'^v. IMAGE EVALUATION TEST TARGET (MT-3) 1.0 1.1 2.5 iiiiii 12.0 18 ■ 1.25 l.4||||,.6 ^ 6" ► others, like the typhoid bacillus, which according to Chautemesse and Widal'" are so susceptible that a loss of virulence is almost immediate. This, after all, is only what is to be expected, if, as seems most probable, the pathogenic property is that which has been latest acquired. A late acquisition is most easily altered, and the tendency to re-acquirement of the same is least evident. Intensification of pathogenic properties can, however, be induced by other means than by passage through animals ; Hueppe's observations would seem to show that the Bacillus Cfaolerte Asiaticee grown ana;robically gains in virulence.*" This principle — that it is difficult to stamp a modification firmly upon a species of bacteria, as, indeed, upon any animal or vegetable species — is exemplified throughout the long series of attempts that have been made to produce new races of micro-organisms, and now, when I proceed to recount some of the more successful of these attempts the principle will be seen to be constantly in evidence. MODIFICATIONS OF LONGER DURATION. From the transient modifications which I have noted above it is easy to pass to a series of instances in which the return to the normal only follows after an increasing number of generations, in which numerous generations retain the acquired characteristics, and the original proper- ties only gradually reassert themselves. Take, for example, almost any of the chromogenic bacteria. If a minute quantity of a high-coloured typical growth be removed and spread over the cut surface of a sterilised potato it will be found that the individual colonies which result present differences in pigment production ; the majority reproduce the type, but some are even deeper in colour than the original, others paler, others colourless. If from one of these colourless colonies fresh potato growths be made, a large number of coloured colonies develop, but also a large number of colourless, and the more frequently this selective process is repeated the greater becomes the proportion of the resulting colourless colonies. Yet even after a very great number of growths made in this way there is always a tendency for certain individuals and their progeny to revert to type. Slight alterations in the nutrition of the individuals probably induced the first differences in pigment production, and the 11 induced modificatioua tend to reproduce themselves through many " generations." '"" The same occurs if a culture of one of these non-pathogenic forms be heated for a few minutes to a point approaching that at which immediate death of the special microbe is brought about. After this treatment there may be numerous ** generations " produced, incapable of pigment production. Illustrating this and the preceding order of appearances, I showed a series of specimens at the Nottingham meeting of the British Medical Association in July. The series included cultures of the Bacillus ruber, Plymouth ; Bacillus ruber, Kiel ; Microbacillus prodigiosus, Bacillus indicus and Sarcina orythromyxa. To yet more marlied effects of high temperature I shall revert at a later period. Again, old cultures of most micro-organisms become attenuated, and although these attenuated forms still propagate themselves, it may be weeks — that is to say, almost countless "generations" — before there is a return to primitive appearance and primitive properties. Thus old growths of Koch's Cholera spirillum," or the Finkler-Prior spirillum of Cholera nostras ;" or, again, of the Vibrio Metchnikovi'^ yield colonies which have an absent or greatly reduced power of liquefying gela- tine, and only very slowly and under favourable conditions does this power manifest itself once more. From old cultures of the Bacillus pyoceaneus, the Pink torula and many more chromogenic microbes, I find, in common with other observers, that colourless or faintly coloured growths are obtainable, which for long show no reversion to •' type." It would seem that without there being necessarily any attenuation there may be modifications persisting for long periods. It has been noted by many observers that for some considerable period after Staphy- lucoccus pyogenes aureus has been separated out of pus and cultivated, the colonies are colourless, and that only eventually may the golden yellow pigment become produced. During the last long vacation I was a victim to this order of affairs, for having obtained a micrococcus from some pus gained from a case at the Addenbrooke's Hospital, I made two successive cultures of the pure growth during the course of a week, and the second of these I employed to distribute to the bacteriological class, as the Staphylococcus pyogenes alb us. The cultures made by the class developed the golden-yellow pigment, and proved tliat we were dealing, not with the "albua," but with the "aureus." It may be added that, save for colour difference, the two forms are indistinguishable. THE FIIODUCTION OF RACES. From such cases as these we can pass on .almost imperceptibly to the development of what may be termed races. I will presently discuss the ([ualiticatious that must be applied iu employing this expression. For 12 immediate purposes it is enough to state that a slight modification of environment acting over a sufficient number of " generations " — that is to say, acting for a sufficiently long period — or a powerful stimulus a{)plied temporarily to the individual bacteria of one "generation," may load to modification of the properties of any species which, so far as we can see, are permanent so long as the microbes so acted upon are