Chapter IV: Preface (3)

*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] 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]

[50] The vapour had lost all odour of turnip. Some of the fluid which splashed over was found to be still slightly 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]

*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]

[52] Flask still in my possession, unopened.

_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.

[53] The filtered infusion of turnip was neutralized by liquor potassæ. The cheese (Cheddar) was new and not in the least mouldy.

[54] The fluid itself being somewhat opaque, the first stages of increased turbidity from presence of _Bacteria_ could not be detected.

_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]

[55] This again is a most instructive experiment when compared with Nos. XVI. and XX., in which portions of the same infusion were employed. The results in No. IX. would lead us to believe that a vegetable infusion which does not ferment, does, nevertheless, undergo some changes in molecular composition, and this notion seems to derive confirmation from the present experiment. Some of the same solution which has been kept for a time (twelve days) from contact with atmospheric particles, subsequently, even when fully exposed to the air, undergoes no apparent change for six days, and then, instead of becoming filled with _Bacteria_, swarms only with _Torulæ_. Yet the infusion in this condition was perfectly capable of nourishing _Bacteria_, as I subsequently proved by inoculating it. Why then was it not inoculated by the living _Bacteria_, with which the air is thought by some to be teeming?

*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_.

[56] Some of the same fluid, exposed in a similar flask, without previous boiling, became turbid in eight hours, and lighter in colour; whilst, after twenty hours, the turbidity was extremely well-marked.

_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]

[57] The condition of the fluid, and the nature of its contents, were very similar to that met with in No. XXI.

_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]

[58] Still in my possession, unopened. In all probability the flocculi which formed would be found to be similar in their microscopical, as they certainly were in their naked-eye characters, to those met with in No. XXXV.

*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.

FOURTH SET OF EXPERIMENTS (XXXI.–XXXVII.).

_b. Fluid in contact with ordinary Air and its Particles; Flask Sealed after the Fluid had become Cold._

*No. XXXI.--Healthy Urine* remained in the warm bath for twenty-eight days without undergoing the least change.

*No. XXXII.--Simple Turnip Infusion* remained in the warm bath for twenty-eight days without undergoing any appreciable change.[59] On breaking the neck of the flask, the fluid was found to be quite odourless. With its neck quite open, the flask was replaced in the water-bath. During the first forty-eight hours it underwent no apparent change, though at the end of seventy-two hours a slight general turbidity was noticeable, and an examination of a drop of the fluid (still odourless), showed a number of minute but very active _Bacteria_.[60]

[59] Experiment No. 8, recorded in ‘Nature,’ 1870, No. 36, p. 194, may be compared with this and No. XXXIII.

[60] This experiment should be compared with Nos. XVIII. and XXXIII. It seems to show that if some fermentable fluids can be kept for a time under conditions in which they will not ferment, the constitution of the fluid, instead of remaining the same, undergoes a slow alteration by which it is rendered absolutely less fermentable, even when exposed to the most favouring influences.

_c. Fluid in a Bent-Neck Flask, having Eight acute Flexures._

*No. XXXIII.--Simple Turnip Infusion* showed no change after eight days’ immersion in the warm bath. After _eleven_ days, the fluid being still clear, the tube was broken just beyond the second bending from the bulb, and then the flask was re-immersed in the bath. After three days’ exposure, the fluid being still clear, it was boiled in the flask for one minute, when it was noticed that the steam was quite odourless. The flask was then replaced in the water-bath, where it remained for twenty-two days (still with the neck open and broken just beyond its second bending) without showing any change.[61] It was then submitted to examination; the fluid was found to be devoid of all odour, it had a slightly bitter taste, and its re-action was very faintly acid. On microscopical examination no living things were found; there were no _Bacteria_, no _Vibriones_, and no _Torulæ_, only some mere granules, a small amount of amorphous matter, and a few fibres.[62]

[61] After this experiment had been completed, a fresh-filtered infusion of turnip was placed in the same flask (having the neck open just beyond its second bending), and after having been boiled for a few minutes it was immersed in the same water-bath. This fluid became turbid in thirty-six hours, and was then found to contain multitudes of _Bacteria_; and the characteristic odour of the turnip infusion was still appreciable.

