Infective diseases of unicellular organisms.—Intracellular digestion in the Protozoa.—Amoebo-diastase.—Part played by digestion in the defence of the Protozoa against infective parasites.—Defences of the Paramaecia against micro-organisms.—Part played by irritability in defence in the lower organisms.
Immunity of unicellular organisms to toxins.—Acclimatisation of Bacteria to toxic substances.—Protective secretion of membranes by Bacteria.
Adaptation of the Protozoa to saline solutions—of yeasts to poisons—of yeasts to milk-sugar.
Irritability of unicellular organisms and Weber-Fechner’s psycho-physical law.
The immunity of unicellular organisms against infective diseases and against toxic agents is as yet very imperfectly understood. Nevertheless, it will be very useful for us to begin our study of the problem of immunity on these lower organisms, because of their greater general simplicity. It may be affirmed that if the line of comparative pathology had been followed in our study of the etiology of diseases of man and the higher animals, the parasitic nature of these infections would have been established considerably earlier than was the case. Thus, at a period when medical men and veterinary surgeons were content to record the presence of Bacteria in the blood of their patients, without attributing to them the slightest etiological rôle, botanists and zoologists had already proved most definitely that many plants and lower animals were subject to epidemic diseases undoubtedly set up by the parasitism of various exceedingly simple organisms. In the same year, 1855, that Pollender[4] published his first observations on the bacterium found in the blood of animals affected by anthrax though he could not trace the slightest relation between the presence of this organism and the etiology of the disease, the celebrated botanist Alexander Braun[5] issued his work on the genus Chytridium, in which he demonstrated the fact that certain plants and flagellated Infusoria suffer from the invasion of a small mobile parasite which, attaching itself to their body wall, absorbs the contents and so destroys its hosts, causing a very great mortality among them. The cycle of development in the Chytridia, established by Braun, left no doubt as to the accuracy of his view and even renders it possible for us to interpret more accurately the earlier observations of Stein, on the supposed evolution of certain Infusoria, by showing that the changes observed in these organisms were in reality due to an invasion by Chytridia.
Since these observations were made it has been clearly demonstrated that among the unicellular organisms, certain Flagellata and ciliated Infusoria are subject to infective maladies the result of parasitism of the Chytridiaceae, a group of the lower Fungi. Small, mobile, colourless cells attach themselves to the surface of the Protozoa, penetrate into their interior and absorb the greater part of their living content. Sometimes these parasites multiply in a most extraordinary fashion and destroy enormous numbers of the Infusoria. Thus, Nowakowski,[6] who has given a very detailed description of Polyphagus euglenae, the Chytridium of the common green fresh-water Euglena, records the disappearance of the Euglenae from his aquaria glasses: the parasites “were reproduced in such great abundance that ultimately they had completely replaced the Euglenae.”
The Flagellata, subject to infection by Chytridia, are found almost exclusively amongst those genera (Cryptomonas, Chlamydomonas, Haematococcus, Phacus, Volvox, etc.) which are nourished after the fashion of vegetables, that is by the absorption of substances dissolved in the surrounding fluids. It is very remarkable that in the group of ciliated Infusoria this parasitism of the Chytridia is observed almost solely in the encysted forms, that is to say, at a stage when the animalcules, surrounded by their envelope, do not take any nourishment. The invasion by the Chytridia has been demonstrated in the case of the cysts of the Vorticellina, Oxytrichinina, Nassula, etc.[7] These facts indicate that the absence of the digestion of solid aliments, such as occurs in almost all the ciliated Infusoria, constitutes a condition favourable to infection by the Chytridia. Whilst the growth of Volvocina, Euglenae and their allies is almost always interfered with by very destructive parasitic epidemics, the ciliated Infusoria, capable of seizing and digesting lower organisms, may be cultivated and flourish for a very long period. Thus Balbiani[8] has watched one of his cultures of Paramaecium aurelia multiply and thrive in splendid condition for 14 years in succession. Now these Infusoria readily adapt themselves to ordinary water untreated to render it more hygienic. Such water swarms with all sorts of lower organisms, among which are the Chytridia and numerous Bacteria, but the Paramaecia and Infusoria in general feed upon these organisms and contribute largely to the purification of the water. Almost the whole body-contents in a ciliated Infusorian is made up