The modes of origin of lowest organisms

No. LIV. – Ammonic Oxalate Solution.– On the eighth day the fluid showed a very faint opalescence, though there was a well-marked, greyish, flocculent deposit, which was found to be composed of an aggregation of colourless and blackish granules, of a multitude of minute crystalline particles (mostly diamond-shaped), and some rounded or ovoidal, thick-walled, spore-like bodies; amongst which, and enveloped in part by them, were several mycelial tufts of a fungus. A number of minute Bacteria were found distributed throughout the fluid, and also a quantity of minute star-like bodies (crystalline), about 1/12000 in diameter.

No. LV. – Ammonic Carbonate Solution.– On the eighth day the fluid showed a very faint opalescence, and a slight deposit, which was found to be composed principally of amorphous granules. Distributed through the fluid were some small and sluggish Bacteria, though no other organisms were seen.

No. LVI. – Ammonic Tartrate Solution.– After twenty-four hours the fluid showed the faintest opalescence, and in forty-eight hours there was a slight bluish-white turbidity. In seventy-two hours the turbidity was well marked, and there was a very thin pellicle on the surface. When examined microscopically the fluid was found to contain multitudes of very active Bacteria, and the pellicle was also composed of an aggregation of Bacteria. On the thirteenth day the opacity had somewhat increased; there was also a well-marked pellicle, and an obvious deposit. The pellicle was found to be composed of Bacteria, and in the fluid there were multitudes of medium-size Bacteria and Vibriones, with here and there a small Torula cell.71

Ammoniacal Solutions (in vacuo) in Flasks which were hermetically Sealed during Ebullition of their Fluids at a Temperature of 90° F. 72 (Subsequently exposed in water-bath to a Temperature of 75°–85° F.)

No. LVII. – Ammonic Tartrate Solution after sixty hours showed a slight sediment, with bluish flakes attached to sides of flask. In eighty-four hours there was a general bluish opalescence, and on microscopical examination the fluid was found to contain multitudes of Bacteria.

No. LVIII. – Ammonic Tartrate and Sodic Phosphate Solution.– After sixty hours there was a slight general bluish opalescence. In eighty-four hours the general opalescence was not more marked, but there were many flake-like aggregations in the fluid, which on microscopical examination were found to be aggregations of Bacteria.

Ammoniacal Solutions boiled (at 212° F.), and exposed to Air in Flasks whose Open Necks were only loosely covered with Paper Caps: subsequent Inoculation. (Temp. 75°–85° F.)

No. LIX. – Ammonic Tartrate Solution.– The fluid remained quite clear, and free from all trace of turbidity up to the ninth day, when it was inoculated with some living Bacteria. In fifty hours after the inoculation there was a very faint opalescence of the fluid, which, in another 24 hours, had become much more marked. On microscopical examination it was found to contain multitudes of Bacteria.

No. LX. – Ammonic Tartrate and Sodic Phosphate Solution.– After four days the fluid was still quite clear. In seven days no trace of general turbidity, though there was a minute dirty-grey aggregation about 1/14″ in diameter at the bottom of the flask. On the sixteenth day the grey aggregation had very slightly increased in size, though the fluid above was still perfectly clear. The grey mass was removed by a small pipette, and, on microscopical examination, it was found to be composed of an aggregation of minute extraneous fibres, mixed with blackish particles and amorphous granular matter, in which were growing many Torula-cells in all stages of development, and also a minute mycelium composed of branched Leptothrix-like fibres.73 The clear fluid was then inoculated with some living Bacteria, and the bulb of the flask was replaced in the warm bath. After fifty hours the solution showed a bluish turbidity, which, in thirty-six hours more, had increased to a well-marked whitish opacity, and when examined, the fluid was found to be swarming with active Bacteria.

Solutions of Ammonic Tartrate and Sodic Phosphate were heated, in their respective Flasks, for Fifteen Minutes to the Temperatures mentioned below. The Necks of the Flasks were afterwards loosely covered with Paper Caps, whilst the Bulbs were immersed in a Water-Bath kept at a Temperature of 75°–85° F

No. LXI. —

Solution heated to

149° F.