[62] The results of this experiment are most interesting, especially if compared with what takes place when some of the same fluid is neutralized by ammonic carbonate (No. XXXIV.), with what occurs when a similar fluid (as in No. XXX.) is contained in a flask sealed during the continuance of ebullition, or also with what occurred in Nos. XIII. and XXXII. In the present case the second boiling seems to have destroyed what small amount of fermentability there was still remaining in the solution; but in No. IX. fermentation did take place after the second boiling--though this occurred only under the influence of diminished pressure and a higher temperature.

*No. XXXIV.--Turnip Infusion Neutralized by Ammonic Carbonate* in forty-eight hours showed a slight turbidity, which slowly increased during the next two days. In two days more the turbidity was very great, and there was also a considerable amount of sediment. The fluid was then examined microscopically, and found to contain myriads of large but very languid _Bacteria_.

_e. Fluid (in vacuo) in a Flask which had been Sealed during Ebullition._

*No. XXXV.--Healthy Urine* underwent no apparent change for the first twelve days, then (the bulk of the fluid still remaining clear and bright) small greyish white flocculi began to collect at the bottom of the flask, which very slowly increased in quantity during the succeeding twelve days. At the expiration of this time the flocculi were pretty numerous, though the fluid was otherwise bright. The vacuum was ascertained to be still good, and on breaking the flask, the fluid was found to have a slightly acid re-action, though no appreciable odour. When examined microscopically, the flocculi were seen to be made up for the most part of mere granular aggregations (simple, and not in the form of _Bacteria_). Small _Torula_ cells, however, existed in some quantity; also a few necklace-like chains, and a comparatively small number of _Bacteria_, some of which were tolerably active.

*No. XXXVI.--Simple Turnip Infusion* after twenty-four hours showed no sign of change, though in thirty-six hours it was slightly turbid. On the fourth day the turbidity was well-marked and general, though there were no flake-like aggregations. When examined microscopically, the fluid was found to contain multitudes of _Bacteria_.

*No. XXXVII.--Turnip Infusion,[63] Neutralized by Ammonic Carbonate* in twenty-four hours was decidedly turbid. In thirty-six hours the turbidity was more marked, and there was a slight sediment. By the end of forty-eight hours both turbidity and sediment had notably increased. On the fourth day, there was a moderately clear fluid, containing an abundance of curdy or flake-like masses. When the flask was opened, these were found to be made up principally by the aggregation of myriads of _Bacteria_.

[63] Some of same as that which was used (unaltered) in last experiment.

FIFTH SET OF EXPERIMENTS (XXXVIII.–XLVII.).

_Fluids not boiled, but half-filling hermetically Sealed Flasks, containing Ordinary Air._

*No. XXXVIII.--Turnip Infusion* in ten hours showed a slight amount of turbidity. After forty-eight hours this was very well-marked: there was a thick pellicle on the surface, and, in addition, a small amount of deposit. On examination, the fluid and the pellicle were found to contain an abundance of _Bacteria_, _Vibriones_ and _Leptothrix_ filaments.

*No. XXXIX.--Turnip Infusion + 1/20 of Carbolic Acid* after eight days showed no appreciable alteration in appearance,[64] no trace of pellicle or deposit. When examined microscopically, however, the fluid was found to contain some very minute _Bacteria_, though they were by no means abundant.

[64] It had been rendered turbid from the first, by the carbolic acid.

*No. XL.--Hay Infusion* had become quite turbid in twenty-four hours, and several shades lighter in colour. After forty-eight hours the colour of the infusion was still lighter; there was more turbidity, and some sediment. On microscopical examination, the fluid was found to contain an abundance of _Bacteria_, _Vibriones_ and short _Leptothrix_ filaments.

*No. XLI.--Hay Infusion + 1/20 of Carbolic Acid* showed no apparent change[65] after forty-eight hours, and when examined microscopically it revealed no trace of _Bacteria_, or other organisms. The neck of the flask was then again closed. On the twelfth day the fluid had still undergone no change in appearance, and when examined microscopically, it still showed no trace of organisms, though the fluid was--as it had been at the time of the first examination--full of minute, undissolved particles of carbolic acid.

[65] The fluid had been rendered paler and turbid from the first, by the addition of the carbolic acid.

_Fluids boiled for five minutes, and half-filling hermetically Sealed Flasks containing Ordinary Air._

*No. XLII.--Hay Infusion*, after forty-eight hours, showed no change, and continued to remain quite clear and free from deposit until the twelfth day, when it was examined microscopically. No organisms of any kind could be detected.