of a digestive protoplasm into which the captured Bacteria and other lower organisms are conveyed; the nutrient particles becoming surrounded by transparent vacuoles, in which the ingested organisms are killed and digested. The food contained in the vacuoles circulates in the endoplasm of the Infusoria by means of the streaming movements of this layer. The digestive vacuoles become filled with a fluid having a distinctly acid reaction. Formerly, in order to demonstrate this reaction, Infusoria were allowed to ingest small granules of blue litmus which after a certain time became more or less intensely red; but the use of aniline colours has much simplified the study of digestion in microscopic organisms. By introducing a solution of alizarin sulpho-acid into a liquid containing Infusoria, the yellow staining (characteristic of the acid reaction) of the digestive vacuoles can be readily made out. When the Infusoria ingest small clumps of alkaline substances, stained violet by this reagent, the vacuoles take on a red tint, indicating the acidity of their contents[9]. Another aniline colour, neutral red (Neutralroth), introduced into microscopical technique by Ehrlich[10], enables us to demonstrate the acid reaction in the digestive vacuoles even within a few minutes. Thus, in Paramaecia treated with a dilute solution of this reagent, the digestive vacuoles at once assume the deep rose tint, characteristic of an acid reaction. This coloration is observed during the life of the Infusorian, but immediately after death the vacuoles become brownish and then completely lose their colour. This reaction, easily demonstrated, indicates that neutralisation of the acid of the vacuoles by the protoplasm and the surrounding water, both of which are alkaline in reaction, has taken place.
In a medium distinctly acid the Infusoria digest their prey which, in a very great number of cases, consists of Bacteria. These micro-organisms are swallowed and carried into the digestive endoplasm in the living condition; we have evidence of this in the active movements of a certain number of the bacteria; at first they are found isolated in the interior of the vacuoles, but later they collect into more or less compact clumps. These masses of micro-organisms undergoing digestion, when treated with neutral red assume a very deep rose tint, preserving their bacillary form to the end, that is to say up to the extrusion of the effete or waste material. There is, indeed, only very imperfect dissolution not only of the bacilli as a whole but also of their contents. Paramaecia placed amongst cholera vibrios swallow them greedily and in great numbers, digesting them as they would any other micro-organism. I have never been able to see any conversion of vibrios into granules going on within the digestive vacuoles.
All the attempts that have been made in my laboratory to extract a digestive fluid from Paramaecia have failed entirely. Very large quantities of these Infusoria, obtained by filtration of rich cultures, and macerated by different methods, have proved inactive even in the case of those Bacteria which constitute their normal food.
Intracellular digestion in the Infusoria unquestionably takes place as the result of the action of some diastase; but from the impossibility of observing the action in vitro the properties of this diastase, except that it can act in a distinctly acid medium, cannot be determined.
Even less is known concerning the digestion of Rhizopods than concerning that of Infusoria. It has long been recognised that, in the majority of cases, Amoeba, Actinophrys and Rhizopods in general, absorb a nourishment composed of lower plants and animals, which are taken into the protoplasmic body by means of the movements of amoeboid processes, pseudopodia or lobopodia. Once within the Rhizopod the nutritive particles are surrounded by a digestive fluid, in which the presence of acid may be recognised by means of colour reactions. The addition of a drop of Ehrlich’s neutral red to Amoebae in the act of digesting Bacteria at once gives the acid colour reaction (Fig. 1 ). Rhumbler[11] has described very precisely and with much detail the way in which the Amoebae behave when they are incorporating filaments of Oscillaria very much longer than their own bodies. He has also described the digestion that these Algae undergo; a process most characteristic in those cases where a portion only of the filament has been taken into the interior of the Amoeba and there subjected to the digestive action. Whilst the free part of the Oscillaria retains its normal properties and appears of a bluish green colour, the ingested portion progressively changes colour, assuming first a deep green tint, then becoming light yellow, orange yellow, brown and finally reddish brown. Simultaneously the cellulose wall of the Alga begins to soften, and the cells break up into minute fragments which are soon extruded. The food is seldom completely digested and there is always an abundant residual material which is thrown out in the form of solid excreta.