No. LXII.

" " "

158° F.

No. LXIII.

" " "

158° F.

No. LXIV.

" " "

167° F.

No. LXV.

" " "

167° F.

All these solutions remained quite clear and free from any trace of general turbidity for ten days. Each fluid was then inoculated with some living Bacteria, and in the space of thirty-six to seventy-two hours, all had become more or less obviously turbid, and on microscopical examination this turbidity was found in each case to be almost wholly due to the presence of multitudes of Bacteria.

Interpretation of the Experiments: Conclusions as to the Cause of Fermentation, and as to the Occurrence of Archebiosis

These experiments seem to show quite conclusively that M. Pasteur’s explanations are altogether inadequate to account for the occasional preservation of boiled fluids in bent-neck flasks. They show that the preservation, far from being universal, is only occasional, and that preservation or non-preservation of different fluids is almost wholly dependent upon their nature. They lend no countenance, moreover, to his particular theory, that fermentation cannot be initiated without the agency of living ferments, – they are, on the contrary, wholly opposed to this restriction.

The plug of cotton-wool, or the narrow and bent tube may, it is true, protect the boiled fluid from subsequent contact with living “germs”; but that the fluids do not undergo change on account of such deprivation cannot be safely affirmed, when the same means would also filter from the fluid some of the multitudinous particles of organic matter (dead), which the air undoubtedly contains, and which may act as ferments. It must be remembered that the main object of M. Pasteur’s investigation was to determine whether fermentation took place under the agency of mere dead nitrogenous matter, as Liebig and others affirm; or whether it is only initiated by living organisms, as he himself supposes. Obviously, therefore, the same filtration which purified the air from any living organisms would filter from it its nitrogenous particles, which are the other possible ferments: so that no conclusion could be drawn from such experiments, more favourable to the one than to the other of these hypotheses. All that could have been safely affirmed was, that by boiling the fluid, and then protecting it from subsequent contact with everything that could act as a ferment, fermentation would not take place.

Even this however, – as the preceding experiments fully show – cannot be truly affirmed to be a general rule. Some infusions do undergo change, notwithstanding this treatment and deprivation, whilst others do not: that is to say, some still preserve a first degree of fermentability even after boiling, whilst others are reduced by this process to the second degree of fermentability. These latter are unable to initiate changes by virtue of their own inherent instability; molecular re-arrangements require to be set on foot in them by contact with some more unstable substance, which is itself undergoing change.

That such is the correct explanation of the reason why some fluids do not ferment in bent-neck flasks, seems obvious from the discordant results obtained in many other experiments, after the free admission of uncalcined air to the fluids which had been boiled. The fluids were deprived of their virtues in some cases by the heat to which they had been subjected, so that whether they underwent change or not, may have depended upon the accidental presence or absence, in the air which was subsequently admitted to the fluid, of some unheated organic fragments, capable of initiating fermentative changes. If germs were as omnipresent as they have been represented to be, such fluids ought always to have undergone fermentation.