culti- vated under ordinary conditions upon the usual media of bacteriological research. To make this statement perfectly clear, I may give an example. The Bacillus ruber of Kiel grows well upon a piece of sterilised potato at the ordinary temperature (15—25° C), the culture becoming a deep crimson red. If now the culture be kept at a temperature of 37° C. for two days, and a new piece of potato be inoculated from this and kept another two days at the same temperature, and a series of six to ten cultures be made thus ; or if, on the other hand, a potato culture be exposed to a tem- perature of 55° to 57° C. for a few minutes, in either case fresh cultures made in series upon potato at the ordinary temperature may for months remain absolutely colourless. It is scarcely necessary to say that the classical example of this order of phenomena is to be found in tlie remarkable observations of Pasteur, Chamberlaud, and Roux upon the attenuation of the anthrax bacillus.^" The anthrax bacillus may be kept (in broth) at a tempera- ture of 42 "5° to 43° C. for a week or more without there being any noticeable diminution of the virulence of the growth. But after this the virulence slowly and steadily diminishes. Cultures made under ordinary conditions from a growth treated thus for twelve days will no longer, when inoculated, cause the fatal disease in sheep. After 31 days the cultures are so feeble that the very susceptible guinea-pigs survive, and mice alone are killed. In 40 days or so the bacilli subjected to the above- mentioned temperature are enfeebled to the point of death, and no further cultures from the original broth are possible. What is more, each succes- sive growth obtained daily after the eighth day from a culture subjected to the Pasteur process, if periodically resown, under ordinary conditions (in alkaline beef broth at 35° to 37° C ), retains permanently — for months and years — the grade of diminished virulence that had been impressed upon the original culture. Several other methods have been suggested for attenuating the anthrax bacillus, all of them possessing tiie advantage of being more expeditious-""^ but all, save one, labour under the disadvantage that the lowering of virulence produced is mistable, and upon con- tinued cultivation the successive growtlis vary and are liable to regain the pathogenic property of the original culture. Tlio exception is Roux's metho(P in whicli, by the addition of minute quantities of potassium 18 bichromate or carbolic acid to the culture medium, a race is, after a shor time, developed, which has lost the power of spore formation. According to the duration of the action of either antiseptic, and the amount of either which is present, so are the daughter cultures of particular degrees of virulence. These asporogenous races appear to retain their characters indefinitely under normal bacteriological conditions. Here again it must be pointed out that " permanent " races are by no means necessarily attenuated races. Starting from a culture of the bacillus anthracis, so weak that only young mice are affected, it is possible, by passage of the virus through a series of animals whose resis- tance to the disease is in an ascending scale, to gain a series of races of gradually increasing virulence, and as Malm has shown"* it is possible to pass beyond the virulence of the type and obtain a race that will kill not only sheep but the very refractory dog.* Another example to the same effect may be gained from Pasteur and Thullier's researches into swine erysipelas (Rouget des pores, or Rothlauf)."' This fatal and most infectious disease is caused by a minute bacillus, first isolated by Liiffler. Rabbits also succumb to the malady, but not so very readily. It is found that passage through these latter animals causes the virus steadily to augment in strength until a stage is reached most fatal to rabbits, but cultures obtained at this stage induce but a transient illness in pigs, which consequently can be inoculated or *' vacci- nated " against the disease. Here exaltation of virulence for one animal and attenuation of virulence for another go hand in hand. (Similar attenuation for the pig can be produced by keeping cultures at 37°C for a long period.) Equally instructive cases may be gleaned from researches upon non- pathogenic micro-organisms. Let me take first the Microbacill us prodi- giosus. It was found by Wasserzug^* that the addition of antiseptics to the medium of culture, in quantities sufficient to retard growth, led not only to the loss of colour production, but also to accompanying remarkable changes in formj some individuals become much elongated, others develop into actual spirilla. This polymorphism and its extent depend (as I have already shown in the case of the Bacillus pyocyaneus) upon the composition of the medium. Upon returning such altered microbes back to the potato there is a gradual return to the original state. Yet it is possible by slight modification of this method to produce permanent races. Taking beef broth to which tartaric acid has been added, * I may hero call attention to a statement In Professor C. Frtlnkel's " Bakterlenkunde " (edition of 1891, p. 177) " Es ist . . bisher noch nicht goglHckt, eino dauemdo Verstilrkung der Baktorion, eino haltbaro zunabmo ihrer natUrlicben Virulcuz herbeizufUhren, .... auf Thiero. . eino raaubero und andere Wirkungsweiso an don Tag zu legen, als sonst an ihnen bemerkt wurde." While these statements, in face of French researches, were, to s.iy the least, already very debateable a year ago, they certainly now cannot bo regarded as other than quito incorrect. 