Popular Science Monthly/Volume 58/January 1901/The Effect of Physical Agents on Bacterial Life

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THE EFFECT OF PHYSICAL AGENTS ON BACTERIAL LIFE.[1]
By Dr. ALLAN MACFADYEN,

THE JENNER INSTITUTE OF PREVENTIVE MEDICINE.

THE fact that life did not exist upon the earth at a remote period of time, the possibility of its present existence as well as the prospect of its ultimate extinction, can be traced to the operation of certain physical conditions. These physical conditions upon which the maintenance of life as a whole depends are in their main issues beyond the control of man. We can but study, predict and it may be utilize their effects for our benefit. Life in its individual manifestations is, therefore, conditioned by the physical environment in which it is placed. Life rests on a physical basis, and the main springs of its energies are derived from a larger world outside itself. If these conditions, physical or chemical, are favorable, the functions of life proceed; if unfavorable, they cease—and death ultimately ensues. These factors have been studied and their effects utilized to conserve health or to prevent disease. It is our purpose this evening to study some of the purely physical factors, not in their direct bearing on man, but in relation to much lower forms in the scale of life—forms which constitute in number a family far exceeding that of the human species, and of which we may produce at will in a test-tube, within a few hours, a population equal to that of London. These lowly forms of life—the bacteria—belong to the vegetable kingdom, and each individual is represented by a simple cell.

These forms of life are ubiquitous in the soil, air and water, and are likewise to be met with in intimate association with plants and animals, whose tissues they may likewise invade with injurious or deadly effects. Their study is commonly termed bacteriology—a term frequently regarded as synonymous with a branch of purely medical investigation. It would be a mistake, however, to suppose that bacteriology is solely concerned with the study of the germs of disease. The dangerous microbes are in a hopeless minority in comparison with the number of those which are continually performing varied and most useful functions in the economy of nature. Their wide importance is due to the fact that they insure the resolution and redistribution of dead and effete organic matter, which if allowed to accumulate would speedily render life impossible on the surface of the earth. If medicine ceased to regard the bacteria, their study would still remain of primary importance in relation to many industrial processes in which they play a vital part. It will be seen, therefore, that their biology presents many points of interest to scientific workers generally. Their study as factors that ultimately concern us really began with Pasteur's researches upon fermentation. The subject of this evening's discourse, the effect of physical agents on bacterial life, is important not merely as a purely biological question, though this phase is of considerable interest, but also on account of the facts I have already indicated, viz., that micro-organisms fulfil such an important function in the processes of nature, in industrial operations and in connection with the health of man and animals. It depends largely on the physical conditions to be met with in nature whether they die or remain inactive. Further, the conditions favoring one organism may be fatal to another, or an adaptability may be brought about to unusual conditions for their life. To the technologist the effect of physical agents in this respect is of importance, as a knowledge of their mode of action will guide him to the means to be employed for utilizing the micro-organisms to the best advantage in processes of fermentation. The subject is of peculiar interest to those who are engaged in combating disease, as a knowledge of the physical agents that favor or retard bacterial life will furnish indications for the preventive measures to be adopted. With a suitable soil and an adequate temperature the propagation of bacteria proceeds with great rapidity. If the primary conditions of soil and an adequate temperature are not present, the organisms will not multiply, they remain quiescent or they die. The surface layers of the soil harbor the vast majority of the bacteria, and constitute the great storehouse in nature for these forms of life. They lessen in number in the deeper layers of the soil, and few or none are to be met with at a depth of 8-10 feet. As a matter of fact, the soil is a most efficient bacterial filter, and the majority of the bacteria are retained in its surface layers and are to be met with there. In the surface soil, most bacteria find the necessary physical conditions for their growth, and may be said to exist there under natural conditions. It is in the surface soil that their main scavenging functions are performed. In the deeper layers, the absence of air and the temperature conditions prove inimical to most forms.

