No sharp line can be drawn between pathogenic and non-pathogenic Schizomycetes, and some of the most marked steps in the progress of our modern knowledge of these organisms depend on the discovery that their pathogenicity or virulence can be modified--diminished or increased--by definite treatment, and, in the natural course of epidemics, by alterations in the environment. Similarly we are unable to divide Schizomycetes sharply into parasites and saprophytes, since it is well proved that a number of species--facultative parasites--can become one or the other according to circumstances. These facts, and the further knowledge that many bacteria never observed as parasites, or as pathogenic forms, produce toxins or poisons as the result of their decompositions and fermentations of organic substances, have led to important results in the applications of bacteriology to medicine.
[Illustration: FIG. 20.--The ginger-beer plant.
A. One of the brain-like gelatinous masses into which the mature "plant"
condenses.
B. The bacterium with and without its gelatinous sheaths (cf. fig. 19).
C. Typical filaments and rodlets in the slimy sheaths.
D. Stages of growth of a sheathed filament--a at 9 A.M., b at 3 P.M., c at 9 P.M., d at 11 A.M. next day, e at 3 P.M., f at 9 P.M., g at 10.30 A.M.
next day, h at 24 hours later. (H. M. W.)]
[Sidenote: Bacteriosis in plants.]
Bacterial diseases in the higher plants have been described, but the subject requires careful treatment, since several points suggest doubts as to the organism described being the cause of the disease referred to their agency. Until recently it was urged that the acid contents of plants explained their immunity from bacterial diseases, but it is now known that many bacteria can flourish in acid media. Another objection was that even if bacteria obtained access through the stomata, they could not penetrate the cell-walls bounding the intercellular spaces, but certain anaerobic forms are known to ferment cellulose, and others possess the power of penetrating the cell-walls of living cells, as the bacteria of Leguminosae first described by Marshall Ward in 1887, and confirmed by Miss Dawson in 1898. On the other hand a long list of plant-diseases has been of late years attributed to bacterial action. Some, _e.g._ the Sereh disease of the sugar-cane, the slime fluxes of oaks and other trees, are not only very doubtful cases, in which other organisms such as yeasts and fungi play their parts, but it may be regarded as extremely improbable that the bacteria are the primary agents at all; they are doubtless saprophytic forms which have gained access to rotting tissues injured by other agents.
Saprophytic bacteria can readily make their way down the dead hypha of an invading fungus, or into the punctures made by insects, and Aphides have been credited with the bacterial infection of carnations, though more recent researches by Woods go to show the correctness of his conclusion that Aphides alone are responsible for the carnation disease. On the other hand, recent investigation has brought to light cases in which bacteria are certainly the primary agents in diseases of plants. The principal features are the stoppage of the vessels and consequent wilting of the shoots; as a rule the cut vessels on transverse sections of the shoots appear brown and choked with a dark yellowish slime in which bacteria may be detected, _e.g._ cabbages, cucumbers, potatoes, &c. In the carnation disease and in certain diseases of tobacco and other plants the seat of bacterial action appears to be the parenchyma, and it may be that Aphides or other piercing insects infect the plants, much as insects convey pollen from plant to plant, or (though in a different way) as mosquitoes infect man with malaria. If the recent work on the cabbage disease may be accepted, the bacteria make their entry at the water pores at the margins of the leaf, and thence via the glandular cells to the tracheids. Little is known of the mode of action of bacteria on these plants, but it may be assumed with great confidence that they excrete enzymes and poisons (toxins), which diffuse into the cells and kill them, and that the effects are in principle the same as those of parasitic fungi. Support is found for this opinion in Beyerinck's discovery that the juices of tobacco plants affected with the disease known as "leaf mosaic," will induce this disease after filtration through porcelain.
[Sidenote: Symbiosis.]
In addition to such cases as the kephir and ginger-beer plants (figs. 19, 20), where anaerobic bacteria are associated with yeasts, several interesting examples of symbiosis among bacteria are now known. _Bacillus chauvaei_ ferments cane-sugar solutions in such a way that normal butyric arid, inactive lactic acid, carbon dioxide, and hydrogen result; _Micrococcus acidi-paralactici_, on the other hand, ferments such solutions to optically active paralactic acid. Nencki showed, however, that if both these organisms occur together, the resulting products contain large quantities of normal butyl alcohol, a substance neither bacterium can produce alone. Other observers have brought forward other cases. Thus neither _B. coli_ nor the _B. denitrificans_ of Burri and Stutzer can reduce nitrates, but if acting together they so completely undo the structure of sodium nitrate that the nitrogen passes off in the free state.
