Popular Science Monthly/Volume 9/May 1876/Air-Germs and Spontaneous Generation

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AIR-GERMS AND SPONTANEOUS GENERATION.[1]
By P. SCHÜTZENBERGER.

The question of the origin of ferments is intimately connected with that of spontaneous generation. In fact, from the time of Van Helmont and others, who, even in the seventeenth century, gave directions for the production of mice, frogs, eels, etc., the partisans of this mode of generation have, by the progress of the tendency to examine into the causes of things, been driven from the larger animals or plants visible to the naked eye, to the smallest living productions, which we can observe only by the aid of the microscope. But ferments are found among these inferior microscopic organisms. Redi, a member of the Academy of Cimento, showed that the worms in putrefied flesh, which were at first thought to be of spontaneous origin, are only the larvæ from the eggs of flies, and that all that was necessary, to prevent entirely the birth of these larvæ, was to surround the decomposing meat with fine gauze; he was the first to ascertain that parasitic animals are sexual and able to lay eggs.

The invention of the microscope, and the numerous observations by which it was followed, toward the end of the seventeenth, and the commencement of the eighteenth century, gave fresh impulse to the doctrine of spontaneous generation, which had lost all credit in questions concerning the origin of living beings of a higher order.

The question now was how to explain the origin of the various living productions, revealed by the microscope in infusions of vegetable and animal substances, among which no apparent symptom of sexual generation could then be found.

The subject was studied for the first time in a scientific manner by Needham, who published, in 1745, in London, a work on this subject. This observer did for infusoria what had already been done for the higher organisms. He protected, or rather endeavored to protect, vegetable or animal infusions from the action of germs, seeds, or any other agents of multiplication which could come from without. At the same time he destroyed by a physical agent, heat, the germs which might be supposed to exist beforehand in the liquid. Under these conditions, either living beings will be produced in the midst of the infusion, or none will be found there; in the former case, it must be admitted that these organisms are developed in the medium which is suitable to them, without the intervention of any germ; in the second, that the doctrine of spontaneous generation is false. In reality, the question can only be resolved in this manner, and all experimenters who have entered upon it from Needham's time to the present day ought to have made use of it.

The serious and grave difficulty, on which, during this period, all discussions raised between heterogenists and panspermists have turned, is so to arrange the experiments as to remove every suspicion of the intervention of germs brought from without, or preexisting in the liquid.

If the result is negative, if when all precautions that seem to be necessary have been taken, and all causes of error have been removed, there is no formation of infusoria, it will be difficult to raise any serious objection to the inevitable conclusion, provided that the methods employed for the purpose of eliminating the preëxisting germs are not of such a nature as to modify the medium, and to render it unfit for the development and the nutrition of living organisms. If, on the contrary, we still meet with the birth of living beings, the suspicion will always revive that the experiment has been badly performed, and that a contrary result would have been obtained by conducting it more carefully. The heterogenists, therefore, find themselves in a more disadvantageous situation than their opponents, and, notwithstanding the success which they may obtain, they will never convince them.

We think, therefore, that it is useless to give here a detailed account of their minute researches; they must be consulted in the original memoirs. A single experiment which proves, by a negative result, that organic infusions, protected from germs from without, do not give birth to infusoria, is worth more, scientifically speaking, than ten experiments tending to establish the contrary opinion.

If, therefore, we pass over the details of the fundamental experiments of the heterogenists, and speak of those the results of which are conformable to the ideas of the panspermists, it will not be in a spirit of partiality. We are convinced that the latter are the only ones free from all objections, the relative skill of the operators being disregarded, and considered as nothing in the estimate formed. We may, however, say that M. Pasteur's researches may serve as a model for all those who may wish to conduct investigations of this kind, whatever may be the preconceived opinion by which they are guided. By their precision, and the care taken to remove every source of error, they leave nothing to be desired.