Amongst pathogenic bacteria the organisms of lockjaw and of malignant œdema appear to be eminently inhabitants of the soil. As an indication of the richness of the surface soil in bacteria, I may mention that 1 gramme of surface soil may contain from several hundred thousand to as many as several millions of bacteria. The air is poorest in bacteria. The favoring physical conditions to be met with in the soil are not present in the air. Though bacteria are to be met with in the air. they are not multiplying forms, as is the case in the soil. The majority to be met with in air are derived from the soil. Their number lessens when the surface soil is moist, and it increases as the surface soil dries. In a dry season the number of air organisms will tend to increase.

Town air contains more bacteria than country air, whilst they become few and tend to disappear at high levels and on the sea. A shower of rain purifies the air greatly of bacteria. The organisms being, as I stated, mainly derived from the surface of the ground, their number mainly depends on the physical condition of the soil, and this depends on the weather. Bacteria cannot pass independently to the air; they are forcibly transferred to it with dust from various surfaces. The relative bacterial purity of the atmosphere is mainly, therefore, a question of dust. Even when found floating about in the air the bacteria are to be met with in much greater number in the dust that settles on exposed surfaces, e. g., floors, carpets, clothes and furniture. Through a process of sedimentation the lower layers of the air become richer in dust and bacteria, and any disturbance of dust will increase the number of bacteria in the air.

The simple act of breathing does not disseminate disease germs from a patient; it requires an act of coughing to carry them into the air with minute particles of moisture. From the earliest times great weight has been laid upon the danger of infection through air-borne contagia, and with the introduction of antiseptic surgery the endeavor was made to lessen this danger as much as possible by means of the carbolic spray, etc. In the same connection numerous bacteriological examinations of air have been made, with the view of arriving at results of hygienic value. The average number of micro-' organisms present in the air is 500-1000 per 1000 liters; of this number only 100-200 are bacteria, and they are almost entirely harmless forms. The organisms of suppuration have been detected in the air, and the tubercle bacillus in the dust adhering to the walls of rooms. Investigation has not, however, proved air to be one of the important channels of infection. The bactericidal action of sunlight, desiccation and the diluting action of the atmosphere on noxious substances will always greatly lessen the risk of direct aerial infection.

The physical agents that promote the passage of bacteria into the air are inimical to their vitality. Thus, the majority pass into the air not from moist but from dry surfaces, and the preliminary drying is injurious to a large number of bacteria. It follows that if the air is rendered dust-free, it is practically deprived of all the organisms it may contain. As regards enclosed spaces, the stilling of dust and more especially the disinfection of surfaces liable to breed dust or to harbor bacteria, are more important points than air disinfection, and this fact has been recognized in modern surgery. In an investigation, in conjunction with Mr. Lunt, an estimation was arrived at of the ratio existing between the number of dust particles and bacteria in the air. We used Dr. Aitken's dust-counter, which not only renders the air dust particles visible, but gives a means of counting them in a sample of air. In an open suburb of London we found 20,000 dust particles in 1 cubic centimeter of air; in a yard in the center of London about 500,000. The dust contamination we found to be about 900 per cent, greater in the center of London than in a quiet suburb. In the open air of London there was on an average just one organism to every 38,300,000 dust particles present in the air, and in the air of a room, amongst 184,000,000 dust particles, only one organism could be detected.

These figures illustrate forcibly the poverty of the air in microorganisms, even when very dusty, and likewise the enormous dilution they undergo in the atmosphere. Their continued existence is rendered difficult through the influence of desiccation and sunlight. Desiccation is one of nature's favorite methods for getting rid of bacteria. Moisture is necessary for their development and their vital processes, and constitutes about 80 per cent, of their cell-substance. When moisture is withdrawn most bacterial cells, unless they produce resistant forms of the nature of spores, quickly succumb. The organism of cholera air-dried in a thin film dies in three hours. The organisms of diphtheria, typhoid fever and tuberculosis show more resistance, but die in a few weeks or months.

