The fate of bacteria when frozen excited the curiosity of investigators already in the early years of bacteriology, for in 1871 we find Burdon Sanderson recording the fact that water which he had obtained from the purest ice contained microzymes, or, as we now prefer to call them, micro-organisms.

It is quite possible that at the time this announcement was made it may have been received with some scepticism, for it was undoubtedly difficult to believe that such minute and primitive forms of vegetable life, seemingly so scantily equipped for the struggle for existence, should be able to withstand conditions to which vegetable life in more exalted circles so frequently and lamentably succumbs.

The tormented agriculturist realises only too well what havoc is followed by a return in May to that season

Again, with what solicitude those of us who have gardens wait to see what will have survived the iron grip of winter in our favourite flower borders, and how frequently we have to face blanks in the ranks of some of its most cherished occupants! Numerous bacteriologists, however, have now confirmed this fact, the fields of ice and snow have been repeatedly explored for micro-organisms, and it has been shown how even the ice on the summit of Mont Blanc has its complement of bacterial flora, that hailstones as they descend upon the earth contain bacteria, that snow, the emblem of purity, is but a whited sepulchre, and will on demand deliver up its bacterial hosts. Quite apart from its general scientific interest, the bacterial occupation of ice is of importance from a hygienic point of view, and a large number of examinations of ice as supplied for consumption have been made. Thus, Professor Fraenkl and also Dr. Heyroth have submitted the ice-supply of the city of Berlin to an exhaustive bacteriological examination. These investigations showed that the bacterial population of ice as supplied to Berlin is a very variable one, and fluctuates between great extremes, rising to as many as 25,000 bacteria in a cubic centimetre (about twenty drops) of ice-water, and falling to as few as two in the same measure.

There are numerous circumstances which come into play in determining the density of the bacterial population in ice. First, of course, the initial quality of the water from which the ice is derived is a factor of great importance, for the purer the water the fewer will be the bacteria found in the resulting ice.

Again, if the ice field is wind-swept by air bearing an unduly rich complement of bacteria, as may be expected in the vicinity of populous cities, for example, then the ice will reflect in its bacterial contents the undesirable neighbourhood in which it was produced. Water in repose, again, yields purer ice than water in movement during freezing, for during rest opportunity is given for the bacteria present in suspension to subside, the process of sedimentation or deposition of bacteria which takes place under these conditions playing an important part in water-purification; when, however, the water is disturbed by swift currents, or agitated by storms, this process is interrupted, and the bacteria become entangled in the ice and frozen in situ.

The importance attaching to the physical conditions under which ice is produced in enabling an estimate to be formed of the safety or otherwise of the same for consumption may be gathered from the following extract from an American report on the subject:—

We have learnt that ice contains bacteria, that its bacterial contents are to a certain extent dependent upon the bacterial quality of the water before crystallisation, and that an important factor in determining its purity is afforded by the physical conditions prevailing at the time of freezing.

It will be of interest to ascertain in more detail what effect the process of freezing has upon the number of bacteria present in the water—what is the degree of bacterial purification effected during the conversion of water into ice.

Now Professor Uffreduzzi, in his investigations on the ice-supply of Turin, part of which is derived from a much-polluted portion of the River Dora, found that about 90 per cent. less bacteria were present in the ice than were present in the water from which it was produced. In the making of ice, therefore, a remarkable removal of bacteria may be effected which approaches very nearly the degree of bacterial purification which is achieved during the best-conducted sand-filtration of water.

Uffreduzzi's results have been repeatedly confirmed by other researches. Thus, in regard to ice obtained from the River Merrimac, water which contained originally about 38,600 bacteria per cubic centimetre, on its conversion into ice had only from three to six. Sewage, again, containing about a million and a half bacteria per cubic centimetre after being frozen only contained under 74,000. It should be mentioned that this last figure represented the number of bacteria obtained by thawing the outside of the sewage ice-cake; inside the cake there were more found—about 121,000. The difference in these figures is due to the fact that, whereas the outer layers of ice looked quite clear, towards the centre the ice contained sewage sludge and hence more bacteria had become arrested; but in spite of this the bacterial purification effected is very striking, although not sufficient to render the use of ice from such a polluted source either palatable or desirable.

It is, of course, a well-known fact that water possesses the power of purifying itself during its transformation into ice, and that the process of crystallisation not only prevents a considerable proportion of the matters in suspension from becoming embodied in the ice, but also eliminates a large percentage of the matters in solution, the latter being driven from the water which is being frozen into the water beneath. If, therefore, ice in the act of forming can get rid of matters in solution, it is not difficult to understand how it can eject bacteria, which though so minute are yet bodies of appreciable dimension and in suspension. But that there are limits to this power of excluding bacteria, and that, as in the case of other mechanical processes, an overtaxing of the available resources is at once reflected in the inferiority of the product, is shown by the frozen sewage experiment, in which the ice, having had too large a supply of bacteria in the first instance to deal with, was unable to get rid of more than a certain proportion, and was obliged to retain a very considerable number. Hence great as is the degree of purification achieved by ice in forming, yet it must be recognised that its powers in this direction are limited, and that the fact of water being frozen does not necessarily convert a bad water into immaculate ice.

It is worthy of note that the city of Lawrence, in Massachusetts, obtains the greater portion of its ice from a river which in its raw, unpurified condition was rejected for purposes of water-supply in consequence of the numerous and severe epidemics of typhoid fever which accompanied its use. Since the application of sand-filtration to this water, however, the death-rate from typhoid in this city, instead of being abnormally high, has fallen abnormally low, and this improvement is attributed to the excellent quality of the water supplied to the city, and has taken place despite the use which still continues of ice from the polluted river. The authorities consider the city's immunity from typhoid amply justifies their sanctioning the distribution of this river-ice, the freezing of the water having rendered it sufficiently pure to remove all danger to health from its consumption.