14 "Wasserzug found that when the culture was a day or two old there was a great development of spirilla. Later, when through the production of trimethylami the medium became alkaline, the spirilla ceased to be developed ; tliere was a return to the coccus or shortened bacillary form. If now he did not permit the production of the alkaline trimethylamin, ■but made new acid growths before the cultures were more than forty- 'eight hours old, he discovered that the longer he persevered with his series of cultures the longer the microbe took when grown upon potato to return to the coccus form, until finally by such acid growth and subsequent heating to SCC. for a few minutes, all that he could obtain upon sowing on potatoes was, not a micrococcus, but a long bacillus — a permanent race of such. Or let us return again to the Bacillus pyocyaneus which, though patho- genic, may be discussed along with other chromogenic forms. By placing his modifications, to which I have already referred, under special conditions of heat, etc., Gessard found that he could obtain permanent races upon ordinary beef broth, one giving the green fluorescent pigment alone, another the blue pyocyauin, another colour- less.'^ The same observer, studying the bacillus of blue milk (Bacillus cyanogenus or syncyanus) found that by passages through egg albumen he could obtain a race giving a deep pigment (which becomes red with alkalies, blue with acids), another race which is only fluorescent, gained from cultures that are some months old, and a third colourless race, this last, either by taking extremely old cultures or by warming a recent culture to a temperature which does not cause death. Similarly Laurent," working with the Bacillus ruber from Kiel (Bacillus rouge de Kiel), found that exposure of a growth to bright sunlight for three hours gave colonies which with but few exceptions were colourless, and though with further successive cultivations there was manifested a tendency for many of the latter colonies to revert to type and develop red pigment, yet by careful selection he was able to gain an absolutely decolourised race which remained colourless, that is, upon agar-agar and in broth at a temperature of 25° to 35° C, a temperature and media at which and in which the original growth always shows a strong reddish violet tint. On potatoes at the same temperature he was able to make thirty-two successive cultures extending over a year, and in these not the least trace of colouration ever appeared. These facts, if alone they were all that we had to consider, would fully justify the statement that it is possible to evolve artificially species or sub-species of bacteria. But there are other facts which must of necessity be taken into account before we can arrive at any definite conclusion — facts which so far as I know apply at present to micro- organisms alone, though everything points to the belief that they are / 16 )/ operative, and should be considered, in discussing the development of new species and sub-species among higher organisms. In cultivating the bacteria we are confronted with the fact, tiiat suc- cessful culture necessitates the employment of special media of growth. We have to sow the microbes in fluid or solid substances whose composi- tion depends upon the mode of life of the special form studied. And thus there is and can be no common soil in which all forms propagate them- selves with equal facility. We are deprived of what I may term a common base. We cannot, with certainty, say that such and such a form, which we have separated out, is a distinct species, inasmuch as grown upon a common normal soil it presents permanent characteristics. The most that we can do is to approach, as near as possible, to the formation of such a common soil. Thus for many pathogenic and most other bacteria we find that slightly alkaline beef-broth, and beef-broth that has been rendered solid by the addition of gelatine or agar-agar, are media in which growth occurs freely, and in which, certain elementary precautions being taken, a given micro-organism retains its characters for countless " generations," retains them so completely that, in the case of the majority of pathogenic microbes a series of successive cultures, if made with due precautions and sufficient frequency, will yield microbes which will all produce typical symptoms when inoculated into a series of rabbits, guinea-pigs, or other animals, the microbes of the last of the series of successive cultures possessing the same degree of virulence as the first of the series. Where this is the case, are we justified in stating that the species is permanent 1 Upon first consideration one is inclined to say that undoubtedly we are, yet further thought brings in doubt, for this is far from being all. There is another and remarkable order of appearances to be considered, which can be exemplified from every case that I have given of the establishment of permanent races. Take, for instance, the races of anthrax bacilli produced by Pasteur's method. These, undoubtedly, when kept upon our approximation to a common soil, namely, upon beef-broth, preserve a well-defined perma- nency. But select any one of these, and pass it through a series of animals of increasing refractoriness to the disease, and there is evolved a race growing equally well upon the beef-broth, whose virulence has been greatly increased. Or take Gessard's " permanent " modifications of the Bacillus pyocyaneus and the Bacillus cyanogenus respectively; grow any of the former upon the rich culture medium formed of agar-agar, glyce- rine, and peptone, and all develop equally into a race producing large quantities of an intense blue pigment j grow