Bacteria in Daily Life

Such results naturally only served to whet the scientific appetite for more, and the liquefaction of air and of hydrogen placing much lower temperatures at the disposal of investigators, those bacteriologists who were fortunate enough to command a supply were not long in availing themselves of the opportunity thus given them of further testing the vitality of micro-organisms.

Botanists had already shown that exposure to liquid air, which means a temperature of about -190 °C., and to liquid hydrogen, which means a temperature of about -250 °C., did not impair the germination powers of various descriptions of seeds, such as those of musk, wheat, barley, peas, vegetable marrow, and mustard, and that their actual immersion in liquid hydrogen for the space of six hours did not prevent them coming up when sown just as well as ordinary seeds which had not undergone this unique experience; hence the opportunity of submitting other members of the vegetable kingdom to these low temperatures was eagerly sought for by bacteriologists. Dr. Macfadyen found this opportunity in the laboratories of the Royal Institution, and, Professor Dewar having placed a generous supply of liquid air and liquid hydrogen at his disposal, he submitted specimens growing in various culture-materials, such as gelatin, broth, potatoes, etc., of typhoid, diphtheria, cholera, anthrax with spores, and other bacteria, for twenty hours and seven days respectively, to a temperature of about -190 °C. In no instance, however, whether exposed when growing in fluid or solid media, could any impairment of their vitality or the slightest alteration in their structure be observed. Similar results were obtained when liquid hydrogen, or a temperature of about -250 °C., was applied. The question of the retention or otherwise of the disease-producing powers of these bacteria was not investigated, and in this connection much interest attaches to Mr. Swithinbank's investigations on the vitality and virulent properties of that notorious malefactor amongst micro-organisms, the bacillus tuberculosis, when exposed to the temperature of liquid air. The specimens of the consumption bacillus employed were originally obtained from the human subject, and they were exposed for periods varying from six hours to six weeks to -190 °C. In each case the malignant properties of the tubercle bacillus after exposure were tested by their direct inoculation into animals, and the results compared with those which followed similar inoculations made with bacilli which had not been frozen in this manner, but had been grown in ordinary circumstances. In no single case, Mr. Swithinbank tells us, were these frozen tubercle bacilli deprived of their virulence, and the length of exposure, at any rate as far as could be judged after six weeks, appeared to make no difference in this respect. It is true that the pathogenic action of the frozen bacilli appeared to be somewhat retarded – that is, they took rather longer to kill animals than the ordinary unfrozen bacilli – but in every case their inoculation produced the typical tuberculous lesions associated with them.

Of particular interest, however, in view of what has been already discovered about the lethal effect upon bacteria of violent alternations of temperature, are Mr. Swithinbank's observations on the vitality of the tubercle bacillus when exposed to such extreme variations of temperature as are represented by a passage from -190 °C. to that of 15 °C.

The bacillus tuberculosis is admittedly a tough antagonist to deal with, and enjoys an unenviable notoriety for its robust constitution amongst the pathogenic members of the microbial world; hence a knowledge of its behaviour in these trying circumstances, as we now know them to be to bacterial life, becomes of special interest. Great must have been the investigator's satisfaction, then, when he discovered that the vitality of the consumption bacillus had been so seriously impaired by this treatment that its pathogenic properties collapsed, and the animals which were inoculated with these specimens, instead of with the continuously frozen bacilli, suffered no inconvenience, and remained in good health.

But although no appreciable change either in the structure, vitality, or malignant properties of the particular bacteria investigated have been noted as resulting from their exposure to extremely low temperatures, yet there is no doubt that a certain proportion of the individual micro-organisms present – those probably whose constitution is less robust than their more fortunate associates – do succumb under these trying conditions.

