BACSANYI, JANOS
Hungarian poet, founded a patriotic paper, the Magyar Museum, at Kaschau in 1788 with the assistance of Baroti and Kazinczy. The paper was suppressed in 1792, and in .1794 Bacsanyi was imprisoned for a share in the conspiracy of Bishop Martinovich. He spent two years in the Spielberg. His life was uneventful until 1809 when he translated. Napoleon's proclamation in Vienna. He fled to Paris, was surrendered in 1814, but was allowed to live on parole at Linz. His first work, a patriotic poem, "The Valour of the Magyars" appeared in
He died at Linz, May 12,
BACTERIA AND DISEASE. Bacteria are minute one-celled organisms consisting of spherical, oblong, or cylindrical cells, widely distributed in air, water and soil. The action of bacteria as pathogenic agents is in great part merely an instance of their gen eral action as producers of chemical change, yet bacteriology as a whole has become so extensive, and has so important a bearing on subjects widely different from one another, that division of it has become essential. The science will accordingly be treated in this section from the pathological standpoint only. It will be considered under the two following heads, viz., (I) the methods employed in the study; (2) the modes of action of bacteria and the effects produced by them. The facts and theories with regard to immunity against bacterial disease are dealt with in a separate article (see IMMUNITY).
The demonstration by Pasteur that definite diseases could be produced by bacteria, proved a great stimulus to research in the etiology of infective conditions, and the result was a rapid advance in knowledge. An all-important factor in this remarkable progress was the introduction by Koch of solid culture media, of the "plate-method," etc., an account of which he published in 1881. By means of these the modes of cultivation, and especially of separation, of bacteria were greatly simplified. Various modifications have since been made, but the routine methods in bacteriological procedure still employed are in great part those given by Koch. By 1876 the anthrax bacillus had been obtained in pure culture by Koch, and some other patho genic bacteria had been observed in the tissues, but it was in the decade 188o-90 that the most important discoveries were made in this field. Thus the organisms of suppuration, tubercle, glanders, diphtheria, typhoid fever, cholera, tetanus, and others were identified, and their relationship to the individual diseases established. In the last decade of the 19th century the chief discoveries were of the bacilli of influenza (1892), of plague (1894) and of dysentery (1898). Immunity against diseases caused by bacteria has been the subject of systematic research from 188o onwards. The modes by which bacteria produce their effects also became a subject of study, and attention was naturally turned to their toxic products. The earlier work, notably that of Brieger, chiefly concerned ptomaines (vide infra), but no great advance resulted. A new field of inquiry was, however, opened up when by filtration a bacterium-free toxic fluid was obtained which produced the important symptoms of the disease—in the case of diphtheria by Roux and Yersin (1888), and in the case of tetanus a little later by various observers. Research was thus directed towards ascertaining the nature of the toxic bodies in such a fluid, and Brieger and Fraenkel (189o) found that they were proteids, to which they gave the name "toxalbumins." Though subsequent researches have on the whole confirmed these results, it is still a matter of dispute whether these proteids are the true toxins or merely contain the toxic bodies precipitated along with them.
Immunity against toxins also became a subject of investiga tion, and the result was the discovery of the antitoxic action of the serum of animals immunized against tetanus toxin by Behring and Kitazato (1890), and by Tizzoni and Cattani. A similar result was also obtained in the case of diphtheria, and diphtheria anti toxin was introduced as a therapeutic agent in 1894. The tech nique of serum preparation has become since that time greatly elaborated and improved. The laws of passive immunity were shown to hold also in the case of immunity against living organ isms by Pfeiffer (1894), and various anti-bacterial sera have been introduced. Of these the anti-streptococcic serum of Marmorek and anti-plague serum are the best known. The principles of protective inoculation have been developed and practically applied on a large scale, notably by Haffkine in the case of cholera (1893) and plague (1896), and ,more recently by Wright and Semple in the case of typhoid fever. One other discovery of great importance may be mentioned, viz. the agglutinative action of the serum of a patient suffering from a bacterial disease, first described in typhoid fever independently by Widal and by Griin baum in 1896. Thus a new aid was added to medical science, viz. serum diagnosis of disease.
During the present century one of the most important dis coveries was that of Spirochaeta pallida (or Treponema pal lidum) in syphilis by Schaudinn and Hoffman in 1905. Other important discoveries which have been made in the course of re cent years are as follows:— Acute Infectious Jaundice or Weil's Disease.—This disease, which came into some prominence as a war disease, especially on, the Western Front, was found by Inada and Ido (1914) to be due to a motile spirochaetal organism Leptospira icterohaernor rhagiae. In certain localities it appears to be a parasite in the kidneys of wild rats, and infection of food and water by the urine of such rats is the most likely mode of transmission. A similar organism has recently been found to be the cause of a disease in young dogs called The Yellows.
Noguchi after 1919 brought forward ex cellent evidence that another Leptospira (L. icteroides) is responsi ble for yellow fever (q.v.), and the employment of therapeutic serum, prepared by immunizing horses with this leptospira, has very favourably influenced the course of the disease. Also good reports have been received of the use of prophylactic vaccines in lowering the incidence.
McCoy
in the course of examining ground squirrels in California for evidence of plague infection discovered another plague-like disease in these rodents which was not due to B. pestis. In the following year, he and Chapin isolated the specific organism and it was given the name of B. tularense from Tulare co. in California where the epidemic in the ground squirrels was prevalent. During the years preceding the demonstration of the organism, cases of severe lymphangitis in man apparently following insect bites had been observed in the state of Utah. Since the discovery of the parasite several human cases of infection have occurred in some of the American states. The disease is rarely fatal but a very protracted con valescence is the rule. The mode of infection in the field is almost certainly by the bite of the horse-fly (Clirysops discalis) which has fed on infected ground squirrels or jack rabbits. In the lab oratory, also, workers engaged on research into this disease have been attacked, e.g., at the Washington Hygiene Laboratories and at the Lister Institute, London.