Whilst I have found that any given fluid, whose strength is about equal on different occasions, acts in a definite manner when the flask is hermetically sealed after expulsion of all its air and during the continuance of ebullition; and, whilst a like definite result can generally be obtained, when calcined air is admitted to the boiled fluid before the vessel is hermetically sealed; it is found, on the other hand, that the result is in no way predicable when uncalcined air is admitted. Sometimes fermentation takes place, and sometimes in other flasks – sealed at the same time, and subsequently placed under the same conditions – no change whatever occurs. My own experience in this respect accords perfectly with that of M. Pasteur.74 He, however, at once came to the conclusion that the only inference from such facts was that “germs” are not so universally distributed as they had been supposed to be by Bonnet and Spallanzani.75 The unprejudiced inquirer, however, will perceive that M. Pasteur was entitled to come to no such conclusion concerning germs which was not equally applicable to minute fragments or débris of organic matter floating in the air. And, similarly, the evidence which he adduces with regard to the diminution in the number of the fertile flasks when they were filled with some of the still air of the caves of the observatory, or else with some from the peaks of the Jura,76 far away from the haunts of men, had no bearing upon the distribution of germs which was not equally applicable to that of dead organic particles. Such evidence, therefore, was valueless for settling between the rival doctrines of fermentation – it could not possibly help us to decide whether living or dead ferments were necessary. Dead organic particles would sink in still air in the same manner as living organisms;77 and similarly, dead organic particles, have been shown to be less and less numerous in the atmosphere in proportion to the elevation obtained.78 In these latter experiments M. Pasteur made use of yeast-water (alone or sweetened), and of urine – all three of them fluids, which, after having been boiled, are apt to possess only the second degree of fermentability. Shortly afterwards, M. Pouchet, in concert with MM. Joly and Musset,79 repeated these experiments, with the sole difference that they employed strong infusions of hay – which experiment has shown almost invariably to possess the first degree of fermentability. And seeing that all their flasks, after a time, yielded organisms from whatever mountain elevation the air had been taken – the combined evidence tends strongly against the view of M. Pasteur. Since the germs in the fluids and in the flasks, in each set of experiments, had been previously destroyed by ebullition, and since in each set, also, air of the same character had been admitted to the boiled fluids, the different results seemed to show that fermentation or non-fermentation, in such cases, depends wholly upon the quality of the fluids employed.

Other evidence which is so much vaunted by M. Pasteur and his supporters, as to the possibility of inducing fertility in previously sterile flasks, by the addition of a portion of asbestos containing the solid particles filtered from the atmosphere,80 is also equally valueless for confirming the proposition that fermentation is only capable of being initiated by living ferments. The same asbestos which may contain living spores or organisms (“germs”), does undoubtedly contain many decomposable particles and fragments of organic matter.81 The previously barren solution may therefore be rendered fertile by the mere addition of those portions of unstable organic matter, whose molecular mobility has not been impaired by the agency of heat, and which are therefore capable of initiating fermentations. This view is strengthened, as M. Pouchet has pointed out, by the fact that in these cases, instead of meeting some of the various kinds of organisms which are supposed to have representatives in the air, and whose spores or ova may be supposed to have been sown, it is often merely Bacteria which are encountered, – differing in no respect from those that may present themselves in a somewhat similar infusion, which has undergone change in a closed flask without any such hypothetical sowing of living spores or germs. It is more especially important to bear in mind this consideration, when portions of organic matter can always be easily demonstrated amongst such atmospheric dust; whilst living Bacteria, or other organisms, such as are first produced as a result of the supposed sowing of spores, either cannot be demonstrated, or would seem, from other evidence, to be at least very sparingly distributed.82

The fact revealed by M. Pasteur, that some fluids remain unchanged for an indefinite period, after having been boiled in flasks, with long and bent necks, is easily explicable in accordance with the physical theory of fermentation, and now that it has been thoroughly proved that other fluids – submitted to precisely similar conditions – do nevertheless undergo fermentation, this fresh fact is just as completely adverse to the explanations and views of M. Pasteur, as it is thoroughly harmonious with the doctrines of Baron Liebig. The fluids which are capable of being preserved – generally not presenting a high degree of fermentability – do not undergo change, at ordinary atmospheric pressure, after having been boiled, unless they are brought into contact either with some pre-existing living things or with some unaltered organic particles from the atmosphere. Neither of these, however, can gain access to the fluid, in such a vessel; because all the air which enters, after the first inrush into the still almost boiling fluid, has to pass, more or less slowly, through the numerous flexures of the narrow neck of the flask and the two or three strata of fluid which always remain therein.