As the results obtained by M. Pasteur lead him to deny spontaneous generation, his opponents must above all prove that he is mistaken, by adopting the same rigorous experimental conditions. Needham's experiments, which led him to admit and sustain the doctrine of spontaneous generation, consisted essentially in placing organic substances which were capable of decomposition, in vessels hermetically sealed, which were subsequently submitted to a high temperature, in order to destroy the preexisting germs. The work of the English writer attracted great notice on account of the support of Buffon, whose ideas he upheld. Soon after began the great controversy between Needham and Spallanzani, who refuted, by experiment, the conclusions arrived at by Needham.

The controversy turned principally on this point: Spallanzani was not satisfied with heating the hermetically-sealed vessels containing the infusions, for several minutes, merely the time which is required to cook a hen's-egg, and to destroy the germs, as Needham expresses it, but he kept them for the space of an hour in boiling water. He then observed no production of infusoria. But, objects the English observer, from the manner in which he treated and put to the torture his nineteen vegetable infusions, it is evident that he not only much weakened, or perhaps totally destroyed, the vegetative force of the substances infused, but also entirely corrupted, by the exhalations and the odor of the fire, the small portion of air which remained in the empty part of his vessels. It is not, therefore, surprising that his infusions, thus treated, gave no signs of life. Such must necessarily have been the case. This idea, that the action of the temperature of boiling water destroys the vegetative force of infusions, is maintained even at the present day, and has served as an argument to the heterogenists; as they were unable to attack the material correctness of Pasteur's experiments, they did not accept the conclusions which he sought to derive from them.

We find also in the passage just cited, the necessity for the experiments made by Schwann and Helmholtz on calcined air, and for those of Schröder and F. Dusch, on strained air. The objection of a possible change in the air contained in the vial, under the influence of prolonged boiling, in presence of organic substances, was a serious one at the time that it was brought forward; it becomes more so, when we know that the air confined over preserved meats, prepared by Appert's process, contains no oxygen. It was, therefore, absolutely necessary to place the infusions in contact with air in a normal condition, after that boiling had deprived them of their preëxisting germs, avoiding at the same time any new germs brought by the air.

For this purpose, Dr. Schwann heated flasks containing the infusions, until the destruction of the germs was insured; but his flask was not closed: it communicated freely with the surrounding air by mean of a glass tube bent in the form of a U, and heated, in one part of its length, by means of a bath of fusible alloy. Under these conditions, the air may be renewed in the flasks, but the fresh atmospheric air admitted has undergone, like the infusion, the action of heat, which destroys the germs. Schwann's experiment was very decisive, as to broth made from meat; and the negative result (no development of infusoria) was quite satisfactory. But it was not the same with analogous trials on alcoholic fermentation, which gave contradictory results. Ure and Helmholtz repeated and multiplied these experiments with the same success.

To obviate the objection of a possible change by heat, in a mysterious and undefined principle, different from germs, but whose presence in the air was necessary to the production of infusoria, Schultze caused the renewed air to pass through energetic chemical reagents, such as concentrated sulphuric acid. He half filled a glass vessel with distilled water containing various animal and vegetable substances; then stopped the vessel with a cork through which passed two bent tubes, and exposed the apparatus thus arranged to the temperature of boiling water. Then, while the vapor was still escaping through the tubes, he adapted to each of them a Liebig's bulb apparatus, one containing concentrated sulphuric acid, and the other concentrated caustic potash. The high temperature must necessarily have destroyed every living thing, all the germs that might happen to be in the inside of the vessel, or of its appendages, and the communication from without was intercepted by the sulphuric acid on one side and the potassa on the other. Nevertheless, it was easy to renew, by aspiration at the end of the apparatus which contained the potassa, the air thus inclosed, and the fresh quantities of this fluid which were introduced could not carry with them any living germ, for they were forced to pass through a bath of concentrated sulphuric acid. M. Schultze placed the apparatus thus arranged at a well-lighted window, side by side with an open vessel, which contained an infusion of the same organic substances; then he was careful to renew the air in his apparatus several times a day for more than two months, and to examine with the microscope what took place in the infusion. The open vessel was soon found filled with vibrios and monads, to which were soon added polygastric infusoria of a larger size, and even rotifers; but by the most attentive observation he could not discover the least trace of infusoria, confervæ, or mildews, in the infusion contained in the apparatus.