So far we have been considering the effect on bacteria of freezing carried on under more or less natural conditions; but much interesting work of a more detailed character has been carried out with reference to the behaviour of particular varieties of micro-organisms when frozen under more or less artificial conditions.

Thus Dr. Prudden froze various bacteria in water at temperatures ranging from -1° C. to -10° C., and he found that different varieties were very differently affected by this treatment; that, for example, a bacillus originally obtained from water, and introduced in such numbers as represented by 800,000 individuals being present in every twenty drops, after four days' freezing had entirely disappeared, not one having survived. On the other hand, similar experiments in which the typhoid bacillus was used resulted in the latter not only enduring a freezing of four days' duration, but emerging triumphant after it had been carried on for more than 103 days!

In these experiments it should be borne in mind that, as the ice was frozen to a solid block or lump, there was no opportunity for the mechanical committal of the bacteria during freezing to the water beneath; all the bacteria present were imprisoned in the ice, and the fact that the typhoid bacteria were not destroyed by being frozen shows that they can withstand exposure to such low temperatures, although, as we have seen, the other variety of bacillus employed was destroyed.

Dr. Prudden, however, discovered an ingenious method by which even typhoid bacilli were compelled to succumb when frozen. In the course of his investigations he found that bacteria which had offered the stoutest resistance under freezing were extremely sensitive to this treatment if the process was carried on intermittently, or, in order words, if the temperature surrounding them was alternately lowered and raised.

In this manner the bacteria may be said to be subjected to a succession of cold shocks, instead of being permitted to remain in a continuously benumbed condition. The vitality of typhoid bacilli was put to the test under these circumstances, the freezing process being carried on over twenty-four hours, during which time, however, it was three times interrupted by the ice being thawed. The effect on the typhoid bacteria was striking in the extreme; from there being about 40,000 present in every twenty drops, representing the number originally put into the water, there were only ninety at the end of the twenty-four hours; and after a further period of three days, during which this treatment was repeated, not a single bacillus could be found. This signal surrender to scientific tactics forms a marked contrast to the stout resistance maintained for over 103 days under the ordinary methods of attack.

But, although the typhoid bacillus appears to submit and meekly succumb to this plan of campaign, yet the conclusion must not be rashly drawn that all descriptions of bacteria will be equally feeble and helpless in these circumstances.

Doctors Percy Frankland and Templeman have shown that the spore form of the anthrax bacillus is able to successfully challenge all such attempts upon its vitality. Thus when put into water and frozen at a temperature of -20° C., the process being extended over a period of three months and interrupted no fewer than twenty-nine times by thawings, when examined even after this severe series of shocks, it showed no signs of submission and clung to life as tenaciously as ever.

The more sensitive form of anthrax, however, the bacillus, was readily destroyed; for after one freezing its numbers were already so much reduced that it was only with difficulty that even one or two could be found, and after the second freezing every one out of the large number originally present had died.

Renewed interest has been of late revived in the question of the behaviour of bacteria at low temperatures, in consequence of the possibility of obtaining, by means of liquid air and liquid hydrogen, degrees of cold which were undreamt of by the scientific philosophers of fifty years ago. Public interest has also been quickened in such inquiries on account of the important part which low temperatures play in many great commercial developments, their application rendering possible the transport from and to all parts of the world of valuable but perishable foodstuffs, thus encouraging local industries by opening up markets, and bringing prosperity to countries and communities which before were seeking in vain an outlet for their surplus produce.

The application of cold storage for preservation purposes is, however, no novelty; for nature ages ago set us the example, and of this we have been lately reminded afresh by the discovery announced by Dr. Herz of a mammoth in Siberia, which, despite the thousands of years which have elapsed since it was originally overwhelmed and frozen, is described as being in a marvellous state of preservation.

Thus we are told that "most of the hair on the body had been scraped away by ice, but its mane and near foreleg were in perfect preservation and covered with long hair. The hair of the mane was from four to five inches long, and of a yellowish brown colour, while its left leg was covered with black hair. In its stomach was found a quantity of undigested food, and on its tongue was the herbage which it had been eating when it died. This was quite green."

Considering that certainly more than eight thousand years had elapsed since this creature was peacefully consuming what proved to be its last meal, nature's method of cold storage must indeed be regarded as unsurpassable in the excellence of its results.

I believe it was in the year 1884 that the first attempts were made to follow more closely and in greater detail the precise effect upon different bacteria of submitting them to temperatures of such a low degree as -130° C., obtained by means of solid carbonic acid. These experiments were carried out by Pictet and Young, and are recorded in the Comptes Rendus of the Paris Academy of Sciences.

They differ from those which we have so far been considering, inasmuch as the bacteria were not frozen in water, but in culture-material, or, in other words, like the mammoth, whilst enjoying a midday meal!

One of the micro-organisms experimented with was a bacillus known at that time as the rinderpest bacillus, capable of producing disease in animals when inoculated into them and existing both in the spore and bacillar form. Pictet and Young specially state that the spore form was present in the specimens employed by them, and hence the fact that this micro-organism was alive after being frozen and exposed to this low temperature of -130° C. for the space of twenty hours is not, perhaps, so surprising when we bear in mind the remarkable feats of endurance exhibited by spores which have with justification obtained for them a prominent place amongst the so-called curiosities of bacteriology. But of more interest than their mere survival in these circumstances is the fact that, on being restored to animation—or, in other words, released from their ice-prison—these bacteria were discovered to have retained all their pathogenic properties, this period of enforced rigidity having in no way affected their disease-producing powers.

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.