Van Senus showed that the concurrence of two bacteria is necessary before his _B. amylobacter_ can ferment cellulose, and the case of mud bacteria which evolve sulphuretted hydrogen below which is utilized by sulphur bacteria above has already been quoted, as also that of Winogradsky's _Clostridium [v.03 p.0170] pasteurianum_, which is anaerobic, and can fix nitrogen only if protected from oxygen by aerobic species. It is very probable that numerous symbiotic fermentations in the soil are due to this co-operation of oxygen-protecting species with anaerobic ones, _e.g._ _Tetanus_.
[Illustration: FIG. 21.--A plate-culture colony of a species of _Bacillus--Proteus_ (Hauser)--on the fifth day. The flame-like processes and outliers are composed of writhing filaments, and the contours are continually changing while the colony moves as a whole. Slightly magnified.
(H. M. W.)]
[Sidenote: Activity of bacteria.]
Astonishment has been frequently expressed at the powerful activities of bacteria--their rapid growth and dissemination, the extensive and profound decompositions and fermentations induced by them, the resistance of their spores to dessication, heat, &c.--but it is worth while to ask how far these properties are really remarkable when all the data for comparison with other organisms are considered. In the first place, the extremely small size and isolation of the vegetative cells place the protoplasmic contents in peculiarly favourable circumstances for action, and we may safely conclude that, weight for weight and molecule for molecule, the protoplasm of bacteria is brought into contact with the environment at far more points and over a far larger surface than is that of higher organisms, whether--as in plants--it is distributed in thin layers round the sap-vacuoles, or--as in animals--is bathed in fluids brought by special mechanisms to irrigate it. Not only so, the isolation of the cells facilitates the exchange of liquids and gases, the passage in of food materials and out of enzymes and products of metabolism, and thus each unit of protoplasm obtains opportunities of immediate action, the results of which are removed with equal rapidity, not attainable in more complex multi-cellular organisms. To put the matter in another way, if we could imagine all the living cells of a large oak or of a horse, having given up the specializations of function impressed on them during evolution and simply carrying out the fundamental functions of nutrition, growth, and multiplication which mark the generalized activities of the bacterial cell, and at the same time rendered as accessible to the environment by isolation and consequent extension of surface, we should doubtless find them exerting changes in the fermentable fluids necessary to their life similar to those exerted by an equal mass of bacteria, and that in proportion to their approximation in size to the latter. Ciliary movements, which undoubtedly contribute in bringing the surface into contact with larger supplies of oxygen and other fluids in unity of time, are not so rapid or so extensive when compared with other standards than the apparent dimensions of the microscopic field. The microscope magnifies the distance traversed as well as the organism, and although a bacterium which covers 9-10 cm. or more in 15 minutes--say 0.1 mm. or 100 per second--appears to be darting across the field with great velocity, because its own small size--say 5 1 --comes into comparison, it should be borne in mind that if a mouse 2 in.
long only, travelled twenty times its own length, _i.e._ 40 in., in a second, the distance traversed in 15 minutes at that rate, viz. 1000 yards, would not appear excessive. In a similar way we must be careful, in our wonder at the marvellous rapidity of cell-division and growth of bacteria, that we do not exaggerate the significance of the phenomenon. It takes any ordinary rodlet 30-40 minutes to double its length and divide into two equal daughter cells when growth is at its best; nearer the minimum it may require 3-4 hours or even much longer. It is by no means certain that even the higher rate is greater than that exhibited by a tropical bamboo which will grow over a foot a day, or even common grasses, or asparagus, during the active period of cell-division, though the phenomenon is here complicated by the phase of extension due to intercalation of water. The enormous extension of surface also facilitates the absorption of energy from the environment, and, to take one case only, it is impossible to doubt that some source of radiant energy must be at the disposal of those prototrophic forms which decompose carbonates and assimilate carbonic acid in the dark and oxidize nitrogen in dry rocky regions where no organic materials are at their disposal, even could they utilize them. It is usually stated that the carbon dioxide molecule is here split by means of energy derived from the oxidation of nitrogen, but apart from the fact that none of these processes can proceed until the temperature rises to the minimum cardinal point, Engelmann's experiment shows that in the purple bacteria rays are used other than those employed by green plants, and especially ultra-red rays not seen in the spectrum, and we may probably conclude that "dark rays"--_i.e._ rays not appearing in the visible spectrum--are absorbed and employed by these and other colourless bacteria.