This disease was first described in 1913 by Whit more as a "glanders-like disease" occurring in Rangoon, and indeed the clinical relationship to glanders, both in man and in horses, is very close. Their morbid anatomy also is very similar. It runs a more acute course, however, than glanders, and is usually fatal.
This is a prolonged, febrile disease, of which inflammation of the lymphatics and a well-marked rash on the skin, together with wasting, are prominent symptoms. The course of the disease is very irregular and the fever intermittent. A spirochaete has been described and is generally accepted as the causal micro-organism.
During the World War the occurrence of large numbers of infections of wounds led to a close study of the bacteria responsible for them. The anaerobic bacilli in par ticular were investigated anew, and much confusion in their classi fication and nomenclature cleared up. To three species especially, B. perfringens (B. welchii), Vibrion septique (B. oedematis malign) and B. oedematiens, most of the cases of gas gangrene were found to be due. From these three species, moreover, toxins and antitoxins were prepared which were found to be of service clinically.
The methods employed in studying the relation of bacteria to disease are in principle comparatively simple, but experience and great care are necessary in applying them and in interpreting results. In any given disease there are three chief steps, viz. (1) the discovery of a bacterium in the affected tissues by means of the microscope; (2) the obtaining of the bacterium in pure culture; and (3) the production of the disease by inoculation with a pure culture. By means of micro scopic examination more than one organism may sometimes be observed in the tissues, but one single organism by its constant presence and special relations to the tissue changes can usually be selected as the probable cause of the disease, and attempts towards its cultivation can then be made. Such microscopic ex amination requires the use of the finest lenses and the application of various staining methods. In these latter the basic aniline dyes in solution are almost exclusively used, on account of their special affinity for the bacterial protoplasm.
Sometimes a simple watery solution of the dye is sufficient, but very often the best result is obtained by increasing the stain ing power, e.g., by addition of weak alkali, application of heat, etc., and by using some substance which acts as a mordant and tends to fix the stain to the bacteria. Excess of stain is after wards removed from the tissues by the use of decolorizing agents, such as acids of varying strength and concentration, alcohol, etc. Different bacteria behave very differently to stains; some take them up rapidly, others slowly, some resist decolorization, others
are easily decolorized. Sometimes the stain can be removed from
the tissues, leaving the bacteria alone coloured. (See CYTOLOGY.) Dark Ground Illumination.—Much use has been made of this method of studying bacteria, whether in the live or fixed state. Its chief practical applications have been in the demonstra tion of slender objects such as the Spirochaeta pallida in material from syphilitic sores (see VENEREAL DISEASES), the detection of motility and the enumeration of bacteria in emulsions such as vaccines. To obtain the dark ground effect, the microscope re quires to be fitted with a special form of condenser, and a power ful source of illumination is necessary. The rays from the source of light after reflection at the substage mirror can enter only the peripheral portion of the condenser and, owing to their obliquity, are then either wholly reflected from the upper surface of the cover-glass or, according to the type of condenser, refracted be yond the range of the objective lens. Should, however, small objects come in the way, such as bacilli possessing a different re fractility from that of the fluid, the rays are dispersed by them, and are able to enter the objective. Bacilli then appear as white spots on a dark background.
In cultivating bacteria outside the body vari ous media to serve as food material must be prepared and steril ized by heat. The general principle in their preparation is to supply the nutriment for bacterial growth in a form as nearly similar as possible to that of the natural habitat of the organisms —in the case of pathogenic bacteria, the natural fluids of the body. The media are used either in a fluid or solid condition, the latter being obtained by coagulation, or the addition of a gelatinizing agent, and are placed in glass tubes or flasks plugged with cotton wool. To mention examples, blood serum solidified at a suitable temperature is a highly suitable medium, and various media are made with extract of meat as a basis, with the addition of gela tine or agar as solidifying agents and of non-coagulable proteids (commercial "peptone") to make up for proteids lost by coagu lation in the preparation. The reaction of the media must in every case be carefully attended to, a neutral or slightly alkaline reaction being, as a rule, most suitable ; for delicate work it may be necessary to standardize the reaction by tritration methods. The media from the store-flasks are placed in glass test-tubes or small flasks, protected from contamination by cotton-wool plugs, and are sterilized by heat. For most purposes the solid media are to be preferred, since bacterial growth appears as a discrete mass and accidental contamination can be readily recognized. Cultures are made by transferring by means of a sterile platinum wire a little of the material containing the bacteria to the medium. The tubes, after being thus inoculated, are kept at suitable tem peratures, usually either at 3 7 ° C., the temperature of the body, or at about 2o° C., a warm summer temperature, until growth appears. For maintaining constant temperature, incubators (q.v.) with regulating apparatus are used. Subsequent cultures ("subcul tures") may be made by inoculating fresh tubes, and in this way growth may be maintained often for an indefinite period. The simplest case is that in which only one variety of bacterium is present, and a "pure culture" may then be obtained at once. When, however, several species are present together, means must be adopted for separating them. For this purpose the most impor tant is the plate-method of Koch. In this method the bacteria are distributed in a gelatine or agar medium liquefied by heat, and the medium is then poured out on sterile glass plates or in shal low glass dishes, and allowed to solidify. Each bacterium capable of growth gives rise to a colony visible to the naked eye, and if the colonies are sufficiently apart, an inoculation can be made from any one to a tube of culture-medium, and a pure culture obtained. Of course, in applying the method means must be adopted for suitably diluting the bacterial mixture. Another important method consists in inoculating an animal with some fluid containing the various bacteria. A pathogenic bacterium present may invade the body, and may be obtained in pure cul ture from the internal organs. This method applies especially to pathogenic bacteria whose growth on culture media is slow, e.g., the tubercle bacillus. (See also below.) Isolation and Cultivation of Single Bacterial Cells.—For the cultivation of "pure line" strains of micro-organisms it is essential to start from a single microbe. In Barber's method (1904) a single bacillus in an appropriately diluted emulsion is picked up into an extremely fine capillary pipette working on a special holder under microscopic control. In the method adopted by Burri, dilutions of the culture are mixed with india ink and drops of these are laid on gelatine or agar plates. Under the mi croscope these drops are examined, and if one is found containing a single bacillus it is marked and allowed to grow into colony form, or the single organism may be removed directly to a nutrient medium.