The latest researches of Schröder and Von Dusch (1854-1859) tended to raise another objection, the possible change in a special principle in the air, by a reagent as energetic as sulphuric acid. Guided by the experiments of Loëwel, who ascertained that common air, when it had been previously filtered through cotton, was unfit to cause the crystallization of supersaturated solutions of sodium sulphate, they placed one of the tubes of Schultze's apparatus in communication with a tube 1.18 inch in diameter, and from 19.68 to 23.62 inches in length, tilled with cotton-wool. The other tube was connected with an aspirator.

When the liquid, the interior of the flask, and the tubes, had been deprived of air by boiling, the apparatus was removed to its place, and the aspiration continued night and day. The two observers thus proved that meat, to which water had been added, the wort of beer, urine, starch, paste, and the various materials of milk taken separately, remained intact in the filtered air. On the contrary, milk, meat without water, and the yolk of egg, grew putrid as rapidly as in common air.

The result of these experiments is, that there are spontaneous decompositions of organic substances which require nothing but the presence of oxygen gas to cause them to commence; while others need, besides oxygen, the presence in the atmospheric air of those unknown things, which are destroyed by heat or sulphuric acid, or are retained by the cotton.

The two observers do not then decide on the nature of these things, and do not assert categorically that they are germs, and, in reality, nothing allows us to draw these conclusions.

M. Pasteur's experiments have advanced the question another step, by proving that they are really germs of ferments and infusoria, which are destroyed by heat, or arrested by the sulphuric acid or cotton in the experiments alluded to above.

M. Pasteur made a hole in a window-shutter, several metres above the ground, and through this he passed a glass tube .196 inch in diameter, and containing a plug of soluble cotton .39 inch in length, kept in its place by a spiral platinum wire. One of the ends of this tube passed into the street; the other communicated with a continuous aspirator. When the air had passed for a sufficient time, the plug of cotton, more or less soiled by the dust which it had intercepted, was placed in a small tube with the mixture of alcohol and ether, which dissolves gun-cotton. It was left for the space of a day. All the dust was deposited at the bottom of the tube, where it is easy to wash it by decantation, without any loss, if care is taken to separate each washing by an interval of repose of from twelve to twenty hours. The deposit, and the liquid which covers it, are put in a watch-glass together; after the evaporation of the alcohol, the remainder is placed in water, and examined with the microscope. M. Pasteur also made use of ordinary sulphuric acid in order to moisten the dust. This agent had the effect of separating the grains of starch and calcium carbonate, which are always found in greater or less quantities in deposits collected on the plug of cotton.

PSM V09 D113 Corpuscles of dust.jpg
Figs. 1 and 2.—Organic Corpuscles of Dust, mixed with Amorphous Particles.

Figs. 1 and 2 represent organic corpuscles, associated with amorphous particles, as seen through the microscope, under a power of 350 diameters; the liquid containing them was common sulphuric acid.

Fig. 1 applies to dust collected from the 25th to the 26th of June, 1860; Fig. 2 to dust from the very intense fog of January, 1861.

It was not enough to discover with the microscope organic particles mixed with amorphous substances, but it was necessary to prove that these particles really consisted of fertile germs, capable of producing the infusoria which are developed in such abundance in organic liquids exposed to the air. For this purpose, M. Pasteur arranged the experiment in the following manner:

Into a flask capable of containing from 15 to 18 cubic inches, he introduced 6 to 9 cubic inches of albuminous saccharine water, prepared in the following proportions:

Water, 100;
Sugar, 10;
Albuminoid and mineral matter from beer-yeast, .2 to .7.

