On the Development of Certain Microscopic Organisms Occurring in the Intestinal Canal¹

At a time when the association of special parasites with morbid states of their host is readily interpreted as evidence in favour of current theories regarding the parasitic origin of disease, any exact information regarding the true significance of the phenomenon in particular cases may serve a useful purpose. It has, therefore, seemed desirable to endeavour to ascertain the nature of certain parasitic forms, which, in this country at all events, are specially related to cholera and certain other conditions in which the contents of the intestinal canal are of an abnormal character, and to determine the relation which they bear to those conditions. The intestinal contents in such cases, like those in health and in other forms of disease, abound in organisms of various kinds, but are specially characterised by the frequency with which they contain excessive numbers of what may be, provisionally, termed monads and Amœbæ. The excessive development of these bodies certainly bears a definite relation to the existence of abnormal conditions in the host, and the aim of the present paper is to show what the real nature of this relation is.

The account given of the life-history, and of the mutual relations of the various bodies described, is founded on a prolonged course of investigation, including a series of cultivationexperiments, carried out with the aid of various media and extending continuously over a period of more than a year, in order to determine the influence exerted on the course of development by variations in external conditions. To give a full account of all the observations would have occupied excessive space and have tended to obscure the general results of the inquiry in masses of detail. It has, therefore, seemed advisable, as far as possible, to avoid histories of individual experiments, and merely to introduce occasional illustrative cases regarding particular phenomena. All the more general statements are, however, founded on notes recorded during the course of the investigation and not on mere memory of results, and are there-fore free from the source of fallacy, at all events, however much they may be affected by errors of interpretation.

The discovery of the occurrence of monad forms in choleraic and other excreta dates many years back. Davaine appears to have been the first to observe them. In the year 1854 he published an account of his observations regarding their presence in choleraic excreta during the epidemic of 1853, 1854.¹ He states that he encountered them in many cases and frequently in very great abundance, and he records the occurrence of what he regarded as a smaller variety of the same organism in typhoid excreta. Subsequently Lambl described and figured similar bodies present in the gelatinous mucoid discharges occurring in cases of diarrhoea in children,³ and Ekeckrantz recorded their occurrence in cases of diarrhoea.³ In the year 1870 Dr. Timothy Lewis published his observations on the occurrence of monads and amboeboid organisms in choleraic and other excreta,⁴ and Tham recorded the occurrence of monads in chronic diarrhoea.⁵ In the following year an account of my own observations on the same subject was published, in which-various forms of monad and amoeboid forms were described.⁶ In 1875 Marchand recorded the occurrence of monads in a case of typhoid fever,⁷ and, in 1878, Zunker described two forms as occurring in severe cases of intestinal disease.⁸.

The question next presents itself, whether all the observations refer to one specific organism, or whether more than one species is referred to. Leuckart, in the recently published, edition of his work on ‘Human Parasites/believes that the latter is the case, and, while referring the organisms described in most cases to the genus Cercomonas, distinguishes those described by Marchand, and certain of those recorded by Zunker as belonging to the genus Trichomonas. As the result of prolonged observation of the varying forms which the organisms occurring in excreta in this country present, I must confess that I am inclined to believe in the specific identity of the parasite in all the recorded cases.

As previously mentioned, the occurrence of amœbal organisms in choleraic and other excreta was recorded by Dr. Lewis and myself, in the years 1870, 1871.¹ The only other accurately recorded observation regarding them appears to be that of Lösch, published in 1875, in which organisms associated with a dysenteric condition of the large intestine, are assumed to have been the specific canse of the disease, and have received the name of Amœba coll²

In so far as morphological characters are concerned, the monads observed in excretal materials in Calcutta in some cases agree with the genus Cercomonas, and seemingly represent the so-called Cercomonas intestinalis, Dav. In others, however, they seem rather to correspond with the genus Trichomonas, taking the characters of the latter as defined by Stein. This authority denies the presence of any true lateral cilia, and maintains that the appearance of a ciliated border is deceptive, and really due to the rapid emission of successive protrusions from the soft body substance of the organism.⁸ Whether this be true of all the organisms at present united under the generic term Trichomonas I am unable to form any opinion from practical experience; but certainly the phenomena pointed out by Leuckart in reference to Trichomonas bactrachorum are opposed to such a belief. In reference to the organisms with which we are at present concerned, however, I can with the utmost confidence assert that Stein’s description is strictly applicable. This being so, it is clear that, unless the lateral cilioid protrusions are constantly emitted, which is certainly not the case, the same organism must at different times present the characters of both genera, Cercomonas and Trichomonas. Not only does our parasite do so, but it presents a protean series of immediate forms, as well as another series connecting it with the form-genus Amœba. This latter series presents characters practically undistinguishable from those of the swarm-spores of the Myxomycetes, a group of organisms with which the parasite, as we shall find, presents other close points of agreement.