A recent method, elaborated by Barnard, depends on the fact that ultraviolet light destroys bacteria. A single bacillus is marked down, and between it and the entering rays a minute droplet of mercury is placed under microscopic control. The bacillus is thus protected, while the surrounding organisms are killed. It can then be dealt with culturally.
Numerous improvements in cultural methods have been introduced of late years, such as the addition of egg to a nutrient basis of agar, or the use of solid, inspissated mixed white and yolk of egg, especially for the growth of tubercle bacilli. A medium composed of broth and serum, covered with liquid paraffin, and containing a piece of sterile, unheated rabbit's kidney was introduced by Noguchi for the growth of spirochaetes, and has been recommended for other organisms of difficult cul ture.
Anaerobiosis.—Anaerobic culture methods have been im proved by the introduction of various patterns of jars to contain Petri dishes or test-tubes; the object of these is to facilitate the removal of all, or a part, of the oxygen with an air pump, and replace it with hydrogen or other indifferent gas. The removal of the oxygen is also attained by using the catalytic action of spongy platinum (or palladium) to produce slow combination of oxygen with hydrogen. After removing the greater part of the air with a pump and replacing it with hydrogen, the jar is closed and the catalyst contained in a small wire-gauze cage heated by an electric current. The remaining oxygen then combines with hydrogen and this action keeps the platinum hot until the oxygen is used up.
The full description of a particular bacterium implies an ac count not only of its microscopical characters, but also of its growth characters in various culture media, its biological prop erties, and the effects produced in animals by inoculation. To demonstrate readily its action on various substances, certain media have been devised. For example, various sugars—lactose, glucose, saccharose, etc.—are added to test the fermentative action of the bacterium on these substances; litmus is added to show changes in reaction, specially standardized media being used for estimating such changes; peptone solution is commonly em ployed for testing whether or not the bacterium forms indol; sterilized milk is used as a culture medium to determine whether or not it is curdled by the growth. Sometimes a bacterium can be readily recognized from one or two characters, but not infre quently a whole series of tests must be made before the species is determined. As our knowledge has advanced it has become abundantly evident that the so-called pathogenic bacteria are not organisms with special features, but that each is a member of a group of organisms possessing closely allied characters. From the point of view of evolution we may suppose that certain races of a group of bacteria have gradually acquired the power of invading the tissues of the body and producing disease. In the acquisition of pathogenic properties some of their original char acters have become changed, but in many instances this has taken place only to a slight degree, and, furthermore, some of these changes are not of a permanent character. In the case of bacteria we can only judge of organisms being of different species by the stability of the characters which distinguish them, and numerous examples might be given where their characters become modified by comparatively slight change in their environment, The cul tural as well as the microscopical characters of a pathogenic organism may be closely similar to other non-pathogenic mem bers of the same group, and it thus comes to be a matter of ex treme difficulty in certain cases to state what criterion should be used in differentiating varieties. The tests which are applied for this purpose at present are chiefly of two kinds. In the first place, such organisms may be differentiated by the chemical change pro duced by them in various culture media. Thus a very important, and usually very reliable, method of distinguishing between allied races and species of bacteria is that founded on their fermentative action (production of acid or acid and gas) on various carbohy drates and alcohols. Secondly the various serum reactions to be described below have been called into requisition.
In testing the effects of bacteria by inocula tion the smaller rodents, rabbits, guinea-pigs, and mice, are usually employed. One great drawback in certain cases is that such animals are not susceptible to a given bacterium, or that the disease is different in character from that in the human sub ject. In some cases, e.g., Malta fever and relapsing fever, mon keys have been used with success, but in others, e.g., leprosy, none of the lower animals has been found to be susceptible. Dis cretion must therefore be exercised in interpreting negative re sults in the lower animals. For purposes of inoculation young vigorous cultures must be used. The bacteria are mixed with some indifferent fluid, or a fluid culture is employed. The injec tions are made by means of a hypodermic syringe into the sub cutaneous tissue, a vein, one of the serous sacs, or more rarely into some special part of the body. The animal, after injection, must be kept in favourable surroundings, and any resulting symptoms noted. It may die, or may be killed at any time de sired, and then a post-morten examination is made, the condi tions of the organs, etc., being observed and noted.
Though the causal relationship of a bacterium to a disease may be completely established by the methods given, another very important part of bacteriology is concerned with the poisons or toxins formed by bacteria. These toxins may become free in the culture fluid, and the living bac teria may then be got rid of by filtering the fluid. The ef fects of the filtrate are then tested by the methods employed in pharmacology. In other instances the toxins are retained to a large extent within the bacteria, and in this case the dead bac teria are injected as a suspension in fluid. Methods have been introduced for the purpose of breaking up the bodies of bac teria and setting free the intracellular toxins. For this purpose Koch ground up tubercle bacilli in an agate mortar and treated them with distilled water until practically no deposit remained. Rowland and Macfadyen for the same purpose introduced the method of grinding the bacilli in liquid air. At this temperature the bacterial bodies are extremely brittle, and are thus readily broken up. Toxic substances have also been separated by corre sponding methods from the bodies of those who have died of certain diseases, and the action of such substances on animals is in some cases an important point in the pathology of the dis ease.