This fact has been well brought out by Dr. Belli, of the University of Padua, in the experiments which he made with the fowl-cholera bacillus and the anthrax bacillus in the presence of very low temperatures. Thus he exposed a large number of fowl-cholera bacilli in broth to the temperature of liquid air, as many as 396,000 bacilli being present in every twenty drops of the liquid. After exposing them continuously for nine hours to -190 °C., he had the curiosity, after thawing them, to count how many were left alive, and he found that an enormous mortality had taken place amongst them; for, instead of nearly 400,000 bacilli being present in one cubic centimetre, there were only about 9,000. On the other hand, in the broth tubes kept during that time in ordinary surroundings, the bacilli had flourished remarkably, and had greatly increased in numbers. Thus not only had no multiplication amongst these bacilli taken place, which circumstance is always regarded as indicative of their vital condition – not only, then, had their vitality been arrested – but a very large number of them had been actually destroyed in consequence of this severe treatment; but that the residue were not only alive, but unimpaired in their energies on being restored to animation, was proved by their being able to destroy animals, not having parted with any of their malicious propensities. Dr. Belli carried out similar experiments with the bacilli of anthrax and obtained very similar results. With regard to both these varieties of pathogenic bacteria, he mentions that their action upon animals was not quite so rapid as is characteristic of normal specimens of these micro-organisms, thus confirming the experiments in this direction made with frozen tubercle bacilli.

Not content with the exhibition of their powers of endurance, Dr. Belli determined to make yet another demand upon the vitality of these bacilli. For this purpose he immersed them in the liquid air itself, thus bringing them into direct contact with it, effecting this by lowering into the liquid strips of filter-paper soaked in broth containing these bacilli. But, in spite of remaining for the space of eight hours in these surroundings, they emerged triumphant, exhibiting no modification whatever either in their structure or pathogenic properties.

There are doubtless many other trials yet awaiting bacteria, to which they will most certainly be submitted before the limits of their powers of endurance have been adequately tested, but it is difficult to conceive of a severer strain upon their vital resources than the imposition of the conditions of which the above is but a brief sketch.

The triumphs achieved in this direction by micro-organisms are, however, closely approximated by the remarkable record established, according to the recent researches of Dr. Krause, by typhoid, anthrax, tubercle, and some other bacteria of preserving unimpaired not only their vitality but their virulence after having undergone for a period of twenty hours a pressure of no less than that of 500 atmospheres. When we reflect that a pressure of 500 atmospheres is equal to a pressure of about 7,500 pounds to the square inch, and that the normal pressure under which life is maintained upon this planet is approximately that of fifteen pounds to the square inch, this bacterial victory over physical conditions will be more readily appreciated.

The more intimate becomes our knowledge of bacteria, the more must we marvel at the equipment with which they have been provided for enabling them to maintain themselves in the struggle for existence – a struggle which is as severe and as remorseless in this lowly region as it is in those domains the inhabitants of which have risen to far loftier heights on the great ladder of life.

SOME POISONS AND THEIR PREVENTION

Little did the learned Dutchman Leeuwenhoek dream when, more than two hundred years ago, he recorded, in his Arcana Naturæ, that he had found "viva animalcula" in his saliva, that this, the first beginning of bacteriology, would lead, a couple of centuries later, to the inauguration of a new era in the treatment of disease, in which these so-called animalcula, from being considered as curiosities, would come to be regarded as powers for good and evil of the first importance. Protective inoculation or serum therapy, of which the public have lately heard so much in connection with diphtheria, is the direct outcome of bacterial investigations which during the last two decades have been pursued with such zeal in every part of the globe.

The vast domain of immunity, which until recently was an undiscovered country, is now being bit by bit annexed, and in all directions workers are engaged upon opening up new tracts, in overcoming difficulties, in changing chaos into order.

The problems which surround immunity are of so complex and subtle a character that their mastery is by no means either easy or rapid, and many recondite researches appear at frequent intervals on this subject in foreign and other scientific journals, rendering it a difficult matter to keep pace with the new discoveries and the latest theories.

The interest in this country in toxins and anti-toxins not unnaturally centres round that branch of the subject which deals with diphtheria, this disease having of late years figured so prominently in our mortality tables, whilst the production of diphtheria and other anti-toxic serums has been so finely elaborated abroad that it already constitutes an article of commerce, and doubtless helps to swell the exports of our great continental commercial rival.