Some of the fluids which do not undergo change in these bent-neck vessels are, however, by no means notable for possessing a low degree of fermentability. This is the case, for instance, with infusions of turnip, which, under other conditions, have been found to be most prone to undergo fermentation. And, I have found in several cases in which such an infusion had been exposed in a bent-neck vessel, and had remained unchanged for twelve or fourteen days (even though subjected to a temperature of 85°–95° F.), that if the neck of the flask were then broken shortly above the bulb, the solution would still continue without alteration for a week, ten days, a fortnight, or even more – although freely exposed to the air, and therefore to the access of any living germs which might be floating about in the atmosphere.83

The views hitherto expressed with reference to the causes of fermentation and putrefaction, and to the interpretations which M. Pasteur’s experiments are capable of receiving, seem to derive all the additional support that can be needed, from the results of my own experiments with boiled fluids, in sealed flasks, from which all air had been expelled.

Some of the same fluid being taken and divided into three parts, each portion is placed in a separate flask, in which it is boiled for a period of ten minutes. One of the flasks (A) is provided with a long and bent neck, so that the air which re-enters is deprived of its germs and organic particles; another (B) has only a short neck, and to this, the access of germs and organic particles is freely permitted till the fluid has become cool, and then the neck of the flask is hermetically sealed; whilst the last (C) is sealed during ebullition, after all air has been expelled. Now, if Pasteur’s theory of fermentation, and the prevalent notions concerning the universal distribution of “germs” throughout the atmosphere were true, it might be expected that the fluid in B would always rapidly change; that that in A would always remain pure; and that the fluid in C would, similarly, undergo no alteration. The facts, however, are quite the reverse: if a strong turnip infusion be employed, the fluid in A will almost always remain unchanged; that in B will sometimes rapidly change, and at other times will remain quite pure; whilst that in C will almost invariably become turbid in from two to six days. But, even if it were not the case that some fluids, different from those used by M. Pasteur, will almost invariably undergo change in bent-neck vessels, M. Pasteur’s explanation of the cause of the preservation would have been altogether upset by the fact that some of the very fluids which remain pure in the bent-neck apparatus will become fœtid if shut up in vacuo. It is, therefore, of course useless to talk of a particular boiled fluid having been saved from putrefaction on the ground that the living atmospheric germs (whose presence is supposed to be necessary for the initiation of such a process) have been altogether filtered from the re-entering air, when some of the same fluid will putrefy, if placed under different conditions by which it is freed both from the influence of the atmosphere and from its germs —i. e., when, instead of filtering the re-entering air, no air is permitted to enter. Germs and atmospheric particles being equally got rid of in both sets of cases, the great difference between them is that the weight of the atmosphere is also got rid of in my experiments – the fluids being contained in vacuo. Now it has been ascertained by Mr. Sorby, that pressure undoubtedly influences “chemical changes taking place slowly, and therefore, probably due to weak or nearly counterbalanced affinities;” and he also states, in the Bakerian Lecture for 1863,84 that “a considerable number of facts have been described, showing that pressure will more or less influence such chemical actions as are accompanied by an evolution of gas, so that it may cause a compound to be permanent, which otherwise would be decomposed.” If increase of pressure retards, a diminution of pressure will facilitate such chemical changes, so that one can only explain the results which I have obtained, on the ground that many boiled fluids, which will not undergo change when protected from the influence of atmospheric particles (living or not living) at the same time that they are subjected to ordinary or increased pressure, will, on the contrary, pass through such changes when pressure is removed, and the fluids are preserved in vacuo. It is not pretended that this is a rule applicable to all organic fluids – far from it. Diminution of pressure does seem, however, to be a very potential cause of change in some fluids. The extent to which changes of a fermentative character can progress in the absence of atmospheric oxygen, is also evidently subject to much variation, in accordance with the nature of the dissolved fermentable substances.