any of the latter upon broth containing 2 per cent of glucose, and all give rise to the specific blue pigment of the typical bacillus. Again, take Laurent's colourless 16 race of the bacillus " Rouge de Kiel," and place it upon potatoes at a temperature of about 18° instead of 25° to 35° C, and the coloured form reappears. Or to epitomise : It is possible with bacteria to bring about modifica- tions which persist when a return is made to the ordinary media of the bacteriologist (whether these be only our approximation to the normal — beef-broth, potatoes, etc. — or whether they be the bodies of one or other species of animal — mice, sheep, etc.) — so that now cultivated upon these "ordinary" media, the modification preserves its characteristics, and remains markedly different from the initial or type growth upon the same media. And here we have a distinction between the races of bacteria and the races of animals under domesticity, which, returned to the wild state, are said to revert to type. Now with the bacteria, where such races have been developed, reversion to type may still be brought about by a further process, namely, by a further alteration of environ- ment. I know of no case quite analogous to this among the higher animals. It is only within strictly limited conditions of environment that the characteristics of the usual form or of a race cau be preserved ; overstep these limits, make certain alterations in the environment, and type and race alike are liable to vary, although the liability is perhaps the greater for the race to approximate towards the type than for the type to pro- duce well-marked races. The only cases that I cau call to mind where this law apparently does not hold good are both of modifications of the antiirax bacillus. It is possible so to attenuate the anthrax bacillus that a race is developed which is absolutely non-pathogenic even for the most susceptible of small rodents, and so far no efforts have succeeded in re-establishing the virulence of such attenuated forms. Roux's asporo- genous race also would seem thus far to remain asporogenous under all conditions, although variations may be induced in its virulence. Even these cases, however, will probably be found eventually not to be excep- tions to the rule. For these reasons I have throughout spoken of types and races rather than of species and subspecies or varieties, for by employing the more uncommon terms in this connection I have attempted to guard myself against appearing to hold that among the bacteria there is anything beyond a relative permanency. A study of the observations that have been made up to the present time does not lead to the conclusion that new species are readily developed — all that it clearly indicates is that among the bacteria our employment of this term '• species " must be elastic — the limit to which the individual may depart from the type (and from which it may again revert to type), is very far removed. But granting freely that not one of the cases here described records the ir developmont of a new spocics, this much at least must be admitted, namely, that the longer the action of any one factor upon tlie bacterial growth, the longer the time requisite to ensure a return to the typical condition ; the stronger the impress left upon any individual microbe, the longer again the time requisite to ensure a similar return. If, therefore, within the relatively short period of experiment changes so marked, changes persisting through so many generations or cell nniltipli- cations can be brought about, surely it is not difficult to comprehend changes in the natural world, whose action is sufficiently great and is prolonged through a sufficiently long period to produce races of even greater permanency, races which might be termed new species. ON NATURAL RACES. Now apart from these results of experiments, we possess already a series of data whose explanation is easiest on the supposition tliat we are dealing not with forms that are wholly distinct and of separate origin, but with allied forms and natural races. Perhaps the best example is one that has been extensively discussed. Fehleisen's streptococcus ory- sipelatosus, the microbe causing erysipelas, is, in microscopic appearance and in mode of growth, undistingiiishable from the streptococcus which can be isolated from many boils and cases of abscess formation. Yet certainly the two micro-organisms differ in pathogeneity, differ in the symptoms they induce when isolated. Are we to say that here we are dealing with two distinct species 1 A few years ago all bacteriologists answered this question in the affirmative, but now most would reply in the negative, although some, among whom may be included Crook- shank,-' still adhere to the old view. The observations of Eugen Frankel would seem to indicate that truly we are dealing with what are only races^. Frankel obtained from a case of " universal peritonitis " a pure growth of streptococcus. There could be here no question of erysipelas. Taking some of a fifth culture of this, five months after it had been isolated, he inoculated it into a rabbit's ear and produced an exquisite bullous erysipelas, with streptococci in the lymphatics, identical with human erysipelas. The same inoculated into the peritoneal cavity gave rise to a fibrinous or fibrino-puruleut peritonitis. From this it is evident that the difiFerence between erysipelas and pyaemia, in its various forms, depends on mode and locality of infection and partly on the quantity of virus, on the individual, and lastly, on differences in the intensity of virulence of the races of streptococcus.