The neck of the drawn-out neck-flask communicated with a platinum tube, as shown in Fig. 3. In this first stage of the experiment the T-shaped tube with three stopcocks is removed, and its place supplied by a simple India-rubber connecting-piece. The platinum tube is raised to a red heat by means of a small gas-furnace. The liquid is boiled for two or three minutes, and is then allowed to grow completely cold. It is filled with common air, at the ordinary pressure of the atmosphere, but which has been wholly exposed to a red heat; then the neck of the flask is hermetically sealed.

This, being thus prepared and detached, is placed in a stove at a constant temperature of about 86° Fahr.; it may be kept there for any length of time without the least change in the liquid which it contains. It preserves its limpidity, its smell, and its weak acid reaction; even a very slight absorption of oxygen is mainly to be observed. M. Pasteur affirms that he never had a single experiment, which was arranged as described above, which yielded a doubtful result; while water of yeast mixed with sugar, and boiled for two or three minutes, and then exposed to the air, was already in evident process of decomposition in a day or two, and was found to be filled with bacteria and vibrios, or covered with mucors. These experiments are directly opposed to those of Messrs. Pousset, Mantegazzo, Joly, and Musset.

It is therefore clearly proved that sweetened yeast-water, a liquid very liable to be decomposed by the contact of common air, may be preserved for years unaltered when it has been exposed to the action of calcined air, after having been allowed to boil for a few minutes (two or three).[2]

This being determined, M. Pasteur adapted, by means of an India-rubber tube, the closed point of his flask filled with sweetened yeast-water, which had been kept for two or three months in a heated stove, without any development of organisms, to an apparatus arranged like that in Fig. 3.

PSM V09 D115 Pasteur apparatus for germ studies.jpg
Fig. 3.—M. Pasteur's Apparatus For the Introduction of Calcined Air into Flasks Containing Organic Infusion.

The pointed end of the flask passed into a strong glass tube .39 to 46 inch in its inner diameter, within which he had placed a piece of tube of small diameter, open at both ends, free to slip into the larger tube, and inclosing a portion of one of the small plugs of cotton loaded with dust. The larger glass tube is bound to a brass tube in form of a T, furnished with stopcocks, one of which communicates with the air-pump, another with the heated platinum tube, and the third with the flask, by means of the large tube which contains the smaller one with the cotton. These various parts are joined together by means of India-rubber.

The experiment is commenced by exhausting the air, after having closed the stopcock connected with the red-hot metallic tube. This being afterward opened, allows calcined air to enter the tubes slowly; this operation (exhaustion and readmission of calcined air) is repeated several times. The point of the flask is then broken off within the India-rubber, and the small tube containing the dust is allowed to slip into the flask, the neck of which is again sealed with the lamp. As an additional proof, and to obviate all objections, the same arrangements were made with similar flasks, prepared like the preceding, but with this difference that, instead of cotton charged with atmospheric dust, there was substituted a small piece of tube containing calcined asbestos (as an additional precaution, it had been ascertained that calcined asbestos, loaded with atmospheric dust, by the same means as the cotton, gave identical results).

The following are the observations obtained constantly by M. Pasteur:

In all the flasks, into which dust collected from the air was introduced—1. Organic productions began to make their appearance in the liquid after twenty-four, thirty-six, or forty-eight hours at the most. This was precisely the time necessary for the same phenomena to appear in sweetened yeast-water exposed to contact with the atmosphere.

2. The products observed are of the same kind as those which are seen to make their appearance in the liquid when left freely exposed to the air, such as mucors, common mucidines, torulacei, bacteria, and vibrios of the smallest species, the largest of which, the Monas lens, is only .000157 inch in diameter.