Fig. 1. An Amoeba treated with neutral red, 1%.
Although it is fully recognised that, in the Rhizopods, digestion goes on in a medium distinctly but feebly acid, and that the intervention of some soluble ferment is essential, our ideas on this subject were very vague until the publication of the researches of Mouton[12], carried out with great care in the Pasteur Institute. In order that he might obtain exact results Mouton made use of cultures of Amoebae grown on agar, in association with the Bacillus coli which served them as food. The bacilli were ingested in large numbers, became enclosed in vacuoles and were digested by a ferment which Mouton was able to obtain in vitro. To that end he collected large numbers of Amoebae, and, after centrifugalising them in water, treated the deposit with glycerine. On adding alcohol he obtained a precipitate readily soluble in water.
The fluid thus obtained exerted an undoubted digestive action upon albuminoid substances. It readily liquefied gelatine and even attacked, though feebly, albumen coagulated by heat; flakes of fibrin heated to 58° C. remained unaltered. There was present then, in this fluid derived from Amoebae, a proteolytic diastase of feeble activity. On the other hand, this extract contained neither sucrase, capable of inverting cane sugar, nor lipase, capable of digesting fatty matters.
The amoebo-diastase of Mouton must be classified with the trypsins. It is very active in a distinctly alkaline medium and continues the diastatic action even when the medium becomes weakly acid (a feature that corresponds to the reaction observed in Amoebae treated with appropriate staining agents). The amoebo-diastase is affected at as low a temperature as 54° C. and at 60° C. is rendered completely inactive.
A question of especial importance is that concerning the action of the amoebo-diastase upon Bacteria. The numerous experiments of Mouton directed to the solution of this point, and made with living Bacillus coli communis, gave negative results. If, however, these bacilli were previously killed by heat or by chloroform, they were at once attacked by the soluble amoebo-ferment. Opalescent emulsions of these dead bacilli, incapable of undergoing self-digestion of any kind, became transparent after remaining for some time in contact with the extract of Amoebae. The amoebo-diastase, then, undoubtedly digests dead bacilli in vitro, whereas in the body of the Amoebae the ingested bacteria are attacked whilst still living. As a result of these observations it must be concluded that only a fractional part of the diastase is extracted in the solution prepared by Mouton.
This intracellular digestion in the Protozoa serves not merely for the nutrition of these organisms, but also as a protection against infective parasites. The protoplasm of the Infusoria, with its vacuolar secretions, has a general digestive action on everything that comes within its reach. If the internal structures, such as the nuclei and the pulsatile vacuoles, resist this process, it is undoubtedly because they possess a power of defending themselves against the attack of the digestive secretions. Thus, as brought out in the beautiful researches of Maupas[13], the macronucleus of the Paramaecia is, at a certain stage in the life of the Infusorian, completely digested by the protoplasm just as is any other nutrient substance introduced from outside. It must be admitted that in this case the nucleus has ceased to produce the protective substance which, under ordinary conditions, interferes with its being digested.
A struggle similar to that observed between the nucleus and the digestive content of the Protozoa goes on between these latter organisms and infective microbes. All organisms which, in any way whatever, penetrate into the body of an Infusorian or Rhizopod, are brought into contact with the digestive endoplasm of these Protozoa. If the intruders are killed and partially digested by the digestive secretions, or are expelled as excrementitious matter, the Protozoon remains uninjured and continues its normal and routine existence. Here, then, we have an example of natural immunity, due to intracellular digestion. On the other hand, when the foreign parasitic organism resists this digestive action, it instals itself permanently in the body of the Protozoon, and should it reproduce itself in small numbers merely, excrete no poison and, in general, exercise no injurious influence upon its host, the parasite may readily become a commensal. Thus, it is not rare to find in the contents of Infusoria and Radiolaria small vegetable organisms of the genera Zoochlorella or Zooxanthella which not only set up no disease but, owing to their assimilation of carbonic acid, may even be useful to their hosts. There are cases, however, where the parasites act in a manner more or less injurious to the Protozoa containing them; in such cases a true and sometimes fatal infection results.