In this connection the following statistics, published by Dr. Jalzer, of the Mülhaus Hospital, are of interest regarding the mortality from diphtheria before and after the introduction and application of diphtheria anti-toxin. The death-rate from this disease, writes Dr. Jalzer, which in 1892 and 1893 was fully 50 per cent., fell in 1895 to 38·5 per cent., in 1896 to 28·8 per cent., in 1897 to 16 per cent., to 20 per cent. in 1898, 15·15 per cent. in 1899, and 18·75 per cent. in 1900.

So far the efforts which have been made to mitigate human suffering have attracted most attention; but it will be remembered that Pasteur, before he commenced the study of hydrophobia, had already won his laurels in combating disease in the victory he gained over anthrax, the ravages of which so frequently decimated the herds of the French farmer and robbed him of his well-earned return on his capital and labour.

In summoning the brilliant Director of the German Imperial Board of Health to South Africa to investigate the nature of rinderpest, and, if possible, discover a means of protecting cattle from its onslaught, the Cape Government afforded another opportunity for the scientific study of a disease associated with animals, upon the successful mastery and limitation of which the agricultural prosperity of South Africa is so largely dependent, being as it is one of the most fatal and contagious maladies to which cattle are subject. Apart from the great commercial importance attending Dr. Koch's discovery of a device whereby cattle can be immunised or protected from contracting rinderpest when exposed to its contagion, this discovery is of great scientific interest, inasmuch as it has inaugurated a new departure in methods of immunisation.

The previous methods in vogue for inducing immunity in animals from a particular disease consisted in converting the virus itself into a vaccine, as was done by Pasteur in his classical investigations on anthrax and its prevention; and secondly, the employment of anti-toxic serums, in which the virus is not directly inoculated into the animal to be protected, but in which an intermediary is employed between the virus and its victim. This intermediary, or living machine for the generation of the anti-toxin, is usually a horse, which is artificially trained by being given gradually increasing doses of the virus or toxin, until it ultimately withstands doses which in the first instance would infallibly have killed it. When the animal has arrived at this satisfactory stage or condition of complete immunity, some of its blood is from time to time drawn off, and the serum thus obtained constitutes the anti-toxin which now figures so prominently in modern therapeutics. Besides diphtheria-anti-toxic serum there are also those of tetanus, or lock-jaw, plague, the famous anti-venene serum, about the discovery and preparation of which greater detail is given later on, and many others which are still the subject of experimental inquiry.

Now Koch's method for the compassing of rinderpest differed from both the systems above mentioned, inasmuch as he neither employed artificially weakened cultures of the virus, or an anti-toxic rinderpest-serum; instead he took one of the natural secretions of an animal infected with rinderpest, and by injecting this into a healthy animal it was discovered that the latter, as is the case with a vaccine, suffered only local and temporary discomfort, and acquired pronounced immunity from the active virus. The secretion selected by Dr. Koch and his assistant, Dr. Kolle, for this purpose was the gall, and it might be supposed, from the fact that its inoculation into healthy animals did not communicate the disease, that the rinderpest bacteria were absent from the gall. But this is not so, for Dr. Kolle has succeeded in isolating the latter from the gall of infected animals, and, moreover, has proved them on isolation to possess their full complement of virulence. Further investigations made by Koch and Kolle have shown that the explanation of this seeming anomaly is to be found in the fact that the gall of an animal suffering from rinderpest contains a substance which prevents the migration of the rinderpest bacteria, with which it is associated, from the point of inoculation. Hampered in their movements by the controlling influence of this special substance which has been generated in the gall, the bacteria remain rooted to the spot where they are first situate, and only a passing and exceedingly slight local affection results, which on its departure leaves the animal with an immunity from rinderpest lasting some four months. A number of interesting investigations have not unnaturally been stimulated by this remarkable discovery, and researches on the properties inherent in the gall of healthy animals of various kinds have been recently carried out by Dr. Neufeld, of the Institute for Infectious Diseases in Berlin, which are, however, of a too technical nature to deal with here.