Before going further, it may be well to describe the characters of these monads, or, as they may with more propriety be termed, zoospores, a little more closely, even though I have comparatively little to add to the descriptions of them previously given by both Dr. Lewis and myself several years ago. In regard to form, it is difficult to make any definite statement in reference to organisms which in this respect exhibit no constancy, but are continually varying in consequence of both intrinsic and extrinsic influences. The body in most cases is a mere fragment of naked protoplasm, with no differentiated covering, and with hardly, if any, indications of a differentiation of ectosarc and endosarc. Owing to this and to their minute size, it is almost impossible to determine with any certainty many points regarding it when in a state of full activity. Nearly all reagents almost immediately produce destructive changes, leading on rapidly to disintegration and disappearance, and even slight changes in the medium, such as depression of temperature or dilution with water, are sufficient to arrest activity and induce disintegration.

It is almost as difficult to make any definite statement as to the number of flagella which is to be regarded as normal, as it appears to vary from one to three, or even four, in some cases. In such an undifferentiated organism the flagella differ little from pseudopodia, and their formation and retraction may frequently be observed in some of the more sluggish states of the body when movement is comparatively slow. This being the case, it is clear that any constancy in their numbers is hardly to be looked for. The posterior extremity of the body sometimes ends in a point, sometimes is more or less rounded, and frequently is provided with a caudal process, or trailing filament of very varying length and thickness. This appears to be connected with the method of nutrition proper to the organism, which is identical with that described by Cienkowski as prevailing among certain of the Monadinæ.² That portion of the body opposite to the site of the flagella, and therefore the posterior portion in reference to motion, is the point through which nutritive materials are absorbed into the body-mass. The process may sometimes be very clearly observed where the nutritive body, as is occasionally the case, consists of an amoeboid body or of a red blood-corpuscle. When a zoospore is about to be nourished at the expense of an active amoeboid organism, it becomes adherent to it by its posterior extremity. The Amoeba at first continues to progress freely, but soon ceases to do so, assumes a spherical and motionless condition, and is dragged passively about by the energetic fiagellary movements of the zoospore. Gradually a diminution in its bulk becomes manifest, the body of the zoospore at the same time becoming distinctly plumper and more refractive, and as the process continues the whole, or almost the whole, of the Amoeba disappears, and its plunderer swims off to seek a new victim. Sometimes several zoospores unite in plundering one Amoeba, which is jerked irregularly about by their opposed movements. In the case where red blood-corpuscles are the source of nutriment, as is frequently the case in choleraic evacuations, the progress of the process may be followed readily by the colouring of the zoosporebody by the absorbed hsemoglobin. In some cases portions of corpuscles, too, seem to be absorbed en masse, though it is difficult to be quite certain on this point, due to appearances arising from surface adhesion to the transparent bodies of the zoospores.

Owing to the constant movements presented by the parasites when in full activity, it is difficult to come to any definite conclusion regarding the frequency with which they possesss a differentiated nucleus or contractile vesicle. That they do do so in some cases there can be no doubt, but in many the most careful and prolonged examinations fail to reveal the presence of either structure. That the presence or absence of a distinct contractile vesicle is not a matter of essential specific importance, is the conclusion which Hertwig and Lesser seem to arrive at as the result of their study of the Rhizopoda,¹ and the phenomena presented by the organisms at present under consideration certainly corroborate this conclusion. The presence of a contractile vesicle appears to be an inconstant character, determined, in some cases at all events, by conditions of the nutritive medium. With regard to the constancy of a nucleus as a specific character, it is necessary to speak with some reserve, as the presence of such a structure may readily escape observation in such minute organisms as the excretal zoospores. This is more especially likely to occur where a distinct nucleolus is not present and where the nucleus is merely represented by a clear vacuolar area in the body-mass. That these conditions may replace one another in one and the same organism is, as we shall see hereafter, unquestionable, bodies which at one period merely show a clearer nuclear area subsequently showing a conspicuous nucleolus within this. In certain cases, as Dr. Lewis pointed out in 1870, the excretal zoospores do show clear areas which are probably of a nuclear nature, and in others either previous to or after treatment with reagents, and specially with Liquor lodi, a nucleoloid particle is rendered manifest. Without feeling justified in stating it as a positive fact, I am strongly inclined to regard the presence or absence of a nucleus as connected in the present case rather with developmental than specific character. The body of the parasite varies considerably in appearance in different cases, and at different times in one and the same specimen, being sometimes almost homogeneous and in others distinctly granular.