The purple bacteria have thus two sources of energy, one by the oxidation of sulphur and another by the absorption of "dark rays." Stoney (_Scient.
Proc. R. Dub. Soc._, 1893, p. 154) has suggested yet another source of energy, in the bombardment of these minute masses by the molecules of the environment, the velocity of which is sufficient to drive them well into the organism, and carry energy in of which they can avail themselves.
[Illustration: FIG. 22.--Portions of a colony such as that in fig. 21, highly magnified, showing the kinds of changes brought about in a few minutes, from A to B, and B to C, by the growth and ciliary movements of the filaments. The arrows show the direction of motion. (H. M. W.)]
AUTHORITIES.--General: Fischer, _The Structure and Functions of Bacteria_ (Oxford, 1900, 2nd ed.), German (Jena, 1903); Migula, _System der Bakterien_ (Jena, 1897); and in Engler and Prantl, _Die naturlichen Pflanzenfamilien_, I. Th. 1 Abt. a; Lafar, _Technical Mycology_ (vol. i.
London, 1898); Mace, _Traite pratique de bakteriologie_ (5th ed. 1904).
Fossil bacteria: Renault, "Recherches sur les Bacteriacees fossiles," _Ann.
des Sc. Nat._, 1896, p. 275. Bacteria in Water: Frankland and Marshall Ward. "Reports on the Bacteriology of Water," _Proc. R. Soc._, vol. li. p.
183, vol. liii. p. 245, vol. lvi. p. 1; Marshall Ward, "On the Biology of _B. ramosus_," _Proc. R. Soc._, vol. lviii. p. 1; and papers on Bacteria of the river Thames in _Ann. of Bot._ vol. xii. pp. 59 and 287, and vol. xiii.
p. 197. Cell-membrane, &c.: Butschli, _Weitere Ausfuhrungen uber den Bau der Cyanophyceen und Bakterien_ (Leipzig, 1896); Fischer, _Unters. uber den Bau der Cyanophyceen und Bakterien_ (Jena, 1897); Rowland, "Observations upon the Structure of Bacteria," _Trans. Jenner Institute_, 2nd ser. 1899, p. 143, with literature. Cilia: Fischer, "Unters. uber Bakterien,"
_Pringsh. Jahrb._ vol. xxvii.; also the works of Migula and Fischer already cited. Nucleus: Wager in _Ann. Bot._ vol. ix. p. 659; also Migula and Fischer, _l.c._; Vejdovsky, "uber den Kern der Bakterien und seine Teilung," _Cent. f. Bakt._ Abt. II. Bd. xi. (1904) p. 481; _ibid._ "Cytologisches uber die Bakterien der Prager Wasserleitung," _Cent. f.
Bakt._ Abt. II. Bd. xv. (1905); Mencl, "Nachtrage zu den Strukturverhaltnissen von Bakterium gammari" in _Archiv f. Protistenkunde_, Bd. viii. (1907), p. 257. Spores, &c.: Marshall Ward, "On the Biology of _B. ramosus_," _Proc. R. Soc._, 1895, vol. lviii. p. 1; Sturgis, "A Soil Bacillus of the type of de Bary's _B. megatherium_," _Phil. Trans._ [v.03 p.0171] vol. cxci. p. 147; Klein, L., _Ber. d. deutschen bot. Gesellsch._ (1889), Bd. vii.; and _Cent. f. Bakt. und Par._ (1889), Bd. vi.