The fact that in anthrax, one of the first diseases to be fully studied, numerous bacilli are present in the blood of infected animals, gave origin to the idea that the organisms might produce their effect by using up the oxygen of the blood. Such action is now known to be quite sub sidiary. And although effects may sometimes be produced in a mechanical manner by bacteria plugging capillaries of important organs, e.g., brain and kidneys, it is now accepted that all the important results of bacteria in the tissues are due to poisonous bodies or toxins formed by them. Here, just as in the general subject of fermentation, we must inquire whether the bacteria form the substances in question directly or by means of non living ferments or enzymes. With regard to toxin formation the following general statements may be made : In certain instances, e.g., in the case of the tetanus and diphtheria bacilli, the pro duction of soluble toxins can be readily demonstrated by filtering a culture in bouillon germ-free by means of a porcelain filter, and then injecting some of the filtrate into an animal. In this way the characteristic features of the disease can be reproduced. Such toxins being set free in the culture medium are often known as extracellular. In many cases, however, the filtrate, when in jected, produces comparatively little effect, whilst toxic action is observed when the bacteria in a dead condition are used ; this is the case with the organisms of tubercle, cholera, typhoid and many others. The toxins are here manifestly contained within the bodies of the bacteria, i.e., are intracellular, though they may become free on disintegration of the bacteria. The distinction be tween the two varieties, though convenient, must not be pushed too far, as we know little regarding their mode of formation. Although the formation of toxins with characteristic action can be shown by the above methods, yet in some cases little or no toxic action can be demonstrated. This, for example, is the case with the anthrax bacillus; although the effect of this organism in the living body indicates the production of toxins which diffuse for a distance around the bacteria. This and similar facts have suggested that some toxins are only produced in the living body. A considerable amount of work has been done in con nection with this subject, and many observers have found that fluids taken from the living body in which the organisms have been growing, contain toxic substances, to which the name of aggressins has been applied. Fluid containing these aggressins greatly increases the toxic effect of the corresponding bacteria, and may produce death at an earlier stage than ever occurs with the bacteria alone. They also appear to have in certain cases a paralyzing action on phagocytes. Not only are the general symp toms of poisoning in bacterial disease due to toxic substances, but also the tissue changes, many of them of inflammatory nature, in the neighbourhood of the bacteria. Thus diphtheria toxin pro duces inflammatory oedema which may be followed by necrosis; dead tubercle bacilli give rise to a tubercle-like nodule, etc. Furthermore, a bacillus may give rise to more than one toxic body, either as stages in one process of change or as distinct products.
Toxins.—Regarding the chemical nature of toxins less is known than regarding their physiological action. In spite of an enormous amount of work on the subject, no important bacterial toxin has as yet been obtained in a pure condition, and, though many of them are probably of proteid nature, even this cannot be asserted with absolute certainty. Brieger, in his earlier work, found that alkaloids were formed by bacteria in a variety of conditions, and that some of them were poisonous. These alkaloids he called ptomaines. The methods used in the investi gations were, however, open to objection, and it is now recog nized that although organic bases may sometimes be formed, and may be toxic, the important toxins are not of that nature. A later research by Brieger along with Fraenkel pointed to the extracellular toxins of diphtheria, tetanus and other diseases being of proteid nature, and various other observers have arrived at a like conclusion. The general result of such research has been to show that the toxic bodies are, like proteids, precipitable by alcohol and various salts ; they are soluble in water, are some what easily dialysable, and are relatively unstable both to light and heat. Attempts to get a pure toxin by repeated precipitation and solution have resulted in the production of a whitish amor phous powder with highly toxic properties. Such a powder gives a proteid reaction, and is no doubt largely composed of albu moses, hence the name toxalbumoses has been applied. The ques tion has, however, been raised whether the toxin is really itself a proteid, or whether it is not merely carried down with the pre cipitate.
These facts show the great difficulty of the problem, which is probably insoluble by present methods of analysis; the only test, in fact, for the existence of a toxin is its physiological effect. It may also be mentioned that many toxins have now been obtained by growing the particular organism in a proteid-free medium, a fact which shows that if the toxin is a proteid it may be formed synthetically by the bacterium as well as by modification of proteid already present. With regard to the nature of intracellular toxins, there is even greater difficulty in the investigation and still less is known. Many of them probably also of proteid nature, are much more resistant to heat ; thus the intracellular toxins of the tubercle bacillus retain certain of their effects even after ex posure to 100° C. Like the extracellular toxins they may be of remarkable potency; for example, fever is produced in the human subject by the injection into the blood of an extremely minute quantity of dead typhoid bacilli.
We cannot as yet speak definitely with regard to the part played by enzymes in these toxic processes. Certain toxins resemble enzymes in their conditions of precipitation and relative instability, and the fact that in most cases a considerable period intervenes between the time of injection and the occur rence of symptoms has been adduced in support of the view that enzymes are present. In diphtheria Sidney Martin obtained toxic albumoses in the spleen, which he considered were due to the digestive action of an enzyme formed by the bacillus in the membrane and absorbed into the circulation. According to this view, then, a part at least of the directly toxic substance is pro duced in the living body by enzymes present in the so-called toxin obtained from the bacterial culture. Recent researches go to show that enzymes play a greater part in fermentation by living ferments than was formerly supposed, and by analogy it is likely that they are also concerned in the processes of disease. The trend of modern work on this subject is to show that specific enzymes are probably necessary for the first stage in the attack on substances of different chemical or stereochemical constitution, but that the immediate products of this attack are frequently of the same character. These then become changed under the influence of a system of enzymes which is common to a con siderable number of different organisms, yielding final products of a similar nature, but in differing proportions.
Thus, B. coli acts both on mannitol and glucose, probably by the aid of two distinct enzymes, but yields the same final prod ucts from both substances, in different proportions, corresponding to the difference in composition of the two compounds. On the other hand B. lactis aerogenes, which attacks the same two com pounds and produces from them all the products yielded by B. coli, in addition converts a part of one of the intermediate com pounds (probably acetaldehyde) into butylene glycol, which is not produced by B. coli and the formation of which is presumably due to the influence of a specific enzyme.
A bacterial infection when analysed is seen to be of the nature of an intoxication. There is, however, another all-important factor concerned, viz., the multiplication of the living organisms in the tissues; this is essential to, and regu lates, the supply of toxins. It is important that these two essen tial factors should be kept clearly in view, since the means of defence against any disease may depend upon the power either of neutralizing toxins or of killing the organisms producing them. It is to be noted that there is no fixed relation between toxin production and bacterial multiplication in the body, some of the organisms most active as toxin producers having comparatively little power of invading the tissues.