After continuing in full activity for some time, the zoospores sometimes pass into a condition in which they exhibit very free amœboid changes of form, accompanied by frequent retraction and protrusion of flagella. Frequently connected with this stage, but sometimes also occurring as a mere interlude in the condition of maximum activity, processes of multiplication by division take place. Division is preceded by a temporary cessation of activity, the flagella being retracted and the body assuming a more or less spherical form (Plate XVIII, fig. 17 k). The outline soon become oval, a constriction now appears transverse to the long axis of the body and rapidly deepens, and a new flagellum appears at either pole and begins to act with energy (Plate XVIII, fig. 17, h, i). The central contraction continues to increase in depth, and ultimately the two segments remain connected merely by a narrow neck, which, due to their energetic struggles, is soon reduced to a thread (Plate XVIII, fig. 17,/), and finally gives way, so as to allow the two twin zoospores to part company and swim off freely in the medium.

In other cases a retardation of movement is the antecedent to the death of the zoospores, as may frequently be observed when unfavorable alterations are naturally or artificially taking place in the medium. It is in these cases that they come to present features causing them to agree with Stein’s description of those in Trichomonas. The movement ceases to be one of energetic rotatory advance and assumes a jerking character. This jerking is due to the emission of lateral pseudopodial protrusions in rapid succession. Where the emission, as is frequently the case at first, is very rapid, an appearance arises as though the body possessed a lateral row of cilia. As, however, a gradual retardation sets in, the true nature of the phenomenon can be readily determined. It is now seen that distinct, slender pseudopodial processes, often of considerable length, are emitted from the side of the body, and sweeping round in a curve are again retracted. Sometimes two are visible at once, a fresh one beginning to be emitted ere the entire disappearance of its predecessor. The pseudopodia gradually diminish in size as time goes on and finally disappear, the last traces of their formation being represented by mere wave-like undulations of the bodymargin. The flagella have been retracted some time previously, and the zoospore finally remains as a mere rounded or oval particle of protoplasm, which rapidly breaks down into a molecular flake and disappears.

The presence of zoospores is by no means confined to choleraic excreta. They certainly, as a rule, are present in such excreta in much larger amount and much greater activity than in other cases, but in many cases of intestinal disease of other nature they may readily be detected, and even in cases where no abnormal condition exists, they are very frequently present in small numbers. Although this is the case, they may readily escape observation, and this for several reasons. In the first place, they are frequently inactive; and secondly, even where they are not so, the nature of the medium is such as to prevent their free movement. Moreover, they are so easily and prejudicially affected by changes in the medium, that the means employed to facilitate their detection very often defeat their own end. Thus, the addition of water is in almost all cases enough almost immediately to secure the abolition of motion, and very rapidly to lead to disintegration and disappearance of the zoospores. The two media which I have found most adapted to secure the demonstration of their presence are, first, the alkaline fluid of choleraic excreta; and second, a solution of cow dung. In either case, before using the media, it is of course necessary to filter and boil them in order to exclude débris and organisms which they may contain. In my first observations I always employed the choleraic fluid, but latterly I have entirely abandoned this in favour of the solution of cow dung, which seems to be peculiarly favorable to the zoospores.

The presence of the zoospores in the excreta is then a phenomenon not peculiar to cholera, or indeed to any diseased condition. On the other hand, certain diseased conditions of the excreta appear certainly to be incompatible with their presence. As I previously pointed out,¹ cases of acid diarrhoea associated with the presence of growing fungal elements are characterised by the absence of any traces of the zoospores. This in itself is sufficient to show that mere fluidity of the medium is not the only condition necessary for the occurrence of these organisms. That this is the case is also proved by their entire absence in many cases of dysentery. A much more important determinant seems to lie in the reaction of the medium, an acid reaction repressing, and an alkaline one favouring, their presence and development. At the same time, however, certain forms of alkalinity, associated with the excessive development of other organic forms, are almost as repressive as acidity; but, as a rule, there can be no doubt that alkalinity of the medium is one of the necessary conditions for their presence in any considerable numbers, and that any excessive acidity is most destructive to them.