Classification: Marshall Ward, "On the Characters or Marks employed for classifying the Schizomycetes," _Ann. of Bot._, 1892, vol. vi.; Lehmann and Neumann, _Atlas and Essentials of Bacteriology_; also the works of Migula and Fischer already cited. Myxobacteriaceae: Berkeley, _Introd. to Cryptogamic Botany_ (1857), p. 313; Thaxter, "A New Order of Schizomycetes," _Bot. Gaz._ vol. xvii. (1892), p. 389; and "Further Observations on the Myxobacteriaceae," _ibid._ vol. xxiii. (1897), p. 395, and "Notes on the Myxobacteriaceae," _ibid._ vol. xxxvii. (1904), p. 405; Baur, "Myxobakterienstudien," _Arch. f. Protistenkunde_, Bd. v. (1904), p.
92; Smith, "Myxobacteria," _Jour. of Botany_, 1901, p. 69; Quehl, _Cent. f.
Bakt._ xvi. (1896), p. 9. Growth: Marshall Ward, "On the Biology of _B.
ramosus_," _Proc. R. Soc._ vol. lviii. p. 1 (1895). Fermentation, &c.: Warington, _The Chemical Action of some Micro-organisms_ (London, 1888); Winogradsky, "Recherches sur les organismes de la nitrification," _Ann. de l'Inst. Past._, 1890, pp. 213, 257, 760, 1891, pp. 92 and 577; "Sur l'assimilation de l'azote gazeux, &c.," _Compt. Rend._, 12 Feb. 1894; "Zur Microbiologie des Nitrifikationsprozesses," _Cent. f. Bakt._ Abt. II. Bd.
ii. (1896), p. 415; "Ueber Schwefel-Bakterien," _Bot. Zeitg._, 1887, Nos.
31-37; _Beitr. zur Morph. u. Phys. der Bakterien_, H. 1 (1888); "Ueber Eisenbakterien," _Bot. Zeitg._, 1888, p. 261; and Omeliansky, "Ueber den Einfluss der organischen Substanzen auf die Arbeit der nitrifizierenden Organismen," _Cent. f. Bakt._ Abt. II. Bd. v. (1896); Schorler, "Beitr. zur Kenntniss der Eisenbakterien," _Cent. f. Bakt._ Abt. II. Bd. xii. (1904), p. 681; Marshall Ward, "On the Tubercular Swellings on the Roots of Vicia Faba," _Phil. Trans._, 1877, p. 539; Hellriegel and Wilfarth, "Unters. uber die Stickstoffnahrung der Gramineen u. Leguminosen," _Beit. Zeit. d.
Vereins fur die Rubenzuckerindustrie_ (Berlin, 1888); Nobbe and Hiltner, _Landw. Versuchsstationen_ (1899), Bd. 51, p. 241, and Bd. 52, p. 455; Maze, _Annales de l'Institut Pasteur_, t. II, p. 44, and t. 12, p. 1 (1897); Prazmowski, _Land. Versuchsstationen_, Bd. 37 (1890), p. 161, Bd.
38 (1891), p. 5; Frank, _Landw. Jahrb._ Bd. 17 (1888), p. 441; Omelianski, "Sur la fermentation de la cellulose," _Compt. Rend._, 4 Nov. 1895; van Senus, _Beitr. zur Kenntn. der Cellulosegahrung_ (Leiden, 1890); van Tieghem, "Sur la fermentation de la cellulose," _Bull. de la soc. bot. de Fr._ t. xxvi. (1879), p. 28; Beyerinck "Ueber Spirillum desulphuricans, &c.," _Cent. f. Bakt._ Abt. II. Bd. i. (1895), p. 1; Molisch, _Die Pflanze in ihren Beziehungen zum Eisen_ (Jena, 1892). Pigment Bacteria: Ewart, "On the Evolution of Oxygen from Coloured Bacteria," _Linn. Journ._, 1897, vol.
xxxiii. p. 123; Molisch, _Die Purpurbakterien_ (Jena, 1907). Oxydases and Enzymes: Green, _The Soluble Ferments and Fermentation_ (Cambridge, 1899).
Action of Light, &c.: Marshall Ward, "The Action of Light on Bacteria,"
_Phil. Trans._, 1893, p. 961, and literature. Resistance to Cold, &c.: Ravenel, _Med. News_, 1899, vol. lxxiv.; Macfadyen and Rowland, _Proc. R.
Soc._ vol. lxvi. pp. 180, 339, and 488; Farmer, "Observations on the Effect of Desiccation of Albumin upon its Coagulability," _ibid._ p. 329.