*No. XLIII.--Hay Infusion + 1/20 part of Carbolic Acid* showed no apparent change[66] for the first five days, though, on the sixth day, a slight deposit was noticed at the bottom of the flask. The deposit had increased, and was well-marked by the twelfth day, when, on microscopical examination, there were found amongst the granular flakes of the deposit, _Torulæ_ of several varieties of size and shape. Many were spherical, others ovoid, or having an elongated oat-like shape: some were of the ordinary colour, and others were brownish in tint. The variety was most striking. No _Bacteria_ were seen, though there were multitudes of active particles which seemed to differ from the minute spherules of undissolved carbolic acid.

[66] The alteration in colour was less marked than in the similar mixture which had not been boiled, though the turbidity was just as obvious.

_Fluids (in vacuo)--boiled for five minutes, and Flasks Sealed during Ebullition._

*No. XLIV.--Turnip Infusion*, in seventy-two hours, showed a slight turbidity, which gradually increased. On the eighth day there was a considerable quantity of flake-like sediment, and some amount of general turbidity. On the thirteenth day the vacuum was found to be still partly preserved. When the flask was opened the fluid was perceived to have a fœtid odour, and an acid re-action; and, on microscopical examination, multitudes of _Bacteria_ and _Vibriones_ were seen. In the flake-like aggregations also (made up almost wholly of these organisms) there were a number of large thick-walled spores; some already formed, and others in process of formation by coalescence.

*No. XLV.--Turnip Infusion + 1/20 part of Carbolic Acid* showed no increase of turbidity[67] for the thirteen days during which it was kept under observation. Before the flask was opened it was ascertained that the vacuum was well preserved. The odour of the fluid was unaltered, and on microscopical examination no _Bacteria_, or other living things, were found.[68]

[67] This fluid was whitish, and somewhat opaque, from the first.

[68] For other experiments showing a similar sterility, induced by a slight acidification with acetic acid, see ‘Nature,’ 1870, No. 37, pp. 226 and 227.

*No. XLVI.--Hay Infusion*, after forty-eight hours, showed no change, though, in seventy-two hours, there was perceptible a very small amount of a dirty greyish deposit. By the fifth day the deposit had slightly increased, and on the seventh day there was a trace of turbidity in the fluid. It did not undergo much further change, so that, on the twelfth day, the flask was opened. The vacuum was found to have been very slightly impaired; the odour of the fluid was almost natural, and its re-action was slightly acid. On microscopical examination of the deposit, _Bacteria_, _Vibriones_, short _Leptothrix_ filaments, and _Torulæ_, were found, though not in very great abundance.

*No. XLVII.--Hay Infusion + 1/20 part of Carbolic Acid* showed no apparent change for the first four days. On the fifth day there was a small quantity of powder-like sediment, and one dirty greyish-coloured flake. On the seventh day there were more small flakes at the bottom, and a slight general turbidity of the fluid. On the twelfth day, the turbidity and deposit having increased, the flask was opened--after it had been first ascertained that the vacuum had only been slightly impaired. The re-action of the fluid was still strongly acid. On microscopical examination of some of the deposit, there was found, amongst granular flakes and aggregations, a large number of _Torulæ_ cells, of most various shapes and sizes; also in the midst of the granule heaps many large, rounded or ovoidal, densely granular nucleated bodies, whose average size was 1/1500″ in diameter, though there were many of them much larger, and others even less than half this size. Intertwined amongst the granular matter also were a large number of algoid-looking filaments, 1/20000 in diameter, containing segmented protoplasmic contents. There were also in the fluid itself a number of medium-size, unsegmented _Bacteria_, whose movements were somewhat languid.[69]

[69] The results of this experiment, and of No. LXIII. are decidedly opposed to the reality of the germ-killing powers with which carbolic acid has been endowed by Professor Lister and others. I, however, had previously found that specimens of _Torulæ_ and _Bacteria_, obtained from freshly opened flasks, and then mounted as microscopical specimens in a mixture of glycerine and carbolic acid (in the proportion of 15:1), not unfrequently grew and multiplied under such conditions. MM. Béchamp and Estor, also found that _Bacteria_ multiplied in carbolized fluids, and similar facts have been testified to by some Italian observers. But, organic fluids differ much from one another, so that the influence of carbolic acid may well be different upon different fluids. And, accordingly, we find that whilst its addition to, and subsequent boiling with, a hay infusion increases the fermentability of this, precisely the opposite effects are produced when the hay is replaced by a turnip infusion (see No. XLV.). Without wishing to undervalue in the least the system of treatment introduced, and so admirably carried out by Professor Lister, I am strongly of opinion that he explains his results by theories which are almost wholly incorrect.

