The modes of origin of lowest organisms

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 1/2° F.41 The two other flasks similarly prepared were kept at a temperature of 118 1/2°–127 1/2° F. for four hours. In two days, the fluid in the first flask became slightly turbid, whilst in two days more the turbidity was most marked. The fluid in the two other flasks which had been exposed to the temperature of 118 1/2°–127 1/2° F. for four hours, remained quite clear and unaltered during the twelve days in which they were kept in the warm bath under observation. These experiments seem to show, therefore, that the prolongation of the period of exposure to four hours, suffices to lower the vital resistance to heat of Bacteria and Torulæ by 14 1/2°–18° F.

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