This alone is sufficient to account for the extent to which their presence in the excreta in health has escaped notice. In health the excreta, as a rule, present a faintly acid reaction when perfectly fresh, but the degree of acidity increases so rapidly that within a short time the medium becomes quite unadapted to the requirements of the zoospores, which consequently die and disappear. Even where the materials, when fresh, are neutral, or, as is sometimes the case, exhibit a mixed reaction with alkalinity more permanent than acidity, the rapidity of the development of an intensely acid condition is very great, so that, unless examined at once, they may show no traces of zoospores. There is also another circumstance which must be regarded as probably accounting in part for the rarity with which these organisms have been detected in Europe, namely, that a depression in the temperature of the medium, as is the case with many other organisms, exerts a most rapid and prejudicial effect on them. During the hot weather months in India this influence exerts hardly any appreciable effect, but in the cold season it comes into play more or less distinctly.

‘While considering the subject of the influence of the condition of the medium on the vitality of the zoospores, it may be well to examine a little more closely the phenomena attendant on the decomposition of normal alvine excreta in this country. These phenomena exhibit a wonderful uniformity, as shown by the records of very numerous observations conducted at all times of the year, and at intervals of several years’ duration. When exposed to rapid drying, comparatively little change beyond increased acidity has time to take place. When, on the other hand, the materials retain their moisture, as, for example, when they are reserved in an isolated moist chamber, a definite series of phenomena manifest itself with great regularity. The first change appreciable consists, as before said, in a very rapid increase in acidity, so that the material, after the lapse of twentyfour hours, shows an intense and permanent acid reaction. This condition is associated with a change in the colour of the basis, specially when exposed to the air, a darkening and reddening being more or less distinctly manifested. At the close of forty-eight hours the material is intensely and permanently acid. If the surface be examined closely at this stage, it will almost invariably be found to be covered with numerous short, erect hyaline points, which on microscopic examination are resolved into filaments of Oidium lactis, beginning to break up into conidial segments, and arising from a series of elongated horizontal tubes traversing the superficial portion of the basis (Fig. 1). Twenty-four hours later the surface is universally covered with a thick shaggy grey coating consisting of dense masses of conidia.

The following are the notes recorded in reference to these phenomena in one case, which may be taken as typical of the normal course under similar conditions. A portion of perfectly fresh normal alvine excreta was placed in a carefully cleaned capsule in a moist chamber at 12 noon. The reaction of the material was distinctly acid. Microscopically, it consisted of the usual elements. Twenty-four hours later it had acquired a reddish tinge, and the acidity was greatly increased. After another interval of twenty-four hours it was covered by a delicate whitish bloom, due to the presence of myriads of short, erect, projecting fungal filaments, which on microscopic examination were found to present the characteristic features of young conidial filaments of Oidium lactis. The reaction was now violently acid. The average breadth of the fungal elements was 5 μ. Many of the filaments were of considerable length, and showed no traces of division; while in others, all stages of that process were clearly manifested, and numerous free conidia represented results of its completion. After separation the conidia seem, as a rule, to become broader, and their extremities, which are at first in many cases more or less truncate, assume a rounded convex outline. In some cases the filaments appear to divide dichotomously at the extremity. The superficial layers at the basis were full of long, horizontal, sparsely-branched filaments, from which short vertical twigs arose, became aerial, and ultimately split up into conidial segments. After another interval of twenty-four hours the reaction of the basis was transiently but distinctly alkaline, and the surface was clothed with a thick, shaggy, greyish-yellow coating of curious gelatinous, acutely conical tufts, composed of dense masses of oidial conidia and filaments. Most of the conidia were very short and broad, many being nearly spherical. They were full of dense shining protoplasm, only showing at utmost one or two minute vacuolar spaces. On being sown on a suitable medium, they rapidly germinated, undergoing a great increase in size, accompanied by extensive vacuolation ere doing so. The short rounded conidia measured from 9 4 × 6T μ to 65 × 6·0 or 5·5 μ. The longer joints measured 23 × 5·5 μ, and all intermediate forms connected the two series with one another. On the following day the reaction of the basis was distinctly and permanently alkaline, and no farther development of fungi occurred in it.