Among the infective diseases of the Protozoa the one that has been most thoroughly studied is that set up by several representatives of a particular genus of micro-organisms discovered by Johannes Müller in 1856 and made the subject of an investigation carried out in my laboratory by Hafkine[14]. I have already discussed these researches in my work on the comparative pathology of inflammation[15] and need here recapitulate only very briefly. Paramaecia are sometimes affected by needle-shaped or spirillar parasites which penetrate, sometimes into the macronucleus, sometimes into the micronucleus, reproducing prolifically, giving rise to a marked hypertrophy of the affected organs. The Infusorian, in spite of this invasion, may continue to exist and carry on its reproductive processes; it is, thus, enabled in many cases to recover from the disease. On the other hand the Paramaecium into whose body the spores of the parasite are introduced treats them as it would any other ingested foreign body. Not being able to digest them, owing to the resistance offered by the membrane of the spore, the Paramaecium expels them just as it would any other excrementitious matter. The Infusorian behaves in the same way in regard to bacterial endospores.
Hay bacilli, which occur so commonly in the infusions in which the Paramaecia live, are digested in the endoplasmic vacuoles of the latter, but the spores of these bacilli, after a more or less prolonged sojourn in the vacuoles, are expelled with the excrement.
As by far the greater part of the body of a Protozoon is made up of digestive protoplasm, it is natural that infective epidemics should be very rare among these animalcules. The Infusoria and Rhizopods, organisms specially well adapted to live upon the lower Algae and Bacteria, are, practically, never subject to bacterial diseases. The infections observed in the Protozoa are due in most cases to the invasion of the lower Fungi, such as the Chytridia, the Microspheres, the Saprolegniae or the special organisms mentioned as occurring in the nuclei of Paramaecia. Further, these infections are met with most frequently in Protozoa which are incapable of carrying on true intracellular digestion or which are in the encysted stage, at which period the Infusoria, leading a passive existence, neither absorb nor digest nutriment. As an exception to the above general statement I ought to mention the epidemic in Amoebae caused by the Microsphaera[16] and the disease in Actinophrys observed by K. Brandt[17] and attributed to Fungi allied to the genus Pythium. In these two instances we have to do with parasites which live and develop in the interior of the active protoplasm of these Protozoa. Certainly a proportion of the parasites are expelled with the excrementa; but there remain others which instal themselves in the protoplasm, multiply there and cause the death of their hosts. In these cases the digestive action of the protoplasm must be neutralised or paralysed by the secretions of the parasite. This aspect of the question, however, has so far not been considered.
In addition to intracellular digestion and the expulsion of parasites by the excretory function, the resistance offered by Protozoa to infective diseases should, in part, be ascribed to their great irritability. Anyone who will watch the manœuvres of Amoebae or of certain Infusoria in the midst of a rich microscopic flora and fauna, will at once be struck by the preferences which these Protozoa exhibit in the choice of their food. Amoebae are often seen making search for Diatoms only, disdaining all other Algae, or again they may single out one species of Palmellaceae from a very varied flora. The Infusoria also have their likes and dislikes in the matter of food. Many of the ciliated Infusoria choose Bacteria to the exclusion of almost everything else; others, as Nassula, have a special partiality for the Oscillariae. A most striking example of this is afforded in Amphileptus claparedei, a voracious Ciliate, which chooses Vorticellae to the exclusion of all other animalcules; these it devours, and then becomes transformed into a cyst upon the peduncle of the Vorticellae it has devoured. This irritability clearly must control and guide the Protozoa in their relations with other organisms and enable them to escape the invasion of parasites.
In this connection I must mention a very interesting observation made by Salomonsen[18] and communicated to the Paris International Medical Congress in 1900. He was able to demonstrate the fact that almost all the ciliated Infusoria, on becoming aware of the proximity of dead bodies of kindred organisms, rapidly draw away, thus manifesting a very marked negative chemiotaxis. This property must, it is evident, protect them from any contamination by the parasites contained in the bodies of Infusoria that have succumbed to infective diseases.