We shall now consider how bacteria may behave when they have gained entrance to the body, what effects may be produced, and what circumstances may mod ify the disease in any particular case. The extreme instance of bacterial invasion is found in some of the septicaemias in the lower animals, e.g., anthrax septicaemia in guinea-pigs, pneumo coccus septicaemia in rabbits. In such diseases the bacteria, when introduced into the subcutaneous tissue, rapidly gain en trance to the blood stream and multiply freely in it, and by means of their toxins cause symptoms of general poisoning. A widespread toxic action is indicated by the lesions found—cloudy swelling, which may be followed by fatty degeneration, in internal organs, capillary haemorrhages, etc. In septicaemia in the human subject, often due to streptococci, the process is similar, but the organisms are found especially in the capillaries of the internal organs and may not be detectable in the peripheral circulation during life. In another class of disease, the organisms first pro duce some well-marked local lesion, from which secondary exten sion takes place by the lymph or blood stream to other parts of the body, where corresponding lesions are formed. In still another class of diseases the bacteria are restricted to some particular part of the body, and the symptoms are due to toxins which are ab sorbed from it. Thus in cholera the bacteria are practically con fined to the intestine, in diphtheria to the region of the false membrane, in tetanus to some wound.
The effects produced by bacteria may be considered under the following heads : (I) tissue changes pro duced in the vicinity of the bacteria, either at the primary or secondary foci; (2) tissue changes produced at a distance by absorption of their toxins; (3) symptoms. The changes in the vicinity of bacteria are to be regarded partly as the direct result of the action of toxins on living cells, and partly as indicating a reaction on the part of the tissues. (Many such changes are usually grouped together under the heading of "inflammation" of varying degree—acute, subacute and chronic.) Degeneration and death of cells, haemorrhages, serous and fibrinous exudations, leucocyte emigration, proliferation of connective tissue and other cells, may be mentioned as some of the fundamental changes. Acute inflammation of various types, suppuration, granulation tissue formation, etc., represent some of the complex resulting processes. The changes produced at a distance by distribution of toxins may be very manifold—cloudy swelling and fatty degener ation, serous effusions, capillary haemorrhages, various degen erations of muscle, hyaline degeneration of small blood-vessels, and, in certain chronic diseases, waxy degeneration, all of which may be widespread, are examples of the effects of toxins, rapid or slow in action.
The lesions mentioned are in many instances necessarily accompanied by functional disturbances or clinical symptoms, varying according to site, and to the nature and de gree of the affection. In addition, however, there occur in bac terial diseases symptoms to which the correlated structural changes have not yet been demonstrated. Amongst these the most important is fever with increased protein metabolism, at tended with disturbances of the circulatory and respiratory sys tems. Nervous symptoms, somnolence, coma, spasms, convul sions and paralysis are of common occurrence. All such phe nomena, however, are likewise due to the disturbance of the molecular constitution of living cells. Alterations in metabolism are found to be associated with some of these, but with others no corresponding physical change can be demonstrated. The action of toxins on various glands, producing diminished or in creased functional activity, has a close analogy to that of certain drugs. In short, if we place aside the outstanding exception of tumour growth, we may say that practically all the important phenomena met with in disease may be experimentally produced by the injection of bacteria or of their toxins.
The result of the entrance of a virulent bac terium into the tissues of an animal is not a disease with hard and fast characters, but varies greatly with circumstances. With regard to the subject of infection the chief factor is suscepti bility; with regard to the bacterium virulence is all-important. Susceptibility, as is well recognized, varies much under natural conditions in different species, in different races of the same species, and amongst individuals of the same race. It also varies with the period of life, young subjects being more susceptible to certain diseases, e.g., diphtheria, than adults. Further, there is the very important factor of acquired susceptibility. It has been experimentally shown that conditions such as fatigue, starvation, exposure to cold, etc., lower the general resisting powers and in crease the susceptibility to bacterial infection. So also the local powers of resistance may be lowered by injury or depressed vitality. In this way conditions formerly believed to be the causes of disease are now recognized as playing their part in pre disposing to the action of the true causal agent, viz. the bacterium. In health the blood and internal tissues are bacterium-free; after death they offer a most suitable pabulum for various bacteria; but between these two extremes lie states of varying liability to infection. It is also probable that in a state of health organisms do gain entrance to the blood from time to time and are rapidly killed off. The circumstances which alter the virulence of bacteria will be referred to again in connection with immunity, but it may be stated here that, as a general rule, the virulence of an organism towards an animal is increased by sojourn in the tissues of that animal. The increase of virulence becomes especially marked when the organism is inoculated from animal to animal in series, the method of passage. This is chiefly to be regarded as an adap tation to surroundings, though the fact that the less virulent members of the bacterial species will be liable to be killed off also plays a part. Conversely, the virulence tends to diminish on cultivation on artificial media outside the body.
During the production of active immunity (see IMMUNITY) certain substances appear in the blood serum of the animal treated, to which the name of antibodies is given, and these have been the subject of extensive study. It is by means of them that immunity (passive) can be transferred to a fresh animal. The development of antibodies is, however, not peculiar to bac teria, but occurs also when alien cells of various kinds, proteins, ferments, etc., are injected. In fact, organic molecules can be divided into two classes according as they give rise to them or fail to do so. Amongst the latter, the vegetable poisons of known constitution, alkaloids, glucosides, etc., are to be placed. The molecules which lead to the production of antibodies are usually known as antigens, and each antigen has a specific combining affinity for its corresponding antibody, fitting it as a lock does a key. The antigens, as already indicated, may occur in bacteria, cells, etc., or they may occur free in a fluid. Antibodies may be arranged into three main groups. In the first group, the antibody simply combines with the antigen, without, so far as we know, producing any change in it. The antitoxins are examples of this variety. In the second group, the antibody, in addition to combin ing with the antigen, produces some recognizable physical change in it; the precipitins and agglutinins may be mentioned as ex amples. In the third group, the antibody, after it has combined with the antigen, leads to the union of a third body called comple ment (alexine or cytase of French writers), which is present in normal serum. As a result of the union of the three substances, a dissolving or digestive action is often to be observed. This is the mode of action in the case of a haemolytic or bacteriolytic serum. So far as bacterial immunity is concerned, the anti-serum exerts its action either on the toxin or on the bacterium itself ; that is, its action is either antitoxic or anti-bacterial.