* It i^i interesting to note how, in clinical practice, one observes this conclusion borne out ; to observe the succession of cases passing tlirt)iigli ordinary erysipelas and acute ' A paper by Belirintf":' publishod during tlio lust nKintli, confirnm this viow of tho identity of the patUot'OUtc .stieptococci, and shows tli;it an animal londcrud iuunuuo to one of tho various races of those {viUe Unaelsheiui" * ), is.uow immuno to all other imthogouic streptococci. 18 lymphnngitis,''" bullous and phlogmonous oryaipolos, and acuto pycoiuia. Indeed, one is driven to the same conclusion with reference to the strepto- coccus pyogenes that Levy arrived at after several years' work at pyogenic micro-organisms in general.-^' Levy denies that anything can be prognosed from the nature of a microbe causing suppuration. *' The prognosis of tbo process whose origin is referable to a micro-organism, depends upon the virulence of the same, and the degree of virulence of pyogenic micro-organisms is subject to extraordinary change." It may be that similar changes in virulence will explain the pheno- mena displayed in the case of swine erysipelas and mouse sopticasmia, cholera, and the Odessa fowl disease respectively. The micro-organisms of the first pair of diseases are in appearances, size, and method of growth scarce to bo distinguished ; but whereas the one form produces a fatal disease in pigs, the other has no action whatsoever upon the»o animals ; rabbits also are susceptible to swine erysipelas, while they are said not to be affected by mouse septicajmia. Still it is worthy of notice that Lofflor has occasionally obtained fatal results in rabbits with inoculations of the bacillus of the latter disease, and that the bacilli of both diseases show themselves peculiarly fatal to white mice, producing similar effects. Quite recently, Lorenz^' has shown that pigs inoculated with cultures of the bacillus of mouse septicoimia are protected against swine erysipelas, and has described a third form, causing an eruptive disease in pigs, which he terms " Backsteiublattern." The bacillus of this, in mode of growth and properties, is intermediate between the other two, and of these tliree any one, either virulent or properly attenuated, confers immunity against the other two. So again with the second pair of diseases. It is impossible to distinguish with certainty a preparation of the vibrio Metchnikovi from one of Koch's cholera spirillum, and the differences in the mode of growth of the two are not greater than may be determined between two cultures of the cholera spirillum of different origin. Yet vibrio Metschnikovi is pathogenic for pigeons and fowls and only affects guinea-pigs (which are susceptible to inoculations of the cholera spirillum) when inoculated in relatively large quantities. According to Gamaleia^' injections of the vibrio Metschnikovi render guinea-pigs immune to cholera. Pfeiffer denies this, though Gamaliila has reiterated the state- ment, and ascribes Pfeiffer's failure to gain immunity to the fact that he employed attenuated cultures. It must, however, be admitted that Gamaleia's statement still awaits confirmation, and, for the present, it is better to look upon the series of microbes which includes Koch's spirillum of Asiatic cholera, the Finkler-Prlor spirillum of cholera nostras, Deneke's S. tyrogeuum, Miller's spirillum obtained from the mouth, and the vibrio Metchnikovi as a group of very closely allied species. 19 Similarly bo high an authority as Professor Huoppe would class together into one group, as the bacteria of hioraorrhngic septicasmia, a large number of bacteria which microscopically are undistinguiuhable, whose ends stain more deeply than the central region, whose growth upon bacteriological media is similar, and which further are identical in the appearance of the individual colonies. C. Fraenkol would separate these into two groups, one including the bacillus of ferret plague (Eberth and Scliimmelbusch), the bacillus of swine plague (Billings), that of hog cholera (Salmon), that of Danish swine plague (Selander), and the bacillus of the German "schweinepest." All these are motile and ciliated. The other group contains forma that are identical, or almost identical, namely, Luffler'a bacillus of chicken cholera, Schutz's swine plague bacillus, Cornil's bacillus of duck cholera, Gaifky's microbe of rabbit septica)niia, and Kitt and Hueppe's bacterium of " Wildseuche " (deer plague). All these only differ iu regard to the animals which they specially affect."' Here, too, I would mention a most interesting observation of Hueppe and Cartwright Wood. These observers gained from ordinary earth a harmless saprophytic bacilluH, in appearance and mode of growth resem- bling closely the bacillus anthracis. Inoculated into mice, which are of all animals the most susceptible to anthrax, pure growths of the earth bacillus induced immunity against this very fatal disease.