The modes of origin of lowest organisms

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 - 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.] and also with very minute particles which were gradually precipitated[30 - Those particles which come to rest, in such cases, are always in contact with one or other of the contiguous surfaces of glass.] 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 - The specific gravity of the fluid being constant. Where this is dense or viscid, as with glycerine, Brownian movements do not occur at all.] 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 - In the proportion of ten grains of neutral ammonic tartrate, with three grains of neutral sodic phosphate, to an ounce of distilled water.] 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 boiled[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.] and allowed to cool, were similarly inoculated with a drop[34 - The proportion was one drop of the fluid, opaque with organisms, to an ounce of the clear solution.] 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 - Into which a piece of glass tube had been slipped to prevent collapse.] 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 - 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.] 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 - 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.] 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 - 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 (#Page_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 - ‘Nouvelles Expériences,’ etc., 1864, p. 38.] 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 - ‘American Journal of Science and Arts,’ Oct. 1867.] 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 1/2° 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.] 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.

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 - 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.]. 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.

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 - ‘Compt. Rend.,’ t. lxi. p. 1060.] 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 - 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.] 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 vessels[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.] 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 - 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 - 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 - Flask still in my possession, unopened.]

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 (#cn_47)

No. VI. – Turnip Infusion underwent no change during the fifteen days in which it was kept in the bath under observation.48 (#cn_47)

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 (#cn_47)

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 - Flask still in my possession, unopened.] (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 boiled[50 - The vapour had lost all odour of turnip. Some of the fluid which splashed over was found to be still slightly acid.] 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 - This experiment is very interesting in two or three respects. A neck of half the usual length – with only four bendings – sufficed to preserve the fluid for several days; and when this fluid (which had been in the bent-neck apparatus for nine days) was sealed up in the same flask during ebullition, it remained in vacuo for thirteen days without undergoing any apparent change, and then only became turbid under the influence of a higher temperature. Yet some of the same fluid, in a flask which was hermetically sealed during the first ebullition (No. XV.) behaved as such an infusion usually does, and became quite turbid in forty-eight hours.]

d. Fluid in a Flask having a Neck two feet long, bent at right angles shortly above the bulb, and provided with a firm Plug of Cotton-Wool twelve inches in length

No. X. – Urine remained quite bright and clear during the fifteen days in which it was kept under observation in the water-bath.[52 - Flask still in my possession, unopened.]

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 (#cn_51)

No. XII. – Turnip Infusion in four days showed a well-marked turbidity, and also very many flakes of a broken pellicle.52 (#cn_51)

e. Fluid (in vacuo) in a Flask, the Neck of which was hermetically Sealed by means of the Blowpipe Flame during Ebullition

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.

Second Set of Experiments (XVI.–XXI.)

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

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 + 1/2 gr. of Cheese,[53 - The filtered infusion of turnip was neutralized by liquor potassæ. The cheese (Cheddar) was new and not in the least mouldy.] in thirty-six hours showed a well-marked pellicle.[54 - The fluid itself being somewhat opaque, the first stages of increased turbidity from presence of Bacteria could not be detected.] 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.

c. Fluid in a Bent Neck Flask, having Eight acute Flexures