These facts are not so new and exceptional, however, as they may at first sight appear. It has been long known that a boiled fluid extremely prone to change, will not yield infusoria if the vessel in which it is contained is filled with the fluid. Burdach says85: – “Gruithuisen a reconnu que des infusions, même très fécondes d’ailleurs, celles du foin par exemple, ne donnaient point d’infusoires dans des flacons de verre dont le bouchon était assez enfoncé pour toucher à la surface de l’eau.” On the other hand, no experiments with which I am acquainted, in which heated fluids and calcined air have been shut up in closed flasks, have yielded so many positive results as those of Professor Wyman of Cambridge, U.S., – and his were performed under precisely the reverse conditions. Large flasks were used, and only 1/15 – 1/20 of their bulk was filled by the experimental fluids.86 Gruithuisen’s results were explained by Burdach on the ground that a certain amount of air was necessary, and it was also with the view of subjecting his fluids to as large an amount of air (calcined) as possible, that Professor Wyman employed small quantities of fluids in large flasks. These views were dictated by the chemical doctrines of Gay-Lussac and others, to the effect that the oxygen of the air is the initiator or primum movens of fermentative changes.87

Now, without doubting in the least that in some instances this may be the case, it seems to me quite obvious, from my own experiments, that a different interpretation may be given of Gruithuisen’s results – which I have myself verified, – and of the fact that meats and vegetables will often remain unchanged for years after having been heated in closed tins from which all air has been expelled.88

If we ponder only upon the fact that certain fluids, in contact with a very small quantity of air, in an hermetically-closed vessel, will not undergo change; though these same fluids will change when exposed to a much larger quantity of calcined air, there may be strong reason for coming to an opinion similar to that of Gay-Lussac. When, however, it is also ascertained that provisions which have been subjected to the long-continued influence of heat, do not undergo change in closed vessels, if all air has been expelled from the very small space above the level of the provisions; though many organic infusions will putrefy if they occupy only one half, or less, of a hermetically closed vessel from which all air has been similarly expelled by ebullition of its fluid contents, it is impossible that the same explanation can hold good. And at the same time another interpretation is suggested for the first set of facts.

The last-mentioned experiments prove89 that fermentation can take place in vacuo, when the conditions are more favourable than those which present themselves within the almost full tins containing provisions. The change in these latter cases cannot (in the great majority of instances) proceed far90, because there is no adequate space into which residual gases may be emitted. When this emission (which is almost always one of the accompaniments of a fermentative change) has taken place to a slight extent, the meats are in the very best condition for preservation. There is an utter absence of light, an absence of free oxygen, and also an absence of that diminished pressure which my experiments seem to show is favourable to the promotion of many kinds of fermentative change. So that if fermentation does not take place in a closed flask which is full of a boiled infusion of hay,91 it may be owing to the fact that there is no space for the residual gases, and that undue pressure retards many fermentative changes. This is also perfectly compatible with the other fact that the same kind of fluid will undergo change when a small quantity of it is contained in a comparatively large flask – owing to there being, in such a case, plenty of room for residual gases to be effused, before that undue amount of pressure is brought about, in the presence of which such a fluid will no longer ferment or putrefy. Fluids, therefore, whose putrefaction is hindered by increased pressure and favoured by diminution of pressure, may be placed under conditions which are successively more favourable than the last, by putting a gradually smaller and smaller quantity of fluid into a flask, to which calcined air is admitted, and, better still – if the stimulus of oxygen is not absolutely needed in order to incite fermentation in the fluid employed – by only half filling the flask, and procuring a more and more perfect vacuum.

In accordance with the doctrines of Baron Liebig, therefore, my experiments, as well as those of many other investigators, tend to show that fermentative and putrefactive changes are merely processes of chemical re-arrangement, which frequently take place – as it were “spontaneously” – owing to the inherent instability of certain nitrogenous compounds in the presence of free oxygen. My experiments have, however, also revealed the additional fact that, under the combined influence of heat and diminished pressure, some fluids will undergo fermentation even in closed vessels, from which all air has been expelled. They lend no support to the idea that the air is so thickly laden with living germs as some would have us suppose; and in view of the mass of positive information now in our possession concerning the degree of heat which suffices to kill the lowest organisms, they also, as I think, entitle us to come to the conclusion that such organisms are (as the microscopical evidence might lead us to believe) really capable of being evolved de novo. These lowest organisms are, in fact, to be regarded as occasional concomitant products, rather than as invariable or necessary causes of all fermentative changes.