The modes of origin of lowest organisms / including a discussion of the experiments of M. Pasteur

by H. Charlton Bastian

In respect of the movements which they may exhibit, therefore, really living, though languid, Bacteria, cannot always be discriminated from dead Bacteria. Both may only display mere Brownian movements.

It becomes obvious, then, that in doubtful cases we ought not to rely very strongly upon the character of their movements, as evidence of the death of Bacteria—although these may frequently be of so extensive a nature as to render it not at all doubtful whether the Bacteria which display them are living. In the experiments which I am about to relate, we shall be able to pronounce that the Bacteria are living or dead, by reference to the continuance or cessation of their most essentially vital characteristic. If Bacteria fail to multiply in a suitable fluid, and under suitable conditions, we have the best proof that can be obtained of their death.

Having made many experiments with solutions of ammonic tartrate and sodic phosphate, I have almost invariably observed that such solutions—when exposed to the air without having been boiled—become turbid in the course of a few days owing to the presence of myriads of Bacteria and Vibriones, with some Torulæ. These organisms seem to appear and multiply in such a solution almost as readily as they do in an organic infusion. On the other hand, having frequently boiled such solutions, and closed the flasks during ebullition, I have invariably found, on subsequent examination of these fluids, that whatever else may have been met with, Bacteria and Vibriones were always absent. The difference was most notable, and it seemed only intelligible on the supposition that any living Bacteria or dead ferments which may have pre-existed in the solution, were deprived of their virtues by the preliminary boiling. These experiments also seemed to show that such solutions, after having been boiled, and shut up in hermetically-sealed flasks, from which all air had been expelled, were quite incapable of giving birth to Bacteria. The unboiled fluid, exposed to the air, might have become turbid, because it was able to nourish any living Bacteria which it may have contained, or because it was capable of evolving these de novo, under the influence of dead ferments whose activity had not been destroyed by heat. Hence we have a fluid which is eminently suitable for testing the vital resistance of Bacteria,—one which, although quite capable of nourishing and favouring their reproduction, does not appear capable of evolving them, when, after previous ebullition, it is enclosed in a hermetically sealed flask from which all air has been expelled. Three flasks were half-filled with this solution.[32] The neck of the first (a) was allowed to remain open, and no addition was made to the fluid. To the second (b), after it had been boiled and had become cool, was added half a minim of a similar saline solution, which had been previously exposed to the air, and which was quite turbid with Bacteria, Vibriones and Torulæ. From this flask—after its inoculation with the living organisms—the air was exhausted by means of an air-pump, and its neck was hermetically sealed during the ebullition of the fluid, without the flask and its contents having been exposed to a heat of more than 90° F. The third flask (c) was similarly inoculated with living Bacteria, although its contents were boiled for ten minutes (at 212° F.), and its neck was hermetically sealed during ebullition. The results were as follows:—the solution in the first flask (a), became turbid in four or five days; the solution in the second (b), became turbid after thirty-six hours; whilst that in the third flask (c), remained perfectly clear. This latter flask was opened on the twelfth day, whilst its contents were still clear, and on microscopical examination of the fluid no living Bacteria were to be found. This particular experiment was repeated three times with similarly negative results, although on two occasions the fluid was only boiled for one minute instead of ten.

It seemed, moreover, that by having recourse to experiments of the same kind, the exact degree of heat, which is fatal to Bacteria and Torulæ might be ascertained. I accordingly endeavoured to determine this point. Portions of the same saline solution, after having been boiled[33] and allowed to cool, were similarly inoculated with a drop[34] of very turbid fluid, containing hundreds of living Bacteria, Vibriones, and Torulæ. A drying apparatus was fixed to an air-pump, and the flask containing the inoculated fluid was securely connected with the former by means of a piece of tight india-rubber tubing,[35] after its neck had been drawn out and narrowed, at about two inches from the extremity. The flask containing the inoculated fluid was then allowed to dip into a beaker holding water at 122° F., in which a thermometer was immersed. The temperature of the fluid was maintained at this point for fifteen minutes,[36] by means of a spirit lamp beneath the beaker. The air was then exhausted from the flask by means of the pump, till the fluid began to boil; ebullition was allowed to continue for a minute or two, so as to expel as much air as possible from the flask, and then, during its continuance, the narrowed neck of the flask was hermetically sealed by means of a spirit-lamp flame and a blowpipe. Other flasks were similarly prepared, except that they were exposed to successively higher degrees of heat—the fluid being boiled off, in different cases, at temperatures of 131°, 140°, 149°, 158°, and 167° F. All the flasks being similarly inoculated with living Bacteria, Vibriones, and Torulæ, and similarly sealed during ebullition, they differed from one another only in respect to the degree of heat to which they had been submitted. Their bulbs were subsequently placed in a water bath, which during both day and night was maintained at a temperature of from 85° to 95° F. The results have been as follows:—The flasks whose contents had been heated to 122° and 131° F. respectively, began to exhibit a bluish tinge in the contained fluid after the first or second day; and after two or three more days, the fluid in each became quite turbid and opaque, owing to the presence and multiplication of myriads of Bacteria, Vibriones and Torulæ; the fluids in the flasks, however, which had been exposed to the higher temperature of 140°, 149°, 158°, and 167° F., showed not the slightest trace of turbidity, and no diminution in the clearness of the fluid while they were kept under observation—that is, for a period of twelve or fourteen days. One kind of conclusion only is to be drawn from these experiments, the conditions of which were in every way similar, except as regards the degree of heat to which the inoculated fluids were subjected—seeing that the organisms were contained in a fluid, which had been proved to be eminently suitable for their growth and multiplication.[37] If inoculated fluids which have been raised to 122° and 131° F. for ten minutes, are found in the course of a few days to become turbid, then, obviously, the organisms cannot have been killed by such exposure; whilst, if similar fluids, similarly inoculated, which have been raised to temperatures of 140°, 149°, 158°, and 167° F. remain sterile, such sterility can only be explained by the supposition that the organisms have been killed by exposure to these temperatures.