The antitoxic serum when injected before the toxin confers immunity (passive) against it; when in jected after the toxin it has within certain limits a curative action, though in this case its dose requires to be large. The anti toxic property is developed in a susceptible animal by successive and gradually increasing doses of the toxin. In the earlier experi ments on smaller animals the potency of the toxin was modified for the first injections, but in preparing antitoxin for therapeutical purposes the toxin is used in its unaltered condition, the horse being the animal usually employed. The injections are made subcutaneously and afterwards intravenously; and, while the dose must be gradually increased, care must be taken that this is not done too quickly, otherwise the antitoxic power of the serum may fall and the health of the animal suffer. The serum of the animal is tested from time to time against a known amount of toxin, i.e., is standardized. The unit of antitoxin is the amount requisite to antagonize ioo times the minimum lethal dose of a particular toxin to a guinea-pig of 25o grm. weight, the indication that the toxin has been antagonized being that a fatal result does not follow within five days of ter the injection. In the case of diphtheria the antitoxic power of the serum may reach Boo units per cubic centimetre, or even more.
Two important questions must next be considered, viz., how does the antitoxin act? and how is it formed within the body? It is now accepted that antitoxin acts on toxin directly, i.e., chemically or physically; the chief grounds for this view are as follows : (4) The action of anti toxin on toxin, as tested by neutralization effects, takes place more quickly in concentrated than in weak solutions, and more quickly at a warm (within certain limits) than at a cold temper ature. (b) Antitoxin acts more powerfully when injected along with the toxin than when injected at the same time in another part of the body; if its action were on the tissue-cells one would expect that the site of injection would be immaterial. (c) The law of multiples is obeyed, the amount necessary to neutralize five times the lethal dose being determined, 20 times that amount
will neutralize a hundred times the lethal dose. In the case of
physiological antagonism of drugs this relationship does not hold.
(d) In certain instances the toxin can be made to pass through a gelatine membrane, whereas the antitoxin cannot, its molecules being of larger size. If, however, toxin be mixed with antitoxin for some time, it can no longer be passed through, presumably because it has become combined with the antitoxin. (e) When a toxin has some action which can be demonstrated in a test-tube experiment, for example, a dissolving action on red corpuscles, this action may be annulled by previously adding the antitoxin to toxin; in such a case the intervention of the living tissues is excluded.
Since antitoxin has a direct action on toxin, theoretically this may take place in one of two ways. It may produce a disintegra tion of the toxin molecule, or it may combine with it to produce a body whose combining affinities are satisfied. The latter view, first advocated by Ehrlich, may now be regarded as established.
The origin of antitoxin is of course merely a part of the general question regarding the pro duction of anti-substances in general, as these all combine in the same way with their homologous substances and have the same character of specificity. As, however, most of the work has been done with regard to antitoxin production we may consider here the theoretical aspect of the subject. There are three chief possi bilities: (a) that the antitoxin is a modification of the toxin; (b) that it is a substance normally present, but produced in ex cess under stimulation of the toxin; (c) that it is an entirely new product. The first of these, which would imply a process of a very remarkable nature, is disproved by what is observed after bleeding an animal whose blood contains antitoxin. In such a case it has been shown that, without the introduction of fresh toxin, new antitoxin appears, and therefore must be produced by the living tissues. The second theory is the more probable a priori, and if established removes the necessity for the third. It was strongly supported by Ehrlich, who, in his so-called "side-chain" (Seitenkette) theory, explained antitoxin production as an in stance of regeneration.
It may be added that in the case of all anti-substances, which are produced by a corresponding reaction, we have examples of the existence of traces of them in the blood serum under normal conditions. We are, accordingly, justified in definitely concluding that their appearance in large amount in the blood, as the result of active immunization, represents an increased production of molecules which are already present in the body.
In preparing anti-bacterial sera the lines of procedure correspond to those followed in the case of antitoxins, but the bacteria themselves in the living or dead con dition or their maceration products are always used in injections. Sometimes dead bacteria, living virulent bacteria, and living supervirulent bacteria, are used in succession, the object being to arrive ultimately at a high dosage, though the details vary in different instances. The serum of an animal thus actively im munized has powerful protective properties towards another ani mal, the amount necessary for protection being sometimes almost inconceivably small. As a rule it has no action on the corre sponding toxin, i.e., is not antitoxic. In addition to the protective action, such a serum may possess activities which can be dem onstrated outside the body. Of these the most important are (a) bacteriolytic or lysogenic action, (b) agglutinative action, and (c) opsonic action.
(a) Lysogenic Action.—The first of these, lysogenic or bac teriolytic action, consists in the production of a change in the corresponding bacterium whereby it becomes granular, swells up and ultimately may undergo dissolution. It has been established that in lysogenesis there are two substances concerned, one spe cially developed or developed in excess, and the other present in normal serum. The former (Immunkorper of Ehrlich, substance sensibilisatrice of Bordet) is the more stable, resisting a tem perature of 60° C., and though giving the specific character to the reaction cannot act alone. The latter (complement) is fer ment-like and much more labile than the former, being readily destroyed at 6o° C. Furthermore, lysogenic action is not con fined to the case of bacteria but obtains also with other organized structures, e.g., red corpuscles (Bordet, Ehrlich and Morgenroth), leucocytes and spermatozoa (Metchnikoff). That is to say, if an animal be treated with injections of these bodies, its serum ac quires the power of producing more disintegrative effect in them.
The development of the immune body with specific combining affinity thus presents an analogy to antitoxin production, the dif ference being that in lysogenesis another substance is necessary to complete the process. It can be shown that in many cases when bacteria are injected the serum of the treated animal has no bacteriolytic effect, and still an immune body is present, which leads to the fixation of complement ; in this case bacteriolysis does not occur, because the organism is not susceptible to the action of the complement. In all cases the important action is the binding of complement to the bacterium by means of the corre sponding immune body; whether or not death of the bacterium occurs, will depend upon its susceptibility to the action of the particular complement, the latter acting like a toxin or digestive ferment. In the process of immunization complement does not increase in amount ; accordingly the immune serum comes to con tain immune body much in excess of the necessary amount of complement.