That the Oidium in this and other cases owed its origin to fungal elements intrinsic to the basis, and not to extrinsic ones accidentally introduced from without, was proved by the following facts:—1st. Oidium lactis is not a form which tended otherwise to occur spontaneously in any of the localities in which the experiments were conducted. 2nd. Boiling the excreta previous to isolation was invariably followed by a failure in the appearance of the fungus, and this not as the result of any change causing them to become an unsuitable medium for it, as an abundant crop appeared as usual on introducing oidial elements artificially. It cannot, moreover, be assumed that in the experiments on the effect of boiling, the excreta were originally fortuitously an unsuitable medium, as check experiments were tried with unboiled portions of the same material, which constantly resulted in the occurrence of the normal development. That the phenomenon is not one dependent on casual peculiarities of a particular season was shown by its uniformity at intervals of several years’ duration. Besides the experiments on a large scale in ordinary moist chambers, others were tried in which minute fragments of the material were hermetically sealed in wax cells, and the sequence of events in these cases was precisely of a similar nature. There can, I think, be little doubt that the digestive canal in man in this country, normally contains the reproductive elements of Oidium, lactis, just as that of the cow normally contains those of Pilobolus crystallinus and other stercoreous fungi.

The development of the Oidium is, as we have seen, coincident with a great increase in the acidity of the basis, and the question naturally suggests itself, how far the two phenomena are causally connected, and how far the increased acidity is due to a fermentive action dependent on the growth of the fungal elements. That it is partially—but only partially—dependent on this appears to be clear from the result of a series of experiments in which neither Oidium nor any other mould-fungus was developed, and in which, at the same time, a distinct but temporary increase in acidity sometimes manifested itself. The notes recorded regarding one of these cases are as follows:—A portion of fresh acid alvine excretion was boiled and set in a moist chamber. Twenty-four hours later there was a distinct increase in the degree of acidity. On the following day it exhibited a mixed reaction, being faintly and transiently acid when first applied to the test paper, and the acidity passing off and being replaced by permanent alkalinity on drying. On the next day all trace of acidity had disappeared, and a permanent and distinctly alkaline condition was present. In other cases, however, and these constituted a great majority, the reaction at the close of twenty-four hours either remained unaltered, or indicated an increase in alkalinity, and after forty-eight hours’ reservation an alkaline condition was almost invariably strongly pronounced.

The increase in acidity never approached in degree that associated with the development of Oidium, and the phenomenon, where present, may, I believe, be regarded rather as an evidence of diminished manufacture of alkaline products than of any positive increase in acid formation. The reasons for this belief are the following:—The appearance of alkalinity in the materials, whether boiled or unboiled, is associated with an enormous development of bacterial elements. During the stage of acidity normally coinciding with the development of Oidium, bacterial development seems to be suppressed or very greatly retarded, and it is only when the fungal development ceases that it comes actively into play. Prolonged boiling also causes an immediate suppression of bacterial development for the time, and at the same time permanently suppresses the oidial elements. If, then, any volatile acid or alkaline elements are originally present, a development of either acid or alkali may seem to occur, due really to alterations in the relative proportions of the products incident on the escape of volatile compounds and not on any increased formation.

As noted above, while the suppression of fungal elements by boiling is complete and permanent, an abundant development of bacteria almost invariably afterwards occurs, even in cases where the greatest precautions are taken to secure the exclusion of extrinsic elements. The following notes were recorded of the phenomena in one case of this kind:—A portion of normal excreta was boiled for half an hour, and set whilst boiling in a moist chamber. This remained closed for forty-eight hours, and the specimen was then examined. The reaction was alkaline, and the surface of the medium was found to swarm with minute active bacilli (Fig. 2). On the following day the majority of the bacilli had passed into the still condition, and formed a thick, grey, creamy layer covering the surface. The individual rods measured about 3·5 μ in length. They were either scattered singly or were associated in series of two, three, and sometimes of four. Their breadth was about 0·92 μ. Many of the still ones had already passed on into spore formation, and in doing so seemed to become somewhat shorter and thicker (Fig. 3). Subsequently the whole of them assumed this condition, and ultimately the bacillar coating was replaced by a thick gelatinous layer of the free spores. No traces of Oidium or of other fungi ever manifested themselves.

