of the fluid was noticed. On the ninth, the turbidity was rather more marked, though there was no trace of a pellicle; the neck of the flask having been broken, the fluid was found to be odourless and very faintly acid. On microscopical examination, multitudes of languid Bacteria of medium size were found, and also short monilated chains with from two to ten segments. There were no Vibriones, Leptothrix or Torulæ.57
No. XXVII.—Healthy Urine after twenty-four hours showed no change. After eleven days there was still no apparent change, though on the thirteenth a slight amount of flocculent sediment was noticed. This deposit increased in amount, very slowly, during the next fortnight; though afterwards the fluid seemed to undergo no further change, and did not become generally turbid.58
No. XXVIII.—Healthy Urine ( 13) and Filtered Turnip Infusion ( 23) after forty-eight hours showed a very slight turbidity, which, however, became quite marked in another twenty-four hours.
No. XXIX.—Albuminous Urine ( 13) and filtered Turnip Infusion ( 23) after twenty-four hours, showed a slight turbidity, which became much more marked in forty-eight hours; whilst in seventy-two hours there was a considerable deposit at the bottom of the flask.
No. XXX.—Simple Turnip Infusion showed no change in forty-eight hours, though in seventy-two hours there was well-marked turbidity. The turbidity and sediment continued to increase for several days, and both were most marked on the tenth day, when the flask was opened. There was an outrush of gas, having an extremely fœtid odour. The fluid had an acid re-action, and when examined microscopically, multitudes of Bacteria, Vibriones and Leptothrix filaments were found—the movements of the Bacteria being very languid.
No. XXXI.—Healthy Urine remained in the warm bath for twenty-eight days without undergoing the least change.
No. XXXII.—Simple Turnip Infusion remained in the warm bath for twenty-eight days without undergoing any appreciable change.59 On breaking the neck of the flask, the fluid was found to be quite odourless. With its neck quite open, the flask was replaced in the water-bath. During the first forty-eight hours it underwent no apparent change, though at the end of seventy-two hours a slight general turbidity was noticeable, and an examination of a drop of the fluid (still odourless), showed a number of minute but very active Bacteria.60
No. XXXIII.—Simple Turnip Infusion showed no change after eight days’ immersion in the warm bath. After eleven days, the fluid being still clear, the tube was broken just beyond the second bending from the bulb, and then the flask was re-immersed in the bath. After three days’ exposure, the fluid being still clear, it was boiled in the flask for one minute, when it was noticed that the steam was quite odourless. The flask was then replaced in the water-bath, where it remained for twenty-two days (still with the neck open and broken just beyond its second bending) without showing any change.61 It was then submitted to examination; the fluid was found to be devoid of all odour, it had a slightly bitter taste, and its re-action was very faintly acid. On microscopical examination no living things were found; there were no Bacteria, no Vibriones, and no Torulæ, only some mere granules, a small amount of amorphous matter, and a few fibres.62
No. XXXIV.—Turnip Infusion Neutralized by Ammonic Carbonate in forty-eight hours showed a slight turbidity, which slowly increased during the next two days. In two days more the turbidity was very great, and there was also a considerable amount of sediment. The fluid was then examined microscopically, and found to contain myriads of large but very languid Bacteria.
No. XXXV.—Healthy Urine underwent no apparent change for the first twelve days, then (the bulk of the fluid still remaining clear and bright) small greyish white flocculi began to collect at the bottom of the flask, which very slowly increased in quantity during the succeeding twelve days. At the expiration of this time the flocculi were pretty numerous, though the fluid was otherwise bright. The vacuum was ascertained to be still good, and on breaking the flask, the fluid was found to have a slightly acid re-action, though no appreciable odour. When examined microscopically, the flocculi were seen to be made up for the most part of mere granular aggregations (simple, and not in the form of Bacteria). Small Torula cells, however, existed in some quantity; also a few necklace-like chains, and a comparatively small number of Bacteria, some of which were tolerably active.
No. XXXVI.—Simple Turnip Infusion after twenty-four hours showed no sign of change, though in thirty-six hours it was slightly turbid. On the fourth day the turbidity was well-marked and general, though there were no flake-like aggregations. When examined microscopically, the fluid was found to contain multitudes of Bacteria.
No. XXXVII.—Turnip Infusion,63 Neutralized by Ammonic Carbonate in twenty-four hours was decidedly turbid. In thirty-six hours the turbidity was more marked, and there was a slight sediment. By the end of forty-eight hours both turbidity and sediment had notably increased. On the fourth day, there was a moderately clear fluid, containing an abundance of curdy or flake-like masses. When the flask was opened, these were found to be made up principally by the aggregation of myriads of Bacteria.