As an illustration of the practical use to which Koch's gall immunisation method may be put in dealing with outbreaks of rinderpest, reference to a recent report furnished by the Health Officer of Shanghai may be of interest. Dr. Arthur Stanley describes the outbreak as follows: —

"A large herd of cattle infected with cattle-plague was brought to Shanghai from the Tanyang district, around the Grand Canal, for export to the allied troops in the north of China. The disease spread to an adjacent dairy, most of the cattle dying. On this dairy becoming infected a police cordon was established round it to prevent ingress and egress of cattle and ingress of persons unconnected with the dairy, while the outside infected herd was removed to an isolated part of the settlement. Having been previously convinced of the futility of police cordons in the prevention of cattle-plague, I was not surprised to find, within a short time, that the disease had spread, by the meeting together of cattle-coolies at a common tea-house, to three other dairies at a distance of a quarter, a half, and two miles from the original source of infection.

"As the animals are not, as a rule, taken away from the immediate vicinity of the dairy, there being no grazing fields, and as neither fodder nor dung is taken from one dairy to another, it is practically certain the infection was carried by the dairy-coolies.

"Immediately on this second series of dairies becoming infected it was resolved to apply the gall immunisation method of Koch as being the means at hand. About 1,500 cubic centimetres were collected from the gall-bladder of a rinderpest animal, and 10 cubic centimetres were injected into the dewlap of each of the twenty remaining cattle in the dairy.

"The injection caused slight local swelling and tenderness, but no constitutional symptoms and no alteration in the milk-supply, an important matter in a dairy. In all sixty-eight cattle were injected with cattle-plague gall. Of these, seventeen were among isolated uninfected herds; the remaining fifty-one belonged to infected herds, and among the latter eleven died of cattle-plague subsequent to the injection."

Dr. Stanley points out that ten of these animals, judging by the time which elapsed after the injection, when they showed the first symptoms of the disease, must have been already infected when the injections were made; the eleventh animal, however, undoubtedly contracted the disease after and in spite of the injection.

"Considering," continues Dr. Stanley, "the usual excessive mortality during an outbreak of this disease, the result may almost be compared to the success of vaccination against small-pox. Three young bullocks, each having received 20 cubic centimetres of cattle-plague gall, were purposely exposed to severe infection. They remained well, while unprotected animals around them died of the disease."

In the domain of immunity there is, however, no more fascinating or interesting story than that which deals with the discovery and elaboration of a cure for snake-bites, a discovery which, while attracting but comparatively little attention in this country, should prove of paramount importance to our fellow-subjects in the great Indian Empire. The significance to India of Professor Calmette's discovery of a specific cure for snake-poison may be gathered, indeed, from the statistics which have been compiled of the number of deaths attributed by Indian officials to this cause alone, amounting, it is said, to some 22,000 annually.

The Pasteur Institute in Paris has despatched many pioneers of science to various quarters of the globe, but perhaps no scientific missionary has produced more fruitful results than has Dr. Calmette. It was while acting in the double official capacity of Médecin de 1st Classe du Corps de Santé des Colonies and Director of the Bacteriological Institute of Saïgon, in Cochin China, in the autumn of 1891, that Calmette first commenced his experiments on the neutralisation of serpent venom in the animal system.

He had, indeed, exceptional opportunities in the matter of serpent venom wherewith to carry out his investigations, for during the rainy season a village in the neighbourhood of Bac-Lieu (Cochin China) had been attacked by a band of most venomous serpents.

These creatures, driven by the floods into the very huts of the natives for shelter, created a terrible panic, and no fewer than forty individuals were bitten by them. The panic was certainly not without justification, for these serpents belonged to the species known as naja tripudians, or cobra de capello, renowned for the deadly nature of their venom, and widely distributed over India, Burmah, Sumatra, Java, Malacca, and Cochin China; but until Calmette set to work to systematically study the nature of this reptile's venom but little precise or reliable information had been obtained as to its character.