^ ON THE OBSERVED MODIFICATIONS OF PATnOGENIO MICROBEa There is, however, another and perhaps more satisfactory way of approaching this subject of the occurrence of natural races. I refer to the actual differences in mode of growth and properties that are to be distinguished in cultures obtained from diverse typical cases of any given disease. This subject has not been worked out so fully as it deserves, nevertheless certain very interesting observations have been mnde. Thus the Talamon-Friinkel diplococcus of acute croupous pneumonia has been studied by Banti." Bauti describes four varieties of the species diplococcus lanceolatus capsulatus. No. 1 is the typical form described by Frankel and Weichselbaum. No. 2 is identical in form and culture, but causes a " Diplococcus septicflcmia " in rabbits, with small spleen and destruction of the red corpuscles. No. 3 is similarly identical, but produces a septicscmia with moderate enlargement of spleen and diffusion of hsemo- globin, and No. 4 only differs from the rest iu that its virulence outside the body disappears even more rapidly than is the case with the other forms; it produces a mild septicasmia associated with albuminuria, and the animals all recover. All were obtained originally from typical cases of pneumonia, and while in the years 1886, 1887, and 1890, the type 10 form was alono (^iiiiiod from almoat ovory ciiao of ptioumonia — nud thoso wore of a bonign imtiiro — hi 1888 and 1889 tho typo No. 1 was novor isolated, but tho othor viirioties wore present, and tho oases were severe.* FoiV has shown that by inoculation variation may be induced in tho diploooccns. If from a ral)bit that has died of iunculation pneumonia two rabbits bo inoculated, one from tho fresh fibrinous pneumonic exuda- tion, the othor from tho cercbro-spinal fluid, it is found tliat tlie disease diffurs in tlio two. The former siiows inflammatory oodema of the skin, tl»e latter none. So, too, if tho diplococcus bo grown for twenty-four hoiirs ana3robically tho latter typo is produced and its properties persist, although the original virulonco and characters may be restored by simul- taneous inoculation of this form along with the staphylococcus pyogenes aureus and the proteus vulgaris. These studies of Foil find their counterpart in Banti's researches into acute primary meningitis.^' From two oases of this diseaHO Banti isolated a diplococcus not to be distin- guished from that of Talamon-Friinkel, except that it rapidly lost its virulence both inside and outside tho body, so that when the meningeal exudation was inoculated into a rabbit, and from this a further series of passages was made, the sixth rabbit had a mild non-fatal disease. In the case of typhoid, Babes^' has made a long series of obser- vations, and though ho handles the subject with what for him is extreme caution it is difficult to arrive at any other conclusion than that the facts he represents can only be satisfactorily accounted for on the assumption that, granting there be a specific micro-organism for typhoid, and granting his results to bo reliable, this micro-organism is capable of modifications so definite that many races are developed. Babes made numerous cultures from twelve typhoid corpses, and while in nearly every case he found the typical form, in every case ho discovered also varieties. In all ho describes 18 of these, all presenting some slight differences. These were sufficiently permanent to permit him to declare that he never saw one revert to the original form (as determined by an original culture from Berlin), although in many there was a tendency towards the loss of characteristics. While acknowledging that these varieties, with rare exceptions, produce in small animals lesions similar to those due to the typical bacillus, he concludes that they are not specific, and that typical and atypical belong to a group possessing similar conditions of exis- tence. He notes also that the baccillus coli communis possesses many natural varieties. Despite this various cautious conclusion, Gaffky * An obsorvation of Nlkiforotl's may bo quoted In this connection*". From the lung of an influenza patient he obtained a microbe, identical with tho diplococcus pneumonini in every rospeot save that it preserved its virulence for longer periods, and was fully virulent for mice, while rabbits were completely refractory, whereas tho true diplococcus is virulent for both mica and 1. bblts. 11 ^who with Eberth shares the honour of having diacoverod the typhoid bacillus) criticised Babes severely, and su^giiated that all his varieties were duo to secondary " Einwanderung."** To this Biil)es replied^" by showing that if so the varieties hud wiiuderecl in duriug 111 , for his autop- sies wore made upon tho fresh corpse very few hours after death. Surely rather than multiply species to the ono/mous extent fhnt these conclu- sions would demand it is simpler, as it ia more inherently probable, to hold these varieties to be races and of a commun origin — races, it mtiy be, produced by the peculiar conditions of the contest l)etween organism and microbe. At the same time X am not prepared to go as far as Arloing^ who declares that the bacillus of typhoid can be developed from the bacillus coli communis in the presence uf fermenting feocal matter. The subject of the bacillus of typhoid cannot be passed over with- out mentioning also Cassodebat's important paper.*' The relation of epidemics of enteric fever to the water supply has been so clearly traced that all that was wanting to afford an absolute proof of the relationship was tho demonstration of the presence of the specific bacillus in the suspected sources. And this demonstration would seem to have been made by the methods of Vincent, Rodet, Kitasato, Chantemesse and Widal, Thoinot, and yet others. But Cassedebat, working at Marseilles where enteric fever is almost endemic, states that in the water supply — the Durance — he was unable to discover the typical bacillus, although in its place he determined three forms all resembling the bacillus of typhoid in their mode of growth upon potato (which is very charac- teristic), and in the form of the colonies upon agar-agar plates, all equally polymorphous, and staining with difficulty, all possessing the same movements of translation and oscillation, and giving much the same growths upon puncture of gelatine, while in even so minute a characteristic as the arrangement of