No. XXXVIII.—Turnip Infusion in ten hours showed a slight amount of turbidity. After forty-eight hours this was very well-marked: there was a thick pellicle on the surface, and, in addition, a small amount of deposit. On examination, the fluid and the pellicle were found to contain an abundance of Bacteria, Vibriones and Leptothrix filaments.
No. XXXIX.—Turnip Infusion + 120 of Carbolic Acid after eight days showed no appreciable alteration in appearance,64 no trace of pellicle or deposit. When examined microscopically, however, the fluid was found to contain some very minute Bacteria, though they were by no means abundant.
No. XL.—Hay Infusion had become quite turbid in twenty-four hours, and several shades lighter in colour. After forty-eight hours the colour of the infusion was still lighter; there was more turbidity, and some sediment. On microscopical examination, the fluid was found to contain an abundance of Bacteria, Vibriones and short Leptothrix filaments.
No. XLI.—Hay Infusion + 120 of Carbolic Acid showed no apparent change65 after forty-eight hours, and when examined microscopically it revealed no trace of Bacteria, or other organisms. The neck of the flask was then again closed. On the twelfth day the fluid had still undergone no change in appearance, and when examined microscopically, it still showed no trace of organisms, though the fluid was—as it had been at the time of the first examination—full of minute, undissolved particles of carbolic acid.
No. XLII.—Hay Infusion, after forty-eight hours, showed no change, and continued to remain quite clear and free from deposit until the twelfth day, when it was examined microscopically. No organisms of any kind could be detected.
No. XLIII.—Hay Infusion + 120 part of Carbolic Acid showed no apparent change66 for the first five days, though, on the sixth day, a slight deposit was noticed at the bottom of the flask. The deposit had increased, and was well-marked by the twelfth day, when, on microscopical examination, there were found amongst the granular flakes of the deposit, Torulæ of several varieties of size and shape. Many were spherical, others ovoid, or having an elongated oat-like shape: some were of the ordinary colour, and others were brownish in tint. The variety was most striking. No Bacteria were seen, though there were multitudes of active particles which seemed to differ from the minute spherules of undissolved carbolic acid.
No. XLIV.—Turnip Infusion, in seventy-two hours, showed a slight turbidity, which gradually increased. On the eighth day there was a considerable quantity of flake-like sediment, and some amount of general turbidity. On the thirteenth day the vacuum was found to be still partly preserved. When the flask was opened the fluid was perceived to have a fœtid odour, and an acid re-action; and, on microscopical examination, multitudes of Bacteria and Vibriones were seen. In the flake-like aggregations also (made up almost wholly of these organisms) there were a number of large thick-walled spores; some already formed, and others in process of formation by coalescence.
No. XLV.—Turnip Infusion + 120 part of Carbolic Acid showed no increase of turbidity67 for the thirteen days during which it was kept under observation. Before the flask was opened it was ascertained that the vacuum was well preserved. The odour of the fluid was unaltered, and on microscopical examination no Bacteria, or other living things, were found.68
No. XLVI.—Hay Infusion, after forty-eight hours, showed no change, though, in seventy-two hours, there was perceptible a very small amount of a dirty greyish deposit. By the fifth day the deposit had slightly increased, and on the seventh day there was a trace of turbidity in the fluid. It did not undergo much further change, so that, on the twelfth day, the flask was opened. The vacuum was found to have been very slightly impaired; the odour of the fluid was almost natural, and its re-action was slightly acid. On microscopical examination of the deposit, Bacteria, Vibriones, short Leptothrix filaments, and Torulæ, were found, though not in very great abundance.
No. XLVII.—Hay Infusion + 120 part of Carbolic Acid showed no apparent change for the first four days. On the fifth day there was a small quantity of powder-like sediment, and one dirty greyish-coloured flake. On the seventh day there were more small flakes at the bottom, and a slight general turbidity of the fluid. On the twelfth day, the turbidity and deposit having increased, the flask was opened—after it had been first ascertained that the vacuum had only been slightly impaired. The re-action of the fluid was still strongly acid. On microscopical examination of some of the deposit, there was found, amongst granular flakes and aggregations, a large number of Torulæ cells, of most various shapes and sizes; also in the midst of the granule heaps many large, rounded or ovoidal, densely granular nucleated bodies, whose average size was 11500″ in diameter, though there were many of them much larger, and others even less than half this size. Intertwined amongst the granular matter also were a large number of algoid-looking filaments, 120000 in diameter, containing segmented protoplasmic contents. There were also in the fluid itself a number of medium-size, unsegmented Bacteria, whose movements were somewhat languid.69