Dust containing tubercle bacilli may be carried about by air currents, and the bacilli in this way transferred from an affected to a healthy individual. It may, however, be said that drying attenuates and kills most of these forms of life in a comparatively short time. The spores of certain bacteria may, on the other hand, live for many years in a dried condition, e. g., the spores of anthrax bacilli, which are so infective for cattle and also for man (wool-sorters' disease). Fortunately few pathogenic bacteria possess spores, and, therefore, drying by checking and destroying their life is a physical agent that plays an important role in the elimination of infectious diseases. This process is aided by the marked bactericidal action of sunlight. Sunlight, which has a remarkable fostering influence on higher plant life, does not exercise the same influence on the bacteria. With few exceptions we must grow them in the dark in order to obtain successful cultures; and a sure way of losing our cultures is to leave them exposed to the light of day. Direct sunlight is the most deadly agent, and kills a large number of organisms in the short space of one to two hours; direct sunlight proves fatal to the typhoid bacillus in half an hour to two hours; in the diphtheria bacillus in half an hour to one hour, and to the tubercle bacillus in a few minutes to several hours. Even anthrax spores are killed by direct light in three and a half hours. Diffuse light is also injurious, though its action is slower. By exposing pigment-producing bacteria to sunlight colorless varieties can be obtained, and virulent bacteria so weakened that they will no longer produce infection. The germicidal action of the sun's rays is most marked at the blue end of the spectrum, at the red end there is little or no germicidal action. It is evident that the continuous daily action of the sun along with desiccation are important physical agents in arresting the further development of the disease germs that are expelled from the body.

It has been shown that sunlight has an important effect in the spontaneous purification of rivers. It is a well-known fact that a river, despite contamination at a given point, may show little or no evidence of this contamination at a point further down in its course. Buchner added to water 100,000 colon bacilli per cubic centimeter, and found that all were dead after one hour's exposure to sunlight. He also found that in a clear lake the bactericidal action of sunlight extended to a depth of about six feet. Sunlight must, therefore, be taken into account as an agent in the purification of waters, in addition to sedimentation, oxidation and the action of algae.

Air or the oxygen it contains has important and opposite effects on the life of bacteria. In 1861, Pasteur described an organism in connection with the butyric acid fermentation which would only grow in the absence of free oxygen. And since then a number of bacteria, showing a like property, have been isolated and described. They are termed anaerobic bacteria, as their growth is hindered or stopped in the presence of air. The majority of the bacteria, however, are aerobic organisms, inasmuch as their growth is dependent upon a free supply of oxygen. There is likewise an intermediate group of organisms, which show an adaptability to either of these conditions, being able to develop with or without free access to oxygen. Preeminent types of this group are to be met with in the digestive tract of animals, and the majority of disease-producing bacteria belong to this adaptive class. When a pigment-producing organism is grown without free oxygen its pigment production is almost always stopped. For anaerobic forms N and H2 give the best atmosphere for their growth, whilst CO2 is not favorable, and may be positively injurious, as, e. g., in the case of the cholera organism.

The physical conditions favoring the presence and multiplication of bacteria in water under natural conditions are a low altitude, warmth, abundance of organic matter and a sluggish or stagnant condition of the water. As regards water-borne infectious diseases, such as typhoid or cholera, their transmission to man by water may be excluded by simple boiling or by an adequate nitration. The freezing of water, whilst stopping the further multiplication of organisms, may conserve the life of disease germs by eliminating the destructive action of commoner competitive forms. Thus the typhoid bacillus may remain frozen in ice for some months without injury. Employment of ordinary cold is not, therefore, a protection against dangerous disease germs.

As regards electricity, there is little or no evidence of its direct action on bacterial life, the effects produced appear to be of an indirect character, due to the development of heat or to the products of electrolysis.