An important point with regard to the therapeutic application of an anti-bacterial serum is that when the serum is kept in vitro the complement rapidly disappears, and accordingly the comple ment necessary for the production of the bactericidal action must be supplied by the blood of the patient treated. This latter com plement may not suit the immune body, that is, may not be fixed to the bacterium by means of it, or if the latter event does occur, may fail to bring about the death of the bacteria. These circum stances serve, in part at least, to explain the fact that the success attending the use of anti-bacterial sera has been much inferior to that in the case of antitoxic sera.
Bacteriolysis may result from other processes than that just described. Thus it may be produced by the action of "lysozyme" (see ANTISEPTICS) or by that of "bacteriophage." This repre sents a newly observed phenomenon, first described by F. W. Twort (i 9 I 5) and F. d'Herelle (i 9 r 8) . The former observed a lytic change accompanied by translucency of some colonies in cer tain cultures of Staphylococcus and found that this lysis could be communicated to other normal colonies by an extract of the affected culture which had been passed through a Berkefeld filter, and was itself free from bacteria. D'Herelle, starting with a Berkefeld filtrate from the faeces of dysentery convales cents, was able by adding a drop of this to a culture of B. dysenteriae (Shiga) to produce lysis of the bacteria. A drop of the resulting solution after filtration could be used to repeat the phenomenon in a fresh culture, and in this way a renewal of the active principle could be brought about indefinitely. This princi ple d'Herelle called the bacteriophage (Bacteriophagum intes tinale), and he maintains that it is an ultramicroscopic, living organism, which is parasitic in the bacteria and reproduces itself. The solution containing the principle is often active in a dilution of i in ioo million, and five c.c. of broth containing bacteriophage in this dilution, if inoculated with the sensitive culture, will re produce in 24 hours in the incubator a clear solution containing the bacteriophage in the original strength, which again is still active when similarly diluted.
If a tube of suitable diluted phage, inoculated with B. dysen teriae, is incubated for three hours, and a loop of it then inoculated on agar, confluent growth of the bacteria may be obtained with a number of small, round, clear areas where no growth of bacteria has occurred. These spaces d'Herelle called tdches vierges and considered them to represent colonies of the bacteriophage which had devoured the bacteria. Other bacteria are attacked besides B. dysenteriae, e.g., B. typhosus, B. paratyp/iosus and B. coli. Most strains of bacteriophage are specific for certain kinds of bacteria, or even for certain strains of these species. Thus some varieties of bacteriophage will act on "rough" variants of B. dys enteriae and on some strains of B. coli, but not on the correspond ing normal or "smooth" cultures.
After the action of the bacteriophage on a broth culture and the resulting clearing of the original turbidity, it is usual for some bacteria to remain alive which, when cultured on agar, are found to be considerably altered in the appearance of their colo nies. Their agglutinability by salts is also changed. These colo nies are often resistant to the bacteriophage. There is no doubt that bacteriophage may be present in small quantities in cultures, but remain unnoticed, and it has been suggested that many vari ations, occurring apparently spontaneously, are in reality brought about by the presence of unrecognized bacteriophage. D'Herelle has advocated the use of bacteriophage in the prevention and treatment of infective disease, but its value for this purpose is not widely recognized.
The question of the nature of the bacteriophage has stimulated a very large amount of research on account of its theoretical im portance. If the more generally accepted view ascribing the prop erties of the bacteriophage to a non-living enzyme or catalyst were proved, the fact that it appears to infect and multiply in a culture of bacteria might by analogy throw light on some infective diseases associated with ultramicroscopic viruses affecting the higher forms of life. The characteristics which make it less prob able that the bacteriophage is a living entity are its ready filtra bility through a porcelain filter, its resistance to heat (6o° to 65° C.), its resistance to acetone and chloroform and its inability to multiply in the absence of a living bacterial culture.
Another property which may be pos sessed by anti-bacterial serum is that of agglutination. By this is meant the aggregation into clumps of the bacteria uniformly distributed in an indifferent fluid; if the bacterium is motile its movement is arrested during the process. The process is of course observed by means of the microscope, but the clumps soon settle in the fluid and ultimately form a sediment, leaving the upper part clear. This change, visible to the naked eye, is called sedimentation. Charrin and Roger first showed in the case of B. pyocyaneus that when a small quantity of the homologous serum (i.e., the serum of an animal immunized against the bac terium) was added to a fluid culture of this bacillus, growth formed a sediment instead of a uniform turbidity. Gruber and Durham showed that sedimentation occurred when a small quan tity of the homologous serum was added to an emulsion of the bacterium in a small test-tube, and found that this obtained in all cases where Pfeiffer's lysogenic action could be demonstrated. Shortly afterwards Widal and also Grunbaum showed that the serum of patients suffering from typhoid fever, even at an early stage of the disease, agglutinates; the typhoid bacillus—a fact which laid the foundation of serum diagnosis. A similar phenom enon has been demonstrated in the case of Malta fever, cholera, plague, infection with B. coli, "meat-poisoning" due to Gart ner's bacillus, and various other infections. As regards the mode of action of agglutinins, Gruber and Durham considered that it consists in a change in the envelopes of the bacteria, by which they swell up and become adhesive. The view has various facts in its support, but Kruse and Nicolle have found that if a bacte rial culture be filtered germ-free, an agglutinating serum still pro duces some change in it, so that particles suspended in it become gathered into clumps. Duclaux, for this reason, considers that agglutinins are coagulative ferments.