The governor of the district gave orders that as many as possible of the reptiles were to be captured alive and forwarded to the Director of the Bacteriological Institute, and a plucky Annanite actually succeeded in securing ninety specimens, which were forwarded in a barrel to Dr. Calmette.

This formidable gift was received with enthusiasm by the director, who realised the importance and scope of the inquiry, which he at once set himself to systematically work out.

Forty of these reptiles arrived alive, and several were at once sacrificed to secure their venom glands. Each gland, resembling both in size and shape a shelled almond, contains about thirty drops of venom, and in this transparent limpid liquid is embodied a toxin of extraordinary strength. It was, of course, necessary in the first instance to ascertain, within as narrow a limit as possible, the exact degree of toxic power inherent in the venom, and to determine, if possible, the precise lethal dose in respect of each variety of animal experimented upon.

A correct calculation of the quantity of venom required in every case was, however, found to be quite impossible, for so virulent is the poison that a single drop of an emulsion produced by pounding up eight glands in 300 grammes of distilled water is sufficient, when introduced into the vein of a rabbit's ear, to kill it in five minutes. All the mammals to which Calmette administered this cobra venom, such as monkeys, dogs, rabbits, guinea-pigs, rats, succumbed more or less quickly, according to the size of the dose.

Small birds and pigeons die very rapidly, but the domestic fowl is more fortunate, being somewhat less susceptible. Frogs also fall a prey to the venom, but they are far more refractory than rabbits, for it takes thirty hours to kill a frog with a dose of venom which would infallibly destroy a rabbit in ten minutes. Toads, curiously, do not enjoy to the same extent this power of resisting its toxic action, for they die more quickly than frogs, whilst it makes short work of lizards and chameleons. Fish form no exception to the rule, and even invertebrates, such as leeches, are killed by minute traces of venom.

Whilst Calmette has found that the venom of different kinds of reptiles exhibits marked differences in its toxic character, he has also discovered that the venom secreted by one and the same serpent varies considerably, according to the length of time the animal has fasted. He describes how he kept a naja haje (Cleopatra's asp) in his laboratory, which during the whole eight months that it lived never took any food whatever, although it was offered the most diverse dainties. On its arrival it was made to bite on a watch-glass, this being one method adopted for collecting the venom; the liquid was at once dried, and 0·7 milligramme was found to kill a rabbit weighing nearly four pounds in four hours. Two months later on, when the venom was again collected, 0·25 milligramme proved a fatal dose. On the death of the animal, at the end of eight months, the venom extracted from the glands was so toxic that it only required 0·1 milligramme to kill a rabbit of about the same weight as the previous one. The same curious fact was noted in the case of a cobra's venom. Another circumstance which appears to control the degree of toxicity inherent in serpent venom is the interval of time which elapses between two successive bites. The longer the interval the more virulent is the venom; and Calmette points out that these observations are in accordance with what has for a long time been known in France with respect to indigenous vipers – that their bites are far more dangerous and far more fatal in the spring, after the winter period of torpor is over, than in the autumn.

Until quite recently it was thought that the only creatures which could resist the fatal action of this poison were serpents, both poisonous and non-poisonous. Calmette was led to this conclusion because, although he inoculated large doses, as much as ten drops, into cobras, they suffered absolutely no inconvenience, and the same results were obtained with harmless snakes. On repeating these experiments, however, and using much larger quantities of venom, Calmette has found that they do ultimately succumb. That their susceptibility in comparison with other animals is very slight, may be gathered from the fact that a lethal dose of venom for reptiles is roughly estimated to amount to as much as three times the quantity of venom normally present in their respective poison glands. These animals, therefore, although very refractory, are not absolutely immune from the action of venom-toxin.

There are, however, other animals which enjoy a relative although not absolute immunity to snake poison, and amongst these may be mentioned swine, hedgehogs, and the mongoose. Swine, it is well known, will greedily devour reptiles, and in some countries they are specially trained up and employed for this purpose. Of particular interest, however, are some experiments which were carried out to test the traditional immunity towards this toxin ascribed to the mongoose. These animals are very useful in sugar plantations, and are largely employed to keep down the serpents and rats with which they abound, for the carnivorous little mongoose is extremely partial to such prey. Attempts have been made by sugar planters to introduce them into Martinique, where they are not found in the wild state, as in the island of Guadeloupe.