During the great increase of acidity occurring coincidently with the development of Oidium the zoospores rapidly become motionless, disintegrate, and disappear. The rate at which they do so is curiously rapid. Frequently within an hour a portion of the material, which at first showed numerous characteristic and active zoospores, retains no reconisible traces of their presence, and in some cases a period of ten minutes is sufficient to produce most marked changes. That this effect is not to be ascribed to change in temperature of the medium is demonstrated by the fact that when isolated portions are kept saturated with suitable fluids of an alkaline nature, such as the fluid of choleraic excreta or solution of cow-dung, the zoospores retain their activity, and even increase considerably in number, due to processes of division, for hours and even for days. This was most clearly shown by a prolonged series of experiments, in which the phenomena in such saturated portions, isolated beneath cover-glasses, were compared with those occurring in the material from which they were derived when left to undergo the normal course of changes.

A certain degree of concentration of the basis seems also, in most cases, to be essential to the continued life of the zoospores; as while those which remained in the interspaces between the solids of the basis continued in uninterrupted activity, others which found their way by their own movements, or by the action of currents, into the peripheral fluid of the preparations, as a rule, rapidly went through the series of changes previously described, and passed on into disintegration. The changes occurring in the natural, basis seem to be completely fatal to the zoospores, as no reappearance, of them was ever observed to occur after thee medium had passed on into the alkaline condition, though, as we shall subsequently find, it is then thoroughly adapted to them when artificially introduced.

The zoospores are not the only infusorial organisms which are prejudicially affected by the initial fermentive changes occurring in the excreta, for the amoeboid bodies and the bacteria are similarly affected. Leaving the effects produced on the former for future consideration, it may be well here to examine those in the case of the bacteria a little more closely. The first point to note regarding.them is that the phenomena differ from those observed in the case of the zoospores. There is no evidence here of any complete destruction of the organisms. There is merely a temporary suppression of development succeeded by excessive activity of it. The phenomena are parallel to those occurring as the result of prolonged boiling of the medium. Whether, however, we are to regard the subsequent development as due to renewed activity in preformed bacterial elements which have merely passed into temporary inaction due to the state of the medium, or whether we are to suppose that these are destroyed, and are to regard those subsequently appearing as the product of spores, remains an open question. In any case, while there is no reappearance of zoospores, an excessive development of bacterial elements invariably succeeds that of Oidium.

While discussing questions relative to the occurrence of bacteria in the excreta, it may be noted that the results of the present series of observations are entirely opposed, in certain respects, to those at which Nageli appears to have arrived in Enrope. In his work ‘Die niederen Pilze in ihre Beziehungen zu den Infections-krankheiten und der Gesundheitspflege,’ he affirms that, although bacterial elements are constantly present in very large numbers within the digestive canal, they are invariably inactive, and on this view he accounts for the absence of ill effects coincident with a constant source of infection of the system at large, by what he regards as pathogenic agents. He affirms that it is inconceivable that bacteria should enter the system from the digestive canal—” weil sie namlick im Magen undimDarmkanalzuerst dur ch die freien Sauren dann dwrch die Salze der Galle gesehwacht Und bewegungswrfahig gemachl sind.” This statement can, I believe, only be founded on geùeral principles, and not on: actual observations, unless, indeed, the latter give very different results in Europe from what they do in India. In India there can be no doubt that the lower portion of the intestinal canal very frequently, indeed, normally, contains very large numbers of active bacteria. After becoming acquainted with this very sweeping statement of Nägeli’s I made an extended series of special observations on this point. The results arrived at were as follows:—A very large proportion of the alvine excretal matter, on its escape from the body, is composed of immense accumulations of bacteria. In very many cases these are in active motion, and in others begin to move at once, whenever a suitable fluid is employed to dilute the basis. Movement is not confined to cases in which the reaction of the basis is alkaline or neutral; it very frequently is present coincidently with distinct acidity. Fluidity of the materials naturally favours the movement, but the capacity for active movement is in very many cases merely concealed, and not absent when the basis is much concentrated, as is clearly shown by its immediate occurrence on dilution. Depression of temperature of the basis causes temporary cessation of activity, as demonstrated by experiments conducted when the air temperature was comparatively low, in which repeated disappearance and reappearance of movement occurred coincidently with depression of the temperature of the material below that of the body and its subsequent elevation to it, the movements referred to being not, of course, mere molecular movements, which might be ascribed to movements established in the fluid, but active darting progressive ones. The addition of water to the basis at once causes an abolition of movement.

It must, I believe, be due to the effects of depression of temperature and employment of unsuitable media for dilution that active bacteria have been asserted not to be present within the intestinal canal. In certain portions, at all events, of the intestinal canal they are almost invariably present in great numbers in an active condition, and the belief that an incapacity for movements prevents their entrance into the system, therefore, falls to the ground.