Ozone is a powerful disinfectant, and its introduction into polluted water has a most marked purifying effect. The positive effects of the electric current may, therefore, be traced to the action of the chemical products and of heat. I am not aware that any direct action of the X-rays on bacteria has up to the present been definitely proved.

Mechanical agitation, if slight, may favor, and if excessive, may hinder bacterial development. Violent shaking or concussion may not necessarily prove fatal so long as no mechanical lesion of the bacteria is brought about. If, however, substances likely to produce triturating effects are introduced, a disintegration and death of the cells follows. Thus Rowland, by a very rapid shaking of tubercle bacilli in a steel tube, with quartz sand and hard steel balls, produced their complete disintegration in ten minutes.

Bacteria appear to be very resistant to the action of pressure. At 300-450 atmospheres putrefaction still takes place, and at 600 atmospheres the virulence of the anthrax bacillus remained unimpaired. Of the physical agents that affect bacterial life, temperature is the most important. Temperature profoundly influences the activity of bacteria. It may favor or hinder their growth, or it may put an end to their life. If we regard temperature in the first instance as a favoring agent, very striking differences are to be noted. The bacteria show a most remarkable range of temperature under which their growth is possible, extending from zero to 70° C. If we begin at the bottom of the scale we find organisms in the water and in soil that are capable of growth and development at zero. Amongst these are certain species of phosphorescent bacteria, which continue to emit light even at this low temperature. At the Jenner Institute we have met with organisms growing and developing at 34-40° F. The vast majority of interest to us find, however, the best conditions for their growth from 15° up to 37° C. Each species has a minimum, an optimum and a maximum temperature at which it will develop. It is important in studying any 'given species that the optimum temperature for their development he ascertained, and that this temperature he maintained. In this respect we can distinguish three broad groups. The first group includes those for which the optimum temperature is from 15-20° C. The second group includes the parasitic forms, viz., those which grow in the living body, and for which the optimum temperature is at blood heat, viz., 37° C. We have a third group, for which the optimum temperature lies as high as 50-55° C. On this account this latter group has been termed thermophilic on account of its growth at such abnormally high temperatures—temperatures which are fatal to other forms of life. They have been the subject of personal investigation in conjunction with Dr. Blaxall. We found that there existed-in nature an extensive group of such organisms to which the term thermophilic bacteria was applicable. Their growth and development occurred best at temperatures at which ordinary protoplasm becomes inert or dies. The best growths were always obtained at 55-65° C. Their wide distribution was of a striking nature. They were found by us in river water and mud, in sewage and also in a sample of sea water. They were present in the digestive tract of man and animals, and in the surface and deep layers of the soil, as well as in straw and in all samples of ensilage examined. Their rapid growth at high temperatures was remarkable, the whole surface of the culture medium being frequently overrun in from fifteen to seventeen hours. The organisms examined by us (fourteen forms in all) belonged to the group of the Bacilli. Some were motile, some curdled milk and some liquefied gelatin in virtue of a proteolytic enzyme. The majority possessed reducing powers upon nitrates and decomposed proteid matter. In some instances cane sugar was inverted and starch was diastased. These facts well illustrate the full vitality of the organisms at these high temperatures, whilst all the organisms isolated grew best at 55-65° C. A good growth in a few cases occurred at 72° C. Evidence of growth was obtained even at 74° C. They exhibited a remarkable and unique range of temperature, extending as far as 30° of the Centigrade scale.

As a concluding instance of the activity of these organisms we may cite their action upon cellulose. Cellulose is a substance that is exceedingly difficult to decompose, and is, therefore, used in the laboratory for filtering purposes in the form of Swedish filter paper, on account of its resistance to the action of solvents. We allowed these organisms to act on cellulose at 60° C. The result was that in ten to fourteen days a complete disintegration of the cellulose had taken place, probably in CO2 and marsh gas. The exact conditions that may favor their growth, even if it be slow at subthermophilic temperatures, are not yet known—they may possibly be of a chemical nature.