Six specimens of the mongoose were forwarded to Calmette from Martinique, and these particular animals, it was stated, had never been set at liberty since they were imported, so that they had had no previous experience of snakes or venom. On arriving at the laboratory, one of these little creatures was placed in a glass cage along with a large cobra. The cobra, at once rising up and dilating its neck, darted with fury upon the mongoose; but the latter, thanks to its extraordinary agility, escaped being caught, and took refuge, stupefied and terrified for the moment, in a corner of the cage. This stunned condition, however, did not last long, for just as the incensed cobra was preparing to make a fresh attack upon its insignificant little victim, the latter, with wide-open mouth, rushed and jumped upon the head of its enemy, viciously bit through its upper jaw, and broke its skull in a few seconds. Thus, although in size but a little larger than a squirrel, this tiny creature was more than a match for a cobra two yards long.

Artificial inoculations of cobra venom into the mongoose fully substantiated all the observed facts as to its remarkable immunity from this poison. A dose sufficient to kill a large rabbit in three hours was absolutely without effect; only when the venom was introduced in quantities amounting to as much as eight milligrammes was it followed by fatal results. Thanks, therefore, to their extraordinary agility and remarkable power of resisting the effects of this lethal toxin, these little animals are able to battle successfully with the most dangerous reptiles.

The rapidity with which serpent venom becomes absorbed by the system is almost incredible, and is well illustrated by the following experiment. A rat was inoculated with venom near the tip of its tail. One minute later the latter was cut off a short distance above the point of inoculation; but this operation was quite unable to save the animal's life, for even in that brief interval the poison had accomplished its fatal work, and a few hours later claimed its victim.

This rapid diffusion of the venom helps to explain the difficulty which is experienced in arresting the course of the poison by local treatment, for its passage is too rapid to permit of its being overtaken by superficial measures of even the most stringent character. But Calmette points out that local precautions are not to be neglected, for although they cannot nullify the action of the venom, they undoubtedly do delay its progress, and thus create a longer interval or respite, during which an opportunity is afforded for administering the anti-toxin. Before, however, passing on to the investigations which have culminated in the production of a specific antidote for this terrible toxin, there are a few more details which Calmette has furnished as to its character which are of interest. Serpent venom is characterised not only by its intensely virulent properties, but also by the tenacity with which it retains them under diverse circumstances. Thus it may be stored up for a whole year, and yet at the end of that time be as active as ever; and even after several years, although its toxic powers are somewhat reduced, it still retains them to a very appreciable extent.

Unlike the bacterial toxins, this venom toxin can stand exposure to considerable temperatures without injury to its activity, and that of the cobra only suffers after it has been submitted to 98 °Centigrade for twenty minutes. Sensitiveness to temperature varies, however, with the snake from which the venom is derived. Thus the venom of the so-called "tiger-snake" of Australia will stand being exposed for ten minutes to from 100° to 102° degrees Centigrade, and its virulence only disappears when this temperature has been applied for twenty minutes. The venom of the "black snake," another Australian variety, loses its toxicity at a temperature of between 99° and 100 °Centigrade; whilst an exposure to only 80 °Centigrade for ten minutes is sufficient in the case of viper venom, according to Messrs. Phisalix and Bertrand, to profoundly modify its lethal action. A continuous exposure for a fortnight to a temperature of 38 °Centigrade does not affect cobra venom in the least; but if during that same time it has been placed in the sunshine, it entirely loses all its lethal properties. Thus, a pigeon was inoculated with about thirty drops of venom which had been exposed to the sun's rays for fourteen days, and it survived; whilst another pigeon was inoculated with a little over six drops of similar venom which had been kept during this time in the dark, and it died in a quarter of an hour.