Even where present in great numbers and extreme activity, a total disappearance of movement in the bacteria coincides with the development of acid coincident with the appearance of Oidium. Only when the latter has matured, and coincidently with the appearance of alkalinity, do active bacteria again present themselves. When once they begin to appear, however, their development goes on with intense activity, and quickly runs through its various stages to the formation of the so-called “spores.”

The amcebal organisms occurring in the excreta remain to be considered. Like the zoospores, they occur in the excreta during health, as well as in cases of cholera and other morbid conditions affecting the intestinal canal. Their presence seems to be determined by the same conditions as those regulating the presence of the zoospores; only, due to the readiness with which they assume an encysted condition, and thereby are enabled to resist the influence of detrimental conditions (Fig. 4) they may possibly be rather more constantly detected in one or other form than the other bodies are. Owing to their having assumed an encysted state, they may be recognised in media where they could not maintain activity, and even for considerable periods in such as have proved fatal to them, the strong capsule of the cyst preventing the content-protoplasm from undergoing disintegration for some time after its vitality has been destroyed. Due to this, in examining excreta for amcebal organisms, we must be prepared to recognise both still and active conditions, and in regard to bodies representing the former, it is necessary to guard carefully against mistaking them for oily particles, or vice versa.

In both the active and encysted condition they exhibit great variations in size, the variation in this respect being specially marked in active specimens, as in different media and at different times in the same one they not only seem to vary in absolute bulk, due to differences innutrition, but they also vary in the form and extent of the pseudopodial extensions of their substance. In some cases isolated thick pseudopodia alone are emitted from the more on spherical body (Plate XVIII, fig. 19), while in others this condition is exchanged for one in which the body is spread out in all directions into an irregular, constantly changing protoplasmic flake. Between these two extremes a connecting series of intermediate forms exists, and the transition from one to the other through these can frequently be observed to take place in individual specimens. In the encysted condition, when their form is more or less spherical or elliptical, they frequently attain a diameter of 25 μ or even more, and they may range downward from this until the diameter only amounts to 8 μ.

The body-substance is sometimes almost homogeneous, at others more or less distinctly granular, due seemingly to the presence of extraneous nutritive matter. Changeable vacuoles, often of considerable size, may or may not be present; but a true contractile vesicle seems to be almost always, if not invariably, wanting so long as they are retained in the original medium.

As in the case of the zoospores, considerable variation exists in regard to the presence of a defined nucleus. In some cases no recognisable traces of such a structure are present, but in others a permanent clear nuclear area is visible. This may or may not include an evident nucleolus (Fig. 5). When present, the latter may attain a diameter of 7 to 9 μ. It is circular and apparently discoid, but in some cases may appear annular from the presence of a thickened margin.

The degree of movement which the Amœbæ present in different cases varies greatly. In some the movement is extremely energetic, the body forcing its way rapidly between the surrounding masses of debris. In others it is confined to changes of form and slow emissions and retraction of protrusions without change of place, and in still others the only signs of it present are the gradual appearance and disappearance of vacuoles, or other indications of content-change. Any direct multiplication of Amœbæ by division does not appear to occur, or if it do so, must occur with extreme infrequency, as, though carefully watched for in very many cases, it was never seen to take place.

In many normal excreta in which Amœbæ are present in considerable numbers, they are all in a state of inactivity, and more or less completely encysted. In such cases they may frequently be roused to activity by the addition of suitable media which are found in the same liquids which have been already indicated as adapted to the zoosporic bodies. Even when seemingly most distinctly encysted no trace of an envelope is left behind on the emergence of the Amœbæ, the cell-wall apparently undergoing complete resolution during the process. When they have emerged, the Amœbæ do not exhibit such extreme susceptibility to changes in external conditions as the zoospores do, for they may often be seen to make excursions in the peripheral zone of nutritive fluid in diluted preparations without showing any symptoms of immediate detriment. In some cases the still and encysted Amœbæ present in the excreta cannot be roused to activity by an addition of nutritive fluid.