When the water of yeast is replaced by urine, the experiment being conducted exactly in the same manner, we always notice the absence of any change as long as atmospheric dust has not been introduced, while, with the addition of this, numerous organisms are developed, in every respect similar to those which appear and are developed in urine kept in the open air. If, on the contrary, the experiment be repeated with common milk, we may be sure that it will in every case curdle, and become putrid. We shall observe the birth of numerous vibrios of the same species, and bacteria, and the oxygen of the flask will disappear. M. Pasteur thinks that this result, so different from those observed in other liquids, arises only from the fact that milk contains germs of vibrios which resist the boiling heat of water. To prove this, he boiled milk, not at 212° Fahr., or at the usual pressure of the atmosphere, but at 230° Fahr., under a greater pressure, and he found that the flasks thus prepared, and hermetically sealed, could be kept for an indefinite time in the stove, without giving rise to the smallest production of mould or infusoria. The milk preserves its taste, its smell, and all its properties; and the atmosphere of the flask is only slightly modified in its composition. This difference between

PSM V09 D117 Pasteur apparatus for germ resistance study.jpg
Fig. 4.—M. Pasteur's Apparatus for Studying the Resistance of Germs and Spores to the Temperatures more or less Elevated.

milk and urine, or sweetened yeast-water, must be attributed to the alkaline condition of the former medium, whereas the two others are acid. In fact, if we previously neutralize the acid of the sweetened yeast-water, by means of calcium carbonate, we obtain organisms under the same conditions of the experiment as those under which they were not before developed.

These facts led M. Pasteur to make researches on the comparative action of temperature on the fecundity of the spores of the mucidines, and of the germs which exist suspended in the atmosphere.

The following is, in few words, the method followed by him: He passed a small portion of asbestos over the small heads of the moulds which he wished to study; he then placed this asbestos, covered with spores, in a small glass tube, which he introduced into a U-tube (Fig. 4) of larger diameter, in which the smaller tube could move freely; one of the extremities of the U-tube is joined by India-rubber to a metal tube in form of a T, with stopcocks. One of these cocks communicated with the air-pump, another with a red-hot platinum tube. The other extremity has an India-rubber tube which is connected with the flask into which the spores are to be introduced; this flask is hermetically sealed, and has been filled with calcined air, and suitable nutritious liquid previously raised to the boiling-point. Finally, the U-tube dips into a bath of oil, of common water, or saltwater, according to the temperature which we wish to attain. Between' the U-tube and that of platinum, there is a drying-tube with sulphuric pumice-stone. When all the apparatus which precedes the platinum tube has been tilled with calcined air, and the spores have been maintained at the desired temperature for a sufficient time, which maybe varied at pleasure, the point of the flask is broken with a blow of a hammer, without unfastening the India-rubber connecting-pieces which attach the flask to the U-tube; then inclining to a proper angle this latter tube, when removed from its bath, the asbestos with its spores is slipped into the flask. The flask is then hermetically sealed, and is carried to the stove at 68° to 86° Fahr. The experiment with the dust from the air is also made in the same manner with asbestos.

Without any humidity, the fecundity of the spores of Penecillium glaucum is preserved up to 248° Fahr., and even a little above—257° Fahr. It is the same with the spores of the other common mucidines. At 266° Fahr., the power of developing or multiplying is destroyed in all of them. The limits are the same for the dust from the air.

In all these careful experiments, the most scrupulous precautions were taken to prevent the access of the slightest portion of common air. But, say the partisans of heterogenesis, if the smallest portion of common air develops organisms in any infusion whatever, it must necessarily be the case that, if these organisms are not spontaneously generated, there must be germs of a multitude of various productions in this portion of common air, however small it may be; and, if things were so, the ordinary air would be loaded with organic matter, which would form a thick mist in it.