Some of these experiments have been repeated several times with the same results. On three occasions, I have found the fluid speedily become turbid, which had only been exposed to 131° F. for ten minutes, whilst on three other occasions I have found the inoculated fluid remain clear, after it had been exposed to a heat of 140° F. for ten minutes.[38]

In experimenting upon rather higher organisms, with which there is little difficulty in ascertaining, by microscopical examination, whether they are living or dead, I have found that an exposure even to the lower temperature of 131° F. for five minutes, always suffices to destroy all signs of life in Vibrios, Amœbæ, Monads, Chlamydomonads, Euglenæ, Desmids, Vorticellæ, and all other Ciliated Infusoria which were observed, as well as in free Nematoids, Rotifers, and other organisms contained in the fluids which had been heated.

These results are quite in harmony with the observations and experiments of M. Pouchet and of Professor Wyman, as to the capability of resisting heat displayed by Vibriones and all kinds of ciliated infusoria. According to the former,[39] the majority of ciliated infusoria are killed at, or even below, the temperature of 122° F., whilst large Vibriones are all killed at a temperature of 131° F.[40] According to the observations of Professor Wyman, the motions of all ciliated infusoria are stopped at less than 130° F., whilst Vibriones, taken from the most various sources, also seemed to be killed at temperatures between 130°–136.4° F. Similarly, we find Baron Liebig quite recently making the following remarks concerning a species of Torula:—“A temperature of 60° C. [140° F.] kills the yeast cells; after exposure to this temperature in water, they no longer undergo fermentation, and do not cause fermentation in a sugar solution. . . . In like manner, active fermentation in a saccharine liquid is stopped when the liquid is heated to 60° C., and it does not recommence again on cooling the liquid.”

That the organisms in question—being minute naked portions of living matter—should be killed by exposure to the influence of a fluid at these temperatures will perhaps not seem very improbable to those who have attempted to keep their fingers for any length of time in water heated to a similar extent. With watch in hand I immersed my fingers in one of the experimental beakers containing water at 131° F., and found that, in spite of my desires, they were hastily withdrawn, after an exposure of less than five-and-twenty seconds.

Wishing to ascertain what difference there would be if the inoculated fluids were exposed for a very long time, instead of for ten minutes only, to certain temperatures, I prepared three flasks in the same manner—each containing some of the previously boiled solution, which, when cold, had been inoculated with living Bacteria, Vibriones, and Torulæ. These flasks and their contents were then submitted to the influence of the following conditions:—One of them was heated for a few minutes in a beaker containing water at 113° F., and then by means of the air-pump a partial vacuum was procured, till the fluid began to boil. After the remainder of the air had been expelled by the ebullition of the fluid, the neck of the flask was hermetically sealed, and the flask itself was subsequently immersed in the water of the beaker, which was kept for four hours at a temperature between 113° and 118 1/2° F.[41] The two other flasks similarly prepared were kept at a temperature of 118 1/2°–127 1/2° F. for four hours. In two days, the fluid in the first flask became slightly turbid, whilst in two days more the turbidity was most marked. The fluid in the two other flasks which had been exposed to the temperature of 118 1/2°–127 1/2° F. for four hours, remained quite clear and unaltered during the twelve days in which they were kept in the warm bath under observation. These experiments seem to show, therefore, that the prolongation of the period of exposure to four hours, suffices to lower the vital resistance to heat of Bacteria and Torulæ by 14 1/2°–18° F.