Pathogenic Bacteria: Baumgarten, _Pathologische Mykologie_ (1890); Kolle and Wassermann, _Handbuch der pathogenen Mikroorganismen_ (1902-1904); and numerous special works in medical literature. Immunity: Ehrlich, "On Immunity with Special Reference to Cell-life," _Proc. R. Soc._ vol. lxvi.
p. 424; Calcar, "Die Fortschritte der Immunitats- und Spezifizetatslehre seit 1870," _Progressus Rei Botanicae_, Bd. I. Heft 3 (1907). Bacteriosis: Migula, _l.c._ p. 322, has collected the literature; see also Sorauer, _Handbuch der Pflanzenkrankheiten_, I. (1905), pp. 18-93, for later literature. Symbiosis: Marshall Ward, "Symbiosis," _Ann. of Bot._ vol.
xiii. p. 549, and literature.
(H. M. W.; V. H. B.)
II. PATHOLOGICAL IMPORTANCE
The action of bacteria as pathogenic agents is in great part merely an instance of their general 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 three following heads, viz. (1) the methods employed in the study; (2) the modes of action of bacteria and the effects produced by them; and (3) the facts and theories with regard to immunity against bacterial disease.
[Sidenote: Historical summary.]
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 human knowledge. An all-important factor in this remarkable progress was the introduction by Koch of solid culture media, of the "plate-method," &c., 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 pathogenic bacteria had been observed in the tissues, but it was in the decade 1880-1890 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 bacillus of influenza (1892), of the bacillus of plague (1894) and of the bacillus of dysentery (1898). Immunity against diseases caused by bacteria has been the subject of systematic research from 1880 onwards. In producing active immunity by the attenuated virus, Duguid and J. S. Burdon-Sanderson and W. S. Greenfield in Great Britain, and Pasteur, Toussaint and Chauveau in France, were pioneers. The work of Metchnikoff, dating from about 1884, has proved of high importance, his theory of phagocytosis (_vide infra_) having given a great stimulus to research, and having also contributed to important advances. 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 L. 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 P. P. E. Roux and A.
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 (1890) 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. In the United Kingdom the work of Sidney Martin, in the separation of toxic substances from the bodies of those who have died from certain diseases, is also worthy of mention. Immunity against toxins also became a subject of investigation, and the result was the discovery of the antitoxic action of the serum of animals immunized against tetanus toxin by E. Behring and Kitazato (1890), and by Tizzoni and Cattani. A similar result was also obtained in the case of diphtheria. The facts with regard to passive immunity were thus established and were put to practical application by the introduction of diphtheria antitoxin as a therapeutic agent in 1894. The technique of serum preparation has become since that time greatly elaborated and improved, the work of P. Ehrlich in this respect being specially noteworthy. The laws of passive immunity were shown to hold also in the case of immunity against living organisms by R. Pfeiffer (1894), and various anti-bacterial sera have been introduced. Of these the anti-streptococcic serum of A. Marmorek (1895) is one of the best known.
The principles of protective inoculation have been developed and practically applied on a large scale, notably by W. M. W. 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 the case of typhoid fever independently by Widal and by Grunbaum in 1896, though led up to by the work of Pfeiffer, Gruber and Durham and others. Thus a new aid was added to medical science, viz. serum diagnosis of disease. The last decade of the 19th century will stand out in the history of medical science as the period in which serum therapeutics and serum diagnosis had their birth.
In recent years the relations of toxin and antitoxin, still obscure, have been the subject of much study and controversy. It was formerly supposed that the injection of attenuated cultures or dead organisms--vaccines in the widest sense--was only of service in producing immunity as a preventive measure against the corresponding organism, but the work of [v.03 p.0172]
Sir Almroth Wright has shown that the use of such vaccines may be of service even after infection has occurred, especially when the resulting disease is localized. In this case a general reaction is stimulated by the vaccine which may aid in the destruction of the invading organisms. In regulating the administration of such vaccines he has introduced the method of observing the _opsonic index_, to which reference is made below. Of the discoveries of new organisms the most important is that of the _Spirochaete pallida_ in syphilis by Schaudinn and Hoffmann in 1905; and although proof that it is the cause of the disease is not absolute, the facts that have been established constitute very strong presumptive evidence in favour of this being the case. It may be noted, however, that it is still doubtful whether this organism is to be placed amongst the bacteria or amongst the protozoa.