SIXTH SET OF EXPERIMENTS (XLVIII.–LXV.).

_Ammoniacal Solutions, unboiled, and exposed to Ordinary Air in a Corked Bottle._[70] (_Temp. 60°–65° F._)

[70] All the simple ammoniacal solutions were in the proportion of ten grains of the salt to the fluid ounce of distilled water; and to those which also contained sodic phosphate, three grains of this were added. About half an ounce of each solution was put into a one-ounce wide-mouthed bottle, and then tightly corked.

*No. XLVIII.--Ammonic Acetate Solution.*--On the tenth day the fluid was still quite clear, and free from sediment.

*No. XLIX.--Ammonic Oxalate Solution.*--On the tenth day there was no distinct opalescence of the fluid, but a well-marked whitish flocculent deposit. On microscopical examination no _Bacteria_ were found in the fluid, and the deposit was made up by an aggregation of blackish and colourless granules, mixed with a few crystals and a very few _Torula_ cells--all being held together by a sort of mucoid matrix. In the midst of this matter were found two or three very small, much branched, mycelial tufts of a fungus-growth.

*No. L.--Ammonic Carbonate Solution.*--On the tenth day the fluid showed a very faint opalescence, with a small amount of deposit, and a partial non-coherent scum on the surface, which, on microscopical examination, was found to be composed partly of amorphous granules, and partly of minute _Bacteria_, mixed with small necklace-like organisms. The fluid itself contained, in suspension, a few small and sluggish _Bacteria_, with a minute _Torula_ cell here and there.

*No. LI.--Ammonic Tartrate Solution* after twenty-four hours showed the faintest opalescence of the fluid; in forty-eight hours there was a bluish-white turbidity, and in seventy-two hours the turbidity was well marked. When examined microscopically the fluid was found to contain multitudes of very active _Bacteria_. On the thirteenth day the turbidity was not so well marked, though there was a very thin pellicle on the surface, and also the dirty-looking crumpled remains of another pellicle at the bottom, which, on examination, was found to be composed of an aggregation of _Bacteria_. The pellicle on the surface was very thin, and composed only of a single layer of _Bacteria_. In the fluid itself many _Bacteria_ were seen, of medium size, and mostly sluggish in movement, though a few of them exhibited very active rotatory movements. No _Vibriones_, _Leptothrix_, or _Torulæ_, were found.

*No. LII.--Ammonic Tartrate and Sodic Phosphate Solution* after twenty-four hours showed the faintest opalescence; in forty-eight hours there was a bluish-white turbidity, which, in seventy-two hours, had become more marked. When examined microscopically multitudes of _Bacteria_ were found whose movements were very sluggish. On the thirteenth day there was a well-marked whitish turbidity, due to _Bacteria_ and _Vibriones_, a slight amount of deposit, and a firm pellicle which was found to be composed, almost wholly, of long unjointed _Vibriones_ and unsegmented _Leptothrix_ filaments, all of which, when separate, exhibited the most distinct eel-like movements, accompanied by an actual progression from place to place.

_Ammoniacal Solutions, unboiled, and exposed to Air in a Corked Bottle, after Inoculation with a Drop of Fluid containing living_ BACTERIA_and_ TORULÆ. (_Temp._ 60°–65° F.)

*No. LIII.--Ammonic Acetate Solution* after twenty-four hours was faintly opalescent, and in forty-eight hours showed a very slight bluish tint. In seventy-two hours it was in the same state, and, on microscopical examination, the fluid showed no distinct _Bacteria_ or other living things, though there were a number of very minute particles distributed, singly or in small groups, throughout the fluid. On the thirteenth day there was no change in appearance, except that the sediment had somewhat increased in amount. Still, no _Bacteria_ could be found in the fluid or the sediment,--only the above-mentioned particles, and a few somewhat larger, which resembled very minute _Torulæ_. Amongst the sediment, however, there were two or three very small mycelial tufts of a developing fungus.

