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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.
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 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.
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
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
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
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
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
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 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.
c. Fluid in a Bent Neck Flask, having Eight acute Flexures
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 1/2 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 1/3000 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 + 1/2 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.
e. Fluid (in vacuo) in a Flask which had been Sealed during Ebullition
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 + 1/2 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.
Third Set of Experiments (XXII.–XXX.)
a. Fluid exposed to Air in a Flask with a short Open Neck
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.
b. Fluid in contact with Ordinary Air and its Particles; Neck of Flask Sealed after the Fluid had become Cold
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.
c. Fluid in a Bent Neck Flask, having Eight acute Flexures
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 + 1/2 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
e. Fluid (in vacuo) in a Flask, Sealed during Ebullition
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 ( 1/3) and Filtered Turnip Infusion ( 2/3) after forty-eight hours showed a very slight turbidity, which, however, became quite marked in another twenty-four hours.
No. XXIX. – Albuminous Urine ( 1/3) and filtered Turnip Infusion ( 2/3) 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.