Organisms may be gradually acclimatized to temperatures that prove unsuited to them under ordinary conditions. Thus the anthrax bacillus, with an optimum temperature for its development of 37° C, may be made to grow at 12° C, and at 42° C. Such anthrax bacilli proved pathogenic for the frog with a temperature of 12° C, and for the pigeon with a temperature of 42° C.

Let us, in a very few words, consider the inimical action of temperature on bacterial life. An organism placed below its minimum temperature ceases to develop, and if grown above its optimum temperature becomes attenuated as regards its virulence, etc., and may eventually die. The boiling point is fatal for non-sporing organisms in a few minutes. The exact thermal death-point varies according to the optimum and maximum temperature for the growth of the organism in question. Thus, for water bacteria with a low optimum temperature, blood heat may be fatal; for pathogenic bacteria developing best at blood heat, a thermophilic temperature may be fatal (60° C); and for thermophilic bacilli any temperature above 75° C. These remarks apply to the bacteria during their multiplying and vegetating phase of life. In their resting or spore stage the organisms are much more resistant to heat. Thus the anthrax organism in its bacillary phase is killed in one minute at 70° C; in its spore stage it resists this temperature for hours, and is only killed after some minutes by boiling. In the soil there are spores of bacteria which require boiling for sixteen hours to ensure their death. These are important points to be remembered in sterilization or disinfection experiments, viz., whether an organism does or does not produce these resistant spores. Most non-sporing forms are killed at 60° C. in a few minutes, but in an air-dry condition a longer time is necessary. Dry heat requires a longer time to act than moist heat: it requires 140° C. for three hours to kill anthrax spores. Dry heat cannot, therefore, be used for ordinary disinfection on account of its destructive action. Moist heat in the form of steam is the most effectual disinfectant, killing anthrax spores at boiling point in a few minutes, whilst a still quicker action is obtained if saturated steam under pressure be used. No spore, however resistant, remains alive after one minute's exposure to steam at 140° C. The varying thermal death-point of organisms and the problems of sterilization cannot be better illustrated than in the case of milk, which is an admirable soil for the growth of a large number of bacteria. The most obvious example of this is the souring and curdling of milk that occurs after it has been standing for some time. This change is mainly due to the lactic acid bacteria, which ferment the milk sugar with the production of acidity.

Another class of bacteria may curdle the milk without souring it in virtue of a rennet-like ferment, whilst a third class precipitate and dissolve the casein of the milk, along with the development of butyric acid. The process whereby milk is submitted to a heat of 65° to 70° C. for twenty minutes is known as pasteurization, and the milk so treated is familiar to us all as pasteurized milk. Whilst the pasteurizing process weeds out the lactic acid bacteria from the milk, a temperature of 100° C. for one hour is necessary to destroy the butyric acid organisms: and even when this has been accomplished there still remain in the milk the spores of organisms which are only killed after a temperature of 100° C. for three to six hours. It will, therefore, be seen that pasteurization produces a partial, not a complete sterilization of the milk as regards its usual bacterial inhabitants. The sterilization to be absolute would require six hours at boiling point. But for all ordinary practical purposes pasteurization is an adequate procedure. All practical hygienic requirements are likewise adequately met by pasteurization, if it is properly carried out, and the milk is subsequently cooled. Milk may carry the infection of diphtheria, cholera, typhoid and scarlet fevers, as well as the tubercle bacillus from a diseased animal to the human subject. For the purpose of rendering the milk innocuous, freezing and the addition of preservatives are inadequate methods of procedure. The one efficient and trustworthy agent we possess is heat. Heat and cold are the agents to be jointly employed in the process, viz., a temperature sufficiently high to be fatal to organisms producing a rapid decomposition of milk, as well as to those which produce disease in man; this is to be followed by a rapid cooling to preserve the fresh flavor and to prevent an increase of the bacteria that still remain alive. The pasteurized process fulfils these requirements.