*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]

[71] On comparing the corresponding experiments of series XLVIII.-LI. with those of series LIII.-LVI. less difference is found than might have been expected by many. The comparison of the numbers of each series with one another, also reveals the interesting fact, that the mere presence of N, C, O, and H, is not all that is required, even for the growth and nutrition of the lower living things. These elements seem to lapse into the new combinations constituting living matter of various kinds, more easily from certain pre-existing states of combination than from others. Solutions of ammonic tartrate are much more favourable starting points for the new combinations than solutions of ammonic acetate. The comparison of experiment No. LI. with No. LII. is extremely interesting in reference to the dogma that phosphorus is a necessary ingredient in living matter. Solutions of the ammonic tartrate in distilled water have been twice analyzed for me by a skilled chemist, without revealing the least trace either of phosphorus or sulphur. This result is very remarkable when compared with the amount of living matter which may so soon appear in such a solution: the number of the organisms and the rapidity of their evolution, being almost equal to that which occurs in a similar solution to which a phosphate has been added. However much, therefore, phosphorus may aid the development of organisms in many fluids, there is still an important difference between _many_ and _all_, which if more frequently borne in mind, would render universal propositions more scarce (see ‘Journal of Chemical Society,’ March, 1871, pp. 72–74). The truth of the dictum “_Ohne Phosphor gar kein Leben_,” is, I venture to think, far from being proved. If on insufficient evidence (referring only to particular fluids) such a dictum is arrived at; and if then, the presence of organisms in any fluid is to be taken as evidence of the existence of phosphorus (even though this cannot be otherwise substantiated), the case of phosphorus in relation to Life comes to be similar to the case of the much abused germs.

Mutato nomine, de te
Fabula narratur.

_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.)._

[72] The fluids were boiled at the low temperature, with the aid of an air-pump, merely in order to be able to procure a more perfect vacuum in the flasks; these experiments being destined to show whether the simple (uninoculated) solutions would become turbid _in vacuo_--that is to say, without the oxidizing influence of the air--when they had not been exposed to an amount of heat sufficient to destroy any living or dead ferments which they might contain.

*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_.

[73] A deposit of this kind is almost invariably found in such solutions after their fermentability has been lowered by previous boiling. Growth takes place very slowly in these cases, and also when similar boiled fluids are contained _in vacuo_.

_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.

[74] Loc. cit. p. 71.

[75] Loc. cit. pp. 75 and 76.

[76] Loc. cit. pp. 83 and 84.

[77] The subsidence of the atmospheric particles has been ably demonstrated by Professor Tyndall.--See ‘Nature,’ 1870.

[78] See M. Pouchet’s ‘Nouvelles Expériences sur la Génération Spontanée,’ &c., p. 69.

[79] See various communications in ‘Compt. Rend.’ (1863), t. LVII.

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]

[80] Loc. cit., p. 40.

[81] Speaking of experiments in closed flasks, in which the air has been either calcined or filtered, Gerhardt (‘Chimie Organique,’ t. IV. p. 545) says:--“Si dans les premières expériences l’air calciné ou tamisé s’est montré beaucoup moins actif que l’air non soumis à ce traitement, c’est que la chaleur rouge ou le tamisage enlève à l’air non seulement les germes des infusoires et des moisissures, mais encore les débris des matières en décomposition qui y sont suspendues, c’est-à-dire les ferments dont l’activité viendrait s’ajouter à celle de l’oxygène de l’air.”

[82] On what other supposition can one explain the results of experiments LVII.-LXV., and of others alluded to on p. 100?

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]

[83] See notes on pp. 73 and 79. It was not that these fluids were incapable of being inoculated, or that they were unsuitable for the development of the lower forms of life, as was shown by their subsequent fate, and by the fact that they can always speedily be made to become turbid if they are really inoculated with living _Bacteria_. Almost similar facts in opposition to the prevalent Panspermic views have been noted by Professor Cantoni (Rend. del R. Istit. Lombardo, Novembre, 1869). He found (as I have also frequently found) that when fluids had been subjected to the influence of high temperatures, and had subsequently remained sterile in closed flasks, they might be freely exposed to the air for one or two weeks, or more, without becoming turbid--although at any time a general turbidity could be speedily induced, by introducing a few living _Bacteria_ into the fluid.

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.

[84] ‘On the Direct Correlation of Mechanical and Chemical Forces.’--Proceedings of the Royal Society, vol. xii. pp. 539 and 546.