[Sidenote: Methods of study.]
The methods employed in studying the relation of bacteria to disease are in principle comparatively simple, but considerable 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 microscopic 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 examination 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. The methods vary much in detail, though in each case the endeavour is to colour the bacteria as deeply, and the tissues as faintly, as possible. Sometimes a simple watery solution of the dye is sufficient, but very often the best result is obtained by increasing the staining power, _e.g._ by addition of weak alkali, application of heat, &c., and by using some substance which acts as a mordant and tends to fix the stain to the bacteria. Excess of stain is afterwards removed from the tissues by the use of decolorizing agents, such as acids of varying strength and concentration, alcohol, &c. Different bacteria behave very differently to stains; some take them up rapidly, others slowly, some resist decolorization, others are easily decolorized. In some instances the stain can be entirely removed from the tissues, leaving the bacteria alone coloured, and the tissues can then be stained by another colour. This is the case in the methods for staining the tubercle bacillus and also in Gram's method, the essential point in which latter is the treatment with a solution of iodine before decolorizing. In Gram's method, however, only some bacteria retain the stain, while others lose it. The tissues and fluids are treated by various histological methods, but, to speak generally, examination is made either in films smeared on thin cover-glasses and allowed to dry, or in thin sections cut by the microtome after suitable fixation and hardening of the tissue. In the case of any bacterium discovered, observation must be made in a long series of instances in order to determine its invariable presence.
[Sidenote: Cultivation.]
In cultivating bacteria outside the body various media to serve as food material must be prepared and sterilized 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 a process of coagulation, or by 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 gelatine or agar as solidifying agents and of non-coagulable proteids (commercial "peptone") to make up for proteids lost by coagulation 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 titration 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 temperatures, usually either at 37 C., the temperature of the body, or at about 20 C., a warm summer temperature, until growth appears.
For maintaining a constant temperature incubators with regulating apparatus are used. Subsequent cultures or, as they are called, "subcultures," 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 various methods have been devised, the most important being 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 shallow 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 culture from the internal organs.
This method applies especially to pathogenic bacteria whose growth on culture media is slow, _e.g._ the tubercle bacillus.
The full description of a particular bacterium implies an account not only of its microscopical characters, but also of its growth characters in various culture media, its biological properties, 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, &c.--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 employed 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 infrequently 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 characters 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. It is to be noted that 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 cultural as well as the microscopical [v.03 p.0173]
characters of a pathogenic organism may be closely similar to other non-pathogenic members of the same group, and it thus comes to be a matter of extreme 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 produced by them in various culture media, _e.g._ by their fermentative action on various sugars, &c., though in this case such properties may become modified in the course of time. And in the second place, the various serum reactions to be described below have been called into requisition. It may be stated that the introduction of a particular bacterium into the tissues of the body leads to certain properties appearing in the serum, which are chiefly exerted towards this particular bacterium. Such a serum may accordingly within certain limits be used for differentiating this organism from others closely allied to it (_vide infra_).
The modes of cultivation described apply only to organisms which grow in presence of oxygen. Some, however--the strictly _anaerobic_ bacteria--grow only in the absence of oxygen; hence means must be adopted for excluding this gas. It is found that if the inoculation be made deep down in a solid medium, growth of an anaerobic organism will take place, especially if the medium contains some reducing agent such as glucose. Such cultures are called "deep cultures." To obtain growth of an anaerobic organism on the surface of a medium, in using the plate method, and also for cultures in fluids, the air is displaced by an indifferent gas, usually hydrogen.
[Sidenote: Inoculation.]
In testing the effects of bacteria by inoculation 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 subject. In some cases, _e.g._ Malta fever and relapsing fever, monkeys have been used with success, but in others, _e.g._ leprosy, none of the lower animals has been found to be susceptible. Discretion must therefore be exercised in interpreting negative results 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 injections are made by means of a hypodermic syringe into the subcutaneous tissue, into a vein, into 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 desired, and then a post-mortem examination is made, the conditions of the organs, &c., being observed and noted. The various tissues affected are examined microscopically and cultures made from them; in this way the structural changes and the relation of bacteria to them can be determined.
[Sidenote: Separation of toxins.]