We have, then, quite a number of facts which throw light on the natural immunity of the Protozoa against the action of pathogenic micro-organisms. Up to the present, however, we know nothing concerning the existence or the possibility of an acquired immunity among the lower animalculae against infective diseases. We are better informed as to the resistance of unicellular organisms to the action of soluble poisons, which is, in general, much more easily studied than is immunity against the micro-organisms themselves.
As a very large number of the higher animals are sensitive to the toxic action of poisons of bacterial origin, the question has been put, “May not the Infusoria also be poisoned by these micro-organismal products?” With the object of answering this question Gengou[19] has studied the influence of the toxins of tetanus and diphtheria on the ciliated Infusoria. He was unable, however, to bring forward proof that these substances exert any special toxic action on the Paramaecia. These Infusoria withstand, perfectly well, doses of cultures of the diphtheria and the tetanus bacillus grown in broth and deprived of the bacilli by filtration as large as those of ordinary broth alone in which no bacilli have been cultivated. Gengou argues from this that the Paramaecia possess a natural and absolute immunity against these two toxins. When we take into consideration the fact that these poisons act but feebly at ordinary temperatures and are often innocuous to “cold-blooded” animals we may perhaps be tempted to attribute the immunity of the Infusoria to the temperature that was maintained in the incubator whilst Gengou’s experiments were being carried on. Led by this train of thought Mme Metchnikoff tried the action of the blood-serum of eels, which is very toxic, not only for warm-blooded Vertebrates but also for cold-blooded Vertebrates and the Invertebrates, on the Paramaecia, and this at a low or medium temperature. This eel’s serum, however, exerted no greater toxic action than did the blood-serum of other animals.
The microbial toxins are innocuous not only to the ciliated Infusoria but also to many other unicellular organisms. It is now well recognised that these toxins, exposed to the air, are soon inhabited by quite a rich flora of micro-organisms, amongst which Bacteria and Yeasts predominate. I have been able to prove[20] that these organisms are not only unaffected in their normal life by the presence of the toxins of diphtheria or tetanus but that they rapidly bring about the more or less complete destruction of these poisons. Gengou, also, observed that yeasts thrive luxuriantly in these bacterial toxins. The rapid increase of micro-organisms and the destruction of these poisons take place at temperatures varying from 15° to 37° C.
Whilst the lower organisms are refractory to bacterial toxins which in quite small doses are capable of killing man and the higher animals, many micro-organisms manifest a special sensitiveness to certain fluids of animal origin. In a succeeding chapter we shall treat at greater length of this microbicidal property of the humours. Here it is merely necessary to indicate certain facts concerning this property, regarding them solely from the point of view of the immunity of the lower organisms. The most striking example of the bactericidal power of an animal fluid is certainly that afforded in the action of the blood-serum of the rat on the anthrax bacillus. This fact, discovered in 1888 by von Behring[21], led to the conclusion that the blood of the rat contains an organic base capable of killing and dissolving a considerable number of anthrax bacilli. Several observers have confirmed von Behring’s observation and have supplemented it by the fact that the bacillus can be readily accustomed to the toxic action of this serum. Thus Sawtchenko[22], in an investigation carried out in my laboratory, was able, by successive cultures, to accustom the anthrax bacillus to an existence in the pure serum of the rat. In this case, therefore, there has been produced a real acquired immunity of a lower plant against a toxic substance of animal origin. More recently Danysz has demonstrated the same thing and has added several other facts which seem to throw light upon the means by which the bacterium becomes adapted to the poison. He has shown, in a work carried out in the Pasteur Institute[23], that the anthrax bacillus protects itself against the toxic action of the serum by surrounding itself with a thick sheath composed of a kind of mucus which fixes the toxin of the rat’s blood and renders it harmless. This same mucus, but in smaller quantity, is likewise produced in a culture of the bacillus grown in ordinary broth. When such a culture is freed from the contained bacilli by filtration through porcelain and a little of this fluid is added to the rat’s serum, this latter becomes less bactericidal than is a mixture of the same serum with ordinary broth. Danysz suggests that this is to be explained by the presence in the filtrate of a certain quantity of the mucous substance produced by the bacillus, which fixes and neutralises a portion of the “rat toxin.” If, in place of sowing the ordinary bacillus, sensitive to this toxin, we inoculate the broth with an anthrax bacillus which has previously been accustomed to the rat’s serum, we find that the liquid of this culture when filtered neutralises a larger proportion of the toxin. Danysz concludes from this that the acclimatised bacillus has acquired the property of producing more mucus than does the ordinary bacillus and that, for this reason, a greater quantity of this protective substance passes into the fluid of the culture.