The phenomenon of agglutination depends essentially on the union of molecules in the bacteria—the agglutinogens—with the corresponding agglutinins, and is often highly specific. Neverthe less group agglutination occurs and recent work has been devoted largely to dividing apparently identical groups into sub-groups by absorption experiments with specially prepared agglutinating sera. In this way the salmonella, the meningococcal and the dys entery groups have been subdivided into distinct strains. It should also be stated that agglutinins are used up in the process of agglutination, apparently combining with some element of the bacterial structure. In view of all the facts it must be admitted that the agglutinins and immune bodies are the result of corre sponding reactive processes, and are probably related to one an other. The development of all antagonistic substances which confer the special character on antimicrobic sera, as well as anti toxins, may be expressed as the formation of bodies with specific combining affinity for the organic substance introduced into the system—toxin, bacterium, red corpuscle, etc., as the case may be. The facts which have emerged during the recent extensive study of agglutination have given rise to the hypothesis that each bacterium contains a mosaic of antigens, some of which are highly specific and peculiar to each subspecies, some are common to the whole species or group and others appear to be entirely unrelated as regards phylogeny and occur in very diverse bacteria (e.g., Forssmann's nonspecific antigens). It is probable that the identi fication and classification of bacteria by their antigens have their limits, though the data are often extremely valuable.
By opsonic action is meant the effect which a serum has on bacteria in making them more susceptible to phagocytosis by the white corpuscles of the blood (q.v.). Such an effect may be demonstrated outside the body by making a suitable mixture of (a) a suspension of the particular bacterium, (b) the serum to be tested, and (c) leucocytes of a normal animal or person. The mixture is placed in a thin capillary tube and incubated at 37° C. for half an hour; a film preparation is then made from it on a glass slide, stained by a suitable method and then examined microscopically. The number of bacteria contained within a number of, say 5o, leucocytes can be counted and the average taken. In estimating the opsonic power of the serum in cases of disease a control with normal serum is made at the same time and under precisely the same conditions. The average num ber of bacteria contained within leucocytes in the case tested, divided by the number given by the normal serum, is called the phagocytic index. Wright and Douglas showed that under these conditions phagocytosis might occur when a small quantity of normal serum was present, whereas it was absent when normal salt solution was substituted for the serum ; the serum thus con tained substances which made the organisms susceptible to the action of the phagocytosis. They further showed that this sub stance acted by combining with the organisms and apparently producing some alteration in them ; on the other hand it had no direct action on the leucocytes. This opsonin of normal serum is very labile, being rapidly destroyed at 55° C. Various ob servers had previously found that the serum of an animal im munized against a particular bacterium had a special action in bringing about phagocytosis of that organism, and it had been found that this property was retained when the serum was heated at 55° C. It is now generally admitted that at least two distinct classes of substances are concerned in opsonic action, that ther mostable immune opsonins are developed as a result of active immunization and these possess the specific properties of anti substances in general, that is, act only on the corresponding bac terium. On the contrary the labile opsonins of normal serum have a comparatively general action on different organisms. It is quite evident that the specific immune-opsonins may play a very im portant part in the phenomena of immunity, as by their means the organisms are taken up more actively by the phagocytic cells. The opsonic action of the serum has been employed by Sir A. Wright and his co-workers to control the treatment of bacterial infections by vaccines.
This is the name given to a pro cedure by which the presence of antisubstances to bacteria can be recognized in the blood serum of animals. In principle it rests on the discovery by Bordet and Gengou that when a bac teriolytic serum containing bacteriolysin and a suspension of the corresponding bacteria are allowed to interact in the presence of fresh serum, a constituent of the latter (alexin or complement) joins the combination of bacteria and antisubstance, and remains fixed to it, this union resulting in the death and solution of the bacteria. To render this fixation of complement obvious even where lysis of the bacteria is not visible, another system which also needs complement for its consummation is added to the mix ture, and the occurrence or not of the first reaction is determined by the completion or the non-completion of the second reaction.
The most commonly used antigen-antibody system for the second system is a mixture of a suspension in salt solution of the red-blood corpuscles of the sheep and of a serum which is known to be lytic for them but does not contain active comple ment. This latter desideratum can be assured by previously heating the haemolytic serum to 56° C. for 3o minutes. If there is free complement at liberty to join the red corpuscles and anti serum, the red cells break up, and the haemoglobin is set free as a clear solution in the surrounding medium. The colour and transparency of the solution is a readily observed indicator of the presence of free complement and, to some extent, of its amount. This by inference allows an estimate to be made of the reagents present in the first system. If no appropriate bacterial substance or no antisubstance is present, then no complement will be fixed in the first reaction, and consequently it will still be available for haemolysis.
This collection of living cultures from all spheres of bacteriological activity was founded in 1920 at the Lister Institute, London, under the aus pices of the Medical Research Council. It has been of great service to microbiologists in all parts of the world, over 2,000 strains being maintained in 1925. The second edition of the cata logue appeared in 1925 and can be procured from H.M. Sta tionery Office, London.
BIBLIOGRAPHY.—The chief journals devoted to medical bacteriology Bibliography.—The chief journals devoted to medical bacteriology and immunology are:—Jour. Pathology and Bact.; Brit. Jour. Exp. Path.; Jour. Bact. (Chicago) ; Jour. Inf. Dis. (Baltimore) ; Jour. Exp. Medicine; Jour. Immun. (Baltimore) ; Zeit. f. Bact., Abt. i (Jena) ; Zeitsch. f. Hyg., etc. (Leipzig) ; Archiv. f. Hyg.; Zeitsch. f. 1 mmuni tidtsf., etc. (Jena) ; Ann. de l'Instit. Pasteur.
The larger reference books are those of R. Kraus and C. Levaditi, Handbuch der Immunitatsforschung and experimentellen Therapie (Jena, 1914) ; and W. Kolle and H. Hetsch, Die experimentelle Bak teriologie, etc. (1922). Smaller text-books are E. 0. Jordan, Text-Book of General Bacteriology (Philadelphia, 1915) ; J. A. Kolmer, A Prac tical Text-Book of Infection, Immunity and Specific Therapy (1915) ; R. Muir and J. Ritchie, Manual of Bacteriology, 7th ed. (1919) ; R. T. Hewlett, Pathology (1922) ; etc. (R. M. ; J. C. G. L.; J. A. A.)