In conjunction with Dr. Hewlett, I had occasion to investigate in how far the best pasteurizing results might be obtained. We found that 60° to 68° C. applied for twenty minutes weeded out about 90 per cent, of the organisms present in the milk, leaving a 10 per cent, residue of resistant forms. It was found advisable to fix the pasteurizing temperature at 68° C, in order to make certain of killing any pathogenic organisms that may happen to be present. We passed milk in a thin stream through a coil of metal piping, which was heated on its outer surface by water. By regulating the length of the coil, or the size of the tubing, or the rate of flow of the milk, almost any desired temperature could be obtained. The temperature we ultimately fixed at 70° C. The cooling was carried out in similar coils placed in iced water. The thin stream of milk was quickly heated and quickly cooled as it passed through the heated and cooled tubing, and whilst it retained its natural flavor, the apparatus accomplished at 70° C. in thirty seconds a complete pasteurization, instead of in twenty minutes, i. e., about 90 per cent, of the bacteria were killed, whilst the diphtheria, typhoid, tubercle and pus organisms were destroyed in the same remarkably short period of time, viz., thirty seconds. This will serve to illustrate how the physical agent of heat may be employed, as well as the sensitiveness of bacteria to heat when it is adequately employed.

Bacteria are much more sensitive to high than to low temperatures, and it is possible to proceed much further downwards than upwards in the scale of temperature, without impairing their vitality. Some will even multiply at zero, whilst others will remain alive when frozen under ordinary conditions.

I will conclude this discourse by briefly referring to experiments recently made with most remarkable results upon the influence of low temperatures on bacterial life. The experiments were conducted at the suggestion of Sir James Crichton-Browne and Professor Dewar. The necessary facilities were most kindly given at the Royal Institution, and the experiments were conducted under the personal supervision of Professor Dewar. The action of liquid air on bacteria was first tested. A typical series of bacteria was employed for this purpose, possessing varying degrees of resistance to external agents. The bacteria were first simultaneously exposed to the temperature of liquid air for twenty hours (about—190° C). In no instance could any impairment of the vitality of the organisms be detected as regards their growth of functional activities. This was strikingly illustrated in the case of the phosphorescent organisms tested. The cells emit light which is apparently produced by a chemical process of intracellular oxidation, and the phenomenon ceases with the cessation of their activity. These organisms, therefore, furnished a very happy test of the influence of low temperatures on vital phenomena. These organisms when cooled down in liquid air became non-luminous, but on re-thawing the luminosity returned with unimpaired vigor as the cells renewed their activity. The sudden cessation and rapid renewal of the luminous properties of the cells, despite the extreme changes of temperature, was remarkable and striking. In further experiments the organisms were subjected to the temperature of liquid air for seven days. The results were again nil. On re-thawing the organisms renewed their life processes with unimpaired vigor. We had not yet succeeded in reaching the limits of vitality. Professor Dewar kindly afforded the opportunity of submitting the organisms to the temperature of liquid hydrogen—about—250° C. The same series of organisms was employed, and again the result was nil. This temperature is only 21° above that of the absolute zero, a temperature at which, on our present theoretical conceptions, molecular movement ceases and the entire range of chemical and physical activities with which we are acquainted either cease, or, it may be, assume an entirely new role. This temperature again is far below that at which any chemical reaction is known to take place. The fact, then, that life can continue to exist under such conditions affords new ground for reflection as to whether after all life is dependent for its continuance on chemical reactions. We, as biologists, therefore follow with the keenest interest Professor Dewar's heroic attempts to reach the absolute zero of temperature; meanwhile, his success has already led us to reconsider many of the main issues of the problem. And by having afforded us a new realm in which to experiment, Professor Dewar has placed in our hands an agent of investigation from the effective use of which we who are working at the subject at least hope to gain a little further insight into the great mystery of life itself.

  1. Lecture before the Royal Institution of Great Britain.