the cilia, Lijffler's method showed no difiference between No. 1 and the type. (It is not stated how the others appeared under this treatment). But there were differences of rate of growth on potatoes, in gelatine, potato-juice-gelatine, and broth, differ- encesalsointhe intensity of colour of potato cultures, differences in the effect of the bacilli upon media to which aniline dyes had been added, and the result on inoculations, which were far from complete, show that on the wh(4e No. 1 was less virulent for mice than in the type Eberth-Gaffky bacillus. From these differences Cassedebat concludes that he is dealing with pseudo-typhoid forms, and that the results of the bacteriological examination of water must be received with very great caution, if they are to be accepted at all. I have already shown that the Eberth-GafFky bacillus is capable of very considerable modifications, is most susceptible to change of medium, etc., and here, again, rather than accept Casse- 22 debat's conclusions, I would hold that the more satisfactory explanation of his facts is that he dealt with races — natural and fairly permanent races — modifications of the type bacillus of typhoid. Turning now to cholera and its spirillum, it is already a well-known fact — to which attention was, I believe, first called by Zaslein** — that cultures of the spirillum derived from simultaneous epidemics in different localities present recognisable differences — differences in tint, in rate of growth, in power of liquefying gelatine, etc. So there are those who declare that from the appearance of a culture they can state its origin, whether from Cairo or from Berlin, from Naples or Massowah, from Palermo or Marseilles. It has, however, been left to Surgeon-Major Cunningham to give the most remarkable example of these differences.^* At the beginning of 1890, three of the principal hospitals in Calcutta contained cases of cholera, not differing from one another in symptoms or virulence, all giving abundance of cultivable spirilla, but these not of one, but of three "species." Later, other cases were observed, in all sixteen, and from these sixteen ten "species" were obtained a to k. In only four of the cases was the form present, even doubtfully, that described by Koch (Cunningham, bacillus a). In some, three different comma bacilli were present at the same time. The forms described by Cunningham may be divided into two groups. The first contains only one which does not liquefy gelatine, does not show interstitial growth in agar-agar, does not develop " cholera red " on the addition of acids, and morphologically is distinguished by its large size and great variability of form and curvature. This, I feel inclined to consider as a truly different species. The other group contains all the other nine, which liquefy gelatine, are capable of interstitial growth, give the " cholera red " reaction, but all show fairly permanent differences in the rate of liquefaction of gelatine. In six the rate is distinctly rapid, in the rest it is slow. In one, growth upon potato is luxuriant, in others there is rare acclimatisation to this medium, in others again accli- matisation has never shown itself. Here, then, one working where cholera is endemic, and one who is a most capable observer, who had been with Koch when he studied the disease in India, is so affected by the general view that species are constant and distinct, that the only con- clusion that he can draw from these facts is that " whilst denying that the primary cause of cholera is represented by any species of intestinal schizomycete which has yet been discovered" it may be "that those whose development is favoured by the existence of the choleraic condi- tion may exert an important influence on the ultimate outcome of indi- vidual cases of the disease." If we accept the view that the spirillum is specific I do not see any other conclusion from these observations than that here is another well-marked instance of the production of allied races. Only within the last few days news has come from St. Petersburg that the spirillum obtained from patients affected in the cholera epidemic now raging, presents marked differences in size, etc., distinguishing it from Koch's original form ; while from the Institute Pasteur I hear that the microbe, isolated from some of the doubtful cases in Paris, presented no greater departure from type than is to be seen in many of the growths obtained from indubitable cases of cholera. I might enter into the discussion of the relationship between human, bovine, and avian tuberculosis, or again into the differences to be made out between the bacilli of pulmonary and surgical tuberculosis (lupus, tuberculosis of bone and joints, and scrofula), but though much has been done on these subjects there is a want of well confirmed results, and the subject is still too chaotic for safe treatment. I will conclude this relation of the occurrence of natural races with a reference to Rous and Yersin's remarkable and admirable work upon diphtheria."" Apart from proving that the bacillus isolated by Loffler'' is specific, by showing that the sterilised medium of growth inoculated into animals will induce the characteristic symptoms of the disease (including the slowly produced paralyses), these observers made careful studies of the patho- genic qualities of the microbe. They found that, according to their origin, some cultures would kill guinea-pigs in twenty-four hours, some in sixty, some in only three or four days, or even after a longer interval, and they turned their attention to a form or forms, that had been described by Loffler as the bacillus pseudo-diphthericus. Loffler