The formation of a transparent sheath has several times been observed in the anthrax bacillus, notably in cases where this organism happens to be in “a state of defence” against various noxious influences. For example, this sheath is well developed in the anthrax bacillus which invades the blood of lizards, animals which are in general very resistant to anthrax[24]. Under analogous conditions the streptococci which, as a rule, do not produce a mucous sheath, will develop one of exceptional size. The guinea-pig is in general very resistant to the streptococcus against which it exhibits a very effective reaction. Sometimes, however, this immunity gives way; in such instances, as demonstrated by J. Bordet[25], the streptococcus, in order to overcome the natural resistance of the guinea-pig, is found to have surrounded itself with a sheath of a thickness such as is seldom to be met with in the world of bacteria (Fig. 2).
Fig. 2. Streptococcus surrounded by a protective envelope.
Fig. 3. Tubercle bacillus surrounded by a transparent envelope and enclosed in the giant cell of a gerbil.
Analogous facts are also observed in cases where the micro-organism is defending itself against the action of substances enclosed in animal cells. I may cite as an example the tubercle bacillus in the interior of the giant cells of a gerbil (Meriones shawii), where, under the influence of noxious substances contained in these cells, the tubercle bacillus (Fig. 3) envelops itself in a transparent sheath similar to that of the bacillus or of the streptococcus. As the action of the giant cell still does not cease, the tubercle bacillus secretes a second sheath (Fig. 4) and continues to surround itself with quite a series of such envelopes (Fig. 5), thus coming to resemble a palmellaceous Alga surrounded by successive layers of membranes or certain other vegetable cells whose principal means of defence against all kinds of injurious influences consists in the production of these protective membranes.
Fig. 4. Another tubercle bacillus surrounded by two membranes.
Fig. 5. Tubercle bacillus surrounded by a series of concentric layers.
Quite recently Trommsdorf[26], in Buchner’s laboratory in Munich, has carried out a series of experiments on the adaptation of the cholera vibrio and of the typhoid bacillus to the bactericidal substance found in the blood of the rabbit. He has been able to confirm the results of his predecessors and by various experiments has convinced himself that these two micro-organisms are capable of adapting themselves to existence in the defibrinated blood and in the blood-serum of the rabbit.
The immunity, or acclimatisation of injurious organisms to different toxins, presents an undoubted analogy to the phenomena of adaptation shown by these organisms to mineral or organic poisons. It has long been known that the same species of Protozoa are met with in both fresh and salt water and that it is possible to gradually accustom Infusoria and Amoebae to tolerate an amount of sea salt which at first is absolutely fatal to them. This toleration is not acquired unless care be taken to increase the amount of salt very gradually: too abrupt a rise inevitably causing death. By this means Cohn[27] accustomed the fresh-water Euplotes to a life in artificial sea water containing 4% of sodium chloride. In Balbiani’s experiments[28] the fresh-water Monads (Menoidium incurvum and Chilomonas paramaecium) died very quickly on the addition of ½% of this salt; but when it was added in small successive doses (0·05 per day), they readily became accustomed to a concentration of 1%. In the encysted state the Protozoa are even more resistant than in the active state to the different salts that may be added to their normal culture medium. It is probable that the wall of the cyst interferes with the penetration of these substances into the endoplasm. If a small quantity of an aniline dye be added to a fluid containing encysted Infusoria, it is seen that the cyst-membrane becomes very intensely coloured but the body of the Infusorian remains unstained. The membrane absorbs a large amount of colouring matter, after which, being saturated, it ceases to take it up; but it does not allow the dye to penetrate into the endoplasm.
