The modes of origin of lowest organisms

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 - 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 - 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.] Examined microscopically it was found to contain an abundance of Bacteria.

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

No. XXIII. – Urine in eighteen hours showed a distinct pellicle, though there was not much general turbidity. During the next few days the old pellicle fell to the bottom, and a new one formed.

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

No. XXIV. – Urine in forty-eight hours showed no change. After twelve days there was still no general turbidity, though there was a slight flocculent deposit of an uncertain nature. Two days afterwards the flask was broken, when the odour of the fluid was still found to resemble that of fresh urine, and its re-action was acid. The flocculi were made up of granular aggregations, in the midst of which were a few bodies closely resembling Torulæ, though they were somewhat doubtful in nature. Neither Bacteria nor Vibriones could be found. The flask, having a short open neck, was then replaced in the warm bath. In sixteen hours the whole fluid had become turbid; it was also slightly fœtid; and on microscopical examination it was found to be swarming with Bacteria, Vibriones, and Leptothrix.

No. XXV. – Turnip Infusion + 1/2 gr. of Cheese in forty-eight hours showed no change, though in seventy-two hours there was a well-marked pellicle, in which some bubbles of gas were engaged. After ninety-six hours the neck of the flask was broken; the fluid was found to be fœtid, and it had an acid re-action. On microscopical examination, a portion of the pellicle was seen to consist of multitudes of Bacteria, Vibriones, and jointed Leptothrix filaments.

No. XXVI. – Simple Turnip Infusion remained clear, and showed no appreciable change for seven days. On the eighth day a slight general turbidity of the fluid was noticed. On the ninth, the turbidity was rather more marked, though there was no trace of a pellicle; the neck of the flask having been broken, the fluid was found to be odourless and very faintly acid. On microscopical examination, multitudes of languid Bacteria of medium size were found, and also short monilated chains with from two to ten segments. There were no Vibriones, Leptothrix or Torulæ.[57 - 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 - 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 - Experiment No. 8, recorded in ‘Nature,’ 1870, No. 36, p. 194, may be compared with this and No. XXXIII.] 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 - 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 - 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.] 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 - 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 - Some of same as that which was used (unaltered) in last experiment.] 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.

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

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

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

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 - This fluid was whitish, and somewhat opaque, from the first.] 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 - 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 - 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 - 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.] (Temp. 60°–65° F.)

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.