found this in the false membranes tilong with the virulent micro-organism, and pointed out that it is practically uudistinguishable from this last, save that it has no toxic effect upon animals. Hoffmann found it also in the pharyngeal mucous membrane of cases of scarlatina and measles. Its discoverer considered it a separate species, and in this view most observers joined, though Klein and Fliigge admitted that it was an attenuated form. The colonies on coagulated blood serum are identical with those of the diphtherial bacillus ; like that, it grows rapidly at a temperature of 33" to 35° C. ; microscopically, the two arc the same, the staining reactions are similar, and growth in alkaline beef broth is characterised by the same production of acidity followed by increasing alkalinity. The differences are, that it is often shorter when grown on blood serum, that the cultures on broth are more abundant, and so are those on agar- agar. It still grows at 20° to 22° C, a temperature at which the virulent bacillus ceases to proliferate, and the alterations in alkalinity and acidity of both cultures proceed more rapidly. This form is very rare in malignant cases of diphtheria — it is more abundant in benign, and becomes increasingly common as severe diph- theria progresses towards cure. But as I have already indicated, it is to 24 be discovered in non-diphtherial cases. At the Hopital des Enfants Malades, Paris, from the pharyngeal mucous membrane of 45 children not affected with the disease it was obtained in 15 cases, and of 59 healthy school children at Caen, in a school where for long there had been no diphtheria, no less than 26 gave growths of this form, though it must be confessed that the colonies were few and far between. Inocula- tion into guinea-pigs and rabbits never led to fatal results, and yet differences were observable. Some guinea-pigs manifested a very con- siderable a3dema at the point of inoculation, in others a slight oedema was to be seen, others presented no sign of even local disturbance. It is noteworthy that the most marked oedema was developed where cultures were employed whose origin was from cases of measles. (Edema at the point of inoculation is one of the characteristic lesions when virulent diphtheria bacilli are injected, and Roux and Yersin found that if they attenuated virulent cultures either by the action of air and high temperature, or again, if they preserved dried-up false membranes for five months in a cool place and in the dark, they gained colonies, some of which caused no oedema, others a little, one alone caused marked oedema. Such attenuated bacilli, like the bacillus pseudo-diphthericus, grew more abundantly at a lower temperature, and, like this, also rendered broth more rapidly alkaline. In fact, the only point distin- guishing the two was that our observers succeeded in intensifying the virulence of the attenuated form, and failed to do this with the pseudo- diphtherial bacillus. Tn every other respect the proof appears conclusive that this bacillus pseudo-diphthericus is a natural race, or races, of the bacillus diphthericus, that it is no wise a separate species of independent origin. What is more, Roux and Yersin suggest an explanation of the onset of diphtheria — of the natural intensification, that is, of the virus in man. Just as they found that from a case of measles presenting no sign of diphtheria proper they gained a race of the bacillus pseudo-diphthericus, which exhibited distinct, if slight, virulence, so have they noticed cases in which, through the access of an anginal attack, or of measles, diph- theria, which was recovering, gained fresh malignancy and became rapidly fatal. There is another micro-organism frequently found in the mouth and saliva of healthy persons, namely, the Talamon-Frankel diplococcus. May it not be that in both these cases a natural intensification of the virus is brought about by catarrhal and other affections of the upper respiratory and pharyngeal mucous membrane, and that to such intensi- fication, rather than to the entry afresh of virulent micro-organisms, is to be ascribed the onset of these diseases ? 25 ■, ' I am well aware that in setting in order a series of facts and observa- tions upon deviations from the normal the tendency is peculiarly strong to see in such deviations the normal, to see in the normal the excep- tional, and It may be that in the preceding pages I have not dwelt with sufficient emphasis upon the fact that employing well recognised and standard methods the typical forms of bacterial growth are easily and most usually to be separated out from cases of disease. Still I shall feel that these pages have not been written in vain if I succeed in drawing increased attention to the fact that the bacteria are organisms acutely susceptible to changes in environment, that as a species they are far from presenting constant characteristics, and that to a variability which may impress itself upon a greater or less number of generations is to be ascribed, in part, the differences between successive epidemics, between the successive stages of one epidemic, and between individual cases of disease. REFERENCES. 1. Naoeu.— " Die niederen Pilze," Munich, 1877. 2. Lankester. — Quart. Journ, Micr, Science, XIII., 1873, p. 408. 3. KxJRTH. — Botanische Zeitung, 1883. 4. Vincent. — Comptea rendus de la Soe. de Biologic, 1890, No. 5. 6. Fbankel and Simmonds. — Zeitschrift f. Ifi/giene, II., 1887, p. 138. 6. Bdchner. — Centralblatt f. Bakteriologie, IV., 1888, p. 353. 7. Schiller. — Arbeitem aua dem Kais. Oesundheitsamb, V. 8. FsiRvacaKY.— Centralblatt f. 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