Balbiani (loc. cit. p. 580), having compared the action of the salts of sodium with that of the salts of potassium and lithium on Infusoria, comes to the conclusion that the injurious influence of these substances can only be partially explained by osmotic phenomena. In addition to these a purely chemical action must be invoked. He bases his opinion on the fact that the isotonic solutions of the three salts acting on Infusoria of the same species and same origin exert a different influence. The salts of potassium and of lithium act in a much more energetic fashion than do the sodium salts. Consequently, the Protozoa are able to adapt themselves progressively not only to noxious influences of a physiological character but also to those of a chemical nature. Thus Infusoria and Rhizopods can be accustomed to the action of high temperatures, to an intense light, etc. On the other hand they can also be habituated to the toxic actions of true poisons. Davenport and Neal[29] have established the fact that Stentors kept for two days in a weak solution of corrosive sublimate (0·00005%) acquire a tolerance to a dose of this poison four times as great as the lethal dose for individuals previously kept in pure water. The same thing has been observed in connection with the toxic action of quinine. This immunity cannot be attributed to the selection and persistence of those Infusoria which possess a natural resistance to the sublimate. It is really acquired as the result of a direct and gradual chemical influence on the protoplasm of the Stentors which, once adapted, all survive doses which are lethal for the unacclimatised control organisms.
The vegetable micro-organisms, which are much more easily cultivated than are the Protozoa, frequently manifest most characteristic phenomena of acclimatisation. The first systematic researches in this direction were carried out by Kossiakoff[30] in the laboratory of Duclaux. He studied the antiseptic action of borax, of boracic acid, and of corrosive sublimate on the anthrax microbe and several other bacilli (Bacillus subtilis, Thyrotrix scaber and T. tenuis). He found that all these micro-organisms can be gradually accustomed to doses which are absolutely bactericidal to the same species when not so acclimatised. The acclimatised Thyrotrix tenuis withstands almost double the amount of bichloride of mercury that the non-acclimatised bacillus will resist. The ordinary anthrax bacillus will not develop at all if the culture medium contains more than 0·005 of boracic acid whilst the same organism, when accustomed by passage through successive cultures in which this substance is present in gradually increasing proportions, grows well in spite of the presence of 0·007 of the same antiseptic. Since these observations were made similar facts have been demonstrated by several other observers, and the ready acclimatisation of Bacteria to poisons is now generally admitted. Danysz (loc. cit.), with the object of elucidating the mechanism of this adaptation, has studied the action of arsenic acid on the Bacillus anthracis. He has demonstrated that this bacillus will gradually accustom itself to grow in broth containing a quantity of arsenic acid which at first inhibited all development. During this phenomenon of adaptation, which is acquired after a series of passages through media more and more highly arsenicated, the bacillus secretes a coating of mucous substance which protects the sensitive parts of the microbial cell. Here, therefore, is formed something exactly corresponding to what the same observer has demonstrated in anthrax bacilli that have acquired a tolerance for rat’s serum. This analogy extends even to the throwing out of the protective substance into the culture fluid. When one sows an ordinary unadapted bacillus in arsenicated broth to which has been added some of the fluid from a culture of the adapted bacillus, development takes place in a marked fashion. On the contrary when the same material is “seeded” into arsenicated broth of the same composition but to which has been added the filtrate from an unadapted culture, the bacillus does not develop nearly so well. The difference is explained by the presence, in the fluid in which the adapted bacillus had grown, of a certain quantity of the mucous substance which fixes the arsenic and prevents it from acting on the protoplasm of the micro-organisms.