M. Pasteur has shown that there is a great deal of exaggeration in the opinion that even the smallest quantity of air is sufficient to develop multitudes of organisms; that, on the contrary, there is not in the atmosphere a continuous cause of these so-called spontaneous generations; that it is always possible to procure, in any determined place, a limited quantity of common air, having undergone no kind of modification, whether physical or chemical, and nevertheless quite un suited to set up any decomposing action in a liquid eminently putrescible. The method of experimenting is very simple. Into a flask of 15 to 18 cubic inches, 9 cubic inches of a liquid that has a tendency to decomposition are introduced; the neck of the flask is drawn out with the lamp, leaving the point open; then the liquid is boiled till the vapor escaping from the extremity has expelled all the air; at this moment the point of the flask is closed by the lamp, by means of a blowpipe, and it is allowed to grow cool. The flask then contains no air; if we break off the point in any particular place, the air reenters suddenly, carrying into it the germs held in suspension; it is again closed with the lamp, and kept in a stove at a temperature of 68° to 86° Fahr. In the generality of cases, organisms are developed; these organisms are even more varied than if the liquid were freely exposed to the air, which M. Pasteur explains by saying that, in this case, the germs in small number, in a limited volume of air, are not hindered in their development by germs in greater number or more precocious in their fecundity, which are able to occupy the space, and leave no room for them. But it is especially important to notice in the results obtained by this method, what frequently happens many times in each series of trials, that the liquid continues absolutely intact, however long it may have remained in the stove, as if it had been filled with calcined air. This phenomenon is the more striking, and shows itself in more marked proportions, when the air received into the flasks is taken from a greater height. Thus, among twenty flasks opened in the country, eight contained organic productions; out of twenty opened on the Jura, only five contained any; and out of twenty flasks opened at Montanvert, in a rather high wind, blowing from the deepest gorges of the "Glacier des Bois," only one was affected by any change.

PSM V09 D119 Pasteur apparatus for air deprivation.jpg
Fig. 28.—M. Pasteur's Flask to deprive the Air of its Germs.

We may also draw other conclusions from this series of observations. Since the putrescible liquid, which had been previously boiled, and which was contained in the flasks, was filled with organic productions in a great number of instances, after the introduction of a limited quantity of air, the genetic power of the infusions had not been destroyed by the material conditions of the experiments. Besides, this objection, which has been raised ever since the earliest controversies between the heterogenists and the panspermists, has been definitely answered by an experiment made by M. Pasteur; he received in a flask, exhausted and deprived of living germs by the momentary application of a sufficiently high temperature, some blood at the instant that it left the organism, and without allowing this liquid, which is so peculiarly putrescible, to come in contact with air. By permitting air deprived of germs, either by calcination or simple filtration, to enter the flask, and then hermetically sealing it, he found that the blood was preserved for an indefinite period intact, although it had not been exposed to heat.

M. Pasteur has also shown that air may be deprived of germs by its passage through a capillary tube bent upon itself. It is, therefore, sufficient, in most cases, to draw out the neck of the flask so as to form a very long, narrow tube, which is bent in several directions, as, for example, in Fig. 5. When the air originally contained in it has been expelled, and the preëxisting germs killed by prolonged boiling, the flask is allowed to cool slowly.

In closing our account of M. Pasteur's interesting memoir, in which heterogenesis was driven to its last intrenchments, we must add that this learned chemist endeavored to deprive his adversaries of one of their principal arguments. Experiments on spontaneous generation have always been conducted with vegetable or animal infusions; it was supposed by Needham, Buffon, and Pouchet, that organisms were only thus produced at the moment of expiring Nature, when the elements of the beings on which they are developed are entering into new chemical combinations, and are passing fully through the phenomena of fermentation or putrefaction.

In other words, albuminoid matters preserve in some degree a certain reserve of vitality, which would allow them to become organic by contact with oxygen, when the conditions of temperature and humidity are favorable. Starting with the idea that albuminoid substances are only aliments for the germs of infusoria, mucidines, or ferments, M. Pasteur has proved directly that organic substances may be replaced by those which are purely mineral or artificial, or, at least, by substances on which this imaginary vegetative force cannot be supposed to have any influence.

  1. Abridged from "Schützenbergcr on Fermentations," No. XX. of the "International Scientific Series."
  2. M. Pasteur has pointed out a cause of want of success, which has led many experimenters into error; by showing that the mercury of a mercurial trough is a complete receptacle for living organisms, and consequently that all experiments made with such a trough must necessarily induce a development of infusoria.