The Yeasts, also, adapt themselves very readily to antiseptics. This property has even had a practical application. We know that small doses of hydrofluoric acid are capable of preventing the proliferation of the yeast of beer, and Effront[31] has accustomed this plant to live in media containing an amount of hydrofluoric acid which is absolutely inhibitory to the unadapted yeast. Under these conditions the adapted cells undergo a stimulation which causes the production of a greater quantity of alcohol. The yeast, in adapting itself to antiseptic doses (300 mm. of hydrofluoric acid per 100 c.c. of beer wort), acquires a kind of immunity which it did not possess in the first instance. Moreover this acquired property can be hereditarily transmitted to new generations developed in ordinary beer wort to which hydrofluoric acid has not been added. The stimulating action of this substance on the fermentative property does not depend upon the acid reaction of the hydrofluoric acid, for other acids which are non-antiseptic, such as tartaric acid, are incapable of inducing it.
The acquired immunity against hydrofluoric acid is strictly specific, the yeasts that have been adapted to this substance becoming even more susceptible to the action of other poisons.
Duclaux[32] has already insisted on the relations which exist between antiseptics and foods. Formic aldehyde which has a very powerful coagulative and therefore strongly antiseptic action on protoplasm may actually serve as a food for micro-organisms. The Thyrotrix tenuis, studied in this connection by Péré[33], adapts itself to the presence of this aldehyde and utilises it for its nutrition. Here is produced something that recalls the case of the Protozoa that digest parasitic organisms.
It is now a current idea in microbiology that Bacteria and Yeasts which primarily do not make use of certain substances, adapt themselves to use them as nutrient substances. Dienert[34] has published a detailed work on the adaptation of the yeasts to milk-sugar. This sugar is usually disdained by the yeasts that set up the fermentation of glucose; it is not difficult, however, to adapt them to galactose which they then attack and transform into alcohol and carbonic acid.
The Protozoa can be progressively accustomed not only to poisons but also to altered physical conditions. Thus, Dallinger[35] succeeded in raising the temperature of the water in which flagellated Infusoria were growing from 15°·5 to 23° C. without causing their death. By prolonging the experiment over several months, he was even able to habituate them to an existence at a temperature of 70° C. In the opinion of Davenport[36], a view which is shared by many other observers, this resistance to high temperatures was dependent on the abstraction of water from the protoplasm. Dallinger has also observed that in Infusoria that are accustomed to life in hot water, the vacuoles become smaller and smaller and may even actually disappear.
This adaptation, then, is a property that is very general and widespread in the microcosm of the unicellular organisms. It is connected with the intracellular digestion of solid food and with the absorption and transformation of soluble substances. These phenomena, chemical in character, are intimately linked with the irritability of microscopic organisms, which represents one of the fundamental properties of living organisms.
A Protozoon, which is refractory to a parasite, may protect itself by flight or it may devour and digest the parasite; another, which acquires a tolerance in regard to a toxin or to a mineral poison, absorbs, fixes and transforms this substance. Consequently, in all these instances of immunity there is a reaction of the living elements of the organism, this being a direct consequence of the irritability of the protoplasm.
Before an Infusorian retreats from the dead body of an allied organism, before a Protozoon secretes a digestive fluid around the prey it has ingested, before a Bacterium secretes a glairy layer for its defence, etc., these unicellular organisms must receive sensations which provoke the above-mentioned reactions. It is to a celebrated botanist, Pfeffer, that we owe the most important researches on this irritability of unicellular organisms, researches which may be summed up in the general statement that this property is subject to the psycho-physical law of Weber-Fechner. Pfeffer, by the observation of the movements of Bacteria under the influence of increasing stimulations, has established the fact that, conformably to this law, when the stimulus increases in geometrical ratio, the irritability increases in arithmetical ratio, that is to say, the reaction is proportional to the logarithm of the stimulation. In order that a motile bacterium (Bacterium termo), grown in a peptonised solution, may perceive a difference of medium, it is necessary to place it in a peptone solution of five times the original concentration; weaker solutions, in which the concentration is but three or four times greater than the original fluid, do not attract the bacteria at all; consequently these differences are below their chemiotactic sensibility.
The different reactions that are exhibited in the immunity of unicellular organisms, reactions which are dependent on the irritability of their protoplasm, therefore, come undeniably under the category of purely cellular phenomena.