Like the zoospores, the Amœbæ are very rapidly affected by the changes normally occurring in the excreta after their exit from the body. The rate at which this occurs is, perhaps, not quite so rapid as in the cases of the zoospores, but the final result, in so far as the vitality of the organism is concerned, is the same. With the increased acidity and the development of Oidium all activity ceases and the organisms either encyst or break up and disappear. When encystment occurs they remain for long recognisable in the medium, and may often be detected in the latter even after the acid fermentation has run its course, and has been succeeded by the alkaline one. So far as vitality is concerned, the result is the same, however, whether encystment occur or not. The acid stage is fatal to them, and they never revive with the development of the alkaline one. As in the case of the zoospores, so with the Amœbæ, no reappearance ever seems to take place in excreta which have passed through the acid fermentation, unless due to the introduction of extraneous germs, and this although the medium, when once it has become alkaline, is eminently suitable to them.

In addition to those which can be recognised as encysted or still Amœbæ, there is another class of bodies frequently present in the excreta which were for long a subject of investigation ere their true nature could be determined. These bodies are, I believe, identical with certain of the bodies long ago observed by Drs. Swayne and Brittan,¹ and subsequently described by Professor Hallier as spores in his celebrated treatise on the fungoid origin of cholera.¹ They consist of spherical or elliptical cells of various sizes, ranging from 3·5 to 9·2 p in diameter, and frequently characterised by the brightly refractive oily appearance of their contents (Plate XVIII, figs. 20, 21). This latter character is not by any means an invariable one, however, for in other cases they are finely clouded or molecular, and with more or less distinct vacuolation; and the passage from the one condition to the other may readily be observed to take place specially under the influence of changes in the nature of the medium. They appear to consist of a very delicate membranous sac enclosing a mass of varying content-matter. They may either occur scattered singly through the basis or may be associated in groups, and, in the latter case, may sometimes be observed to be connected with one another by a delicate gelatinous, intercellular basis, which, I take it, represents the structure described by Hallier as the sporogenic cyst (Plate XVIII, fig. 20). As a rule, their occurrence is associated with that of zoospores and Amœbæ, but in some excreta they are present apart from such bodies. The number present in the excreta during health varies very considerably. In some cases of intestinal disorder they are present in increased numbers, but never apparently are they so very abundant as in certain cases of cholera.

In the normal excreta in health they are very transitory, disappearing like the zoospores very rapidly with the increasing acidity of the medium, so that specimens which when quite recent showed an abundance of them may, within the course of twenty-four hours, retain no traces of their presence. Like the zoospores, too, they are very susceptible to the influence of other changes in their surroundings, rapidly disappearing when they happened to be washed out into the ring of nutritive fluid surrounding the thicker portion of a preparation. Owing to their rapid disappearance from the unmixed excreta, it is hopeless to attempt their continuous investigation without the aid of suitable nutritive media; and of these, that which I have found to act most satisfactorily is the solution of cow dung which has been already mentioned as adapted to the requirements of the zoospores and Amœbæ. Under the influence of this they may often be preserved for several days, and continued observations of various developmental changes occurring in them may thus be carried out. The results of such cultivations seem to me to have clearly shown that these enigmatic cells are reproductive bodies belonging to the Amœbæ, and forming a connecting link between these and the zoospores.

A suspicion that they really were products of reproductive processes occurring in the Amœbæ was originally aroused by certain cases in which the fresh excreta contained Amœbæ, within which varying numbers of bodies, indistinguishable from them, were present (Fig. 6). As, however, the amœbæ were in some cases still active, and only differed from their compeers in not being provided with a distinct nucleus, it appeared at first very doubtful whether the phenomenon was not due rather to the ingestion of extraneous bodies than to any process of intrinsic development; although, on the other hand, it seemed strange that in isolated cases such an ingestion should have occurred simultaneously in numerous Amœbæ, while in the vast majority of cases in which the latter coexisted with the sporoid cells, no evidence of the occurrence of any such process presented itself. Further observations appeared clearly to show that whatever interpretation ought to be put on the above described phenomenon, the sporoid cells really are developed from the Amœbæ. The process of formation normally occurs coincidently with the cessation of activity in the parent, so that it is possible that in those cases in which sporoid cells were present within active Amœbæ, they may have been derived from without. As, however, the preparations in which the phenomenon was observed had been treated with nutritive fluid, it is quite possible that it was due to an abnormal resumption of activity in Amœbæ which had passed into the preliminary stages of reproductive multiplication. The phenomenon may, in fact, have possibly been parallel to those observed in the sclerotia of the Myxomycetes under the influence of favorable nutritive conditions. The plasmodia of the latter organisms, in passing into the sclerotial state, break up into a multitude of distinct spheres, each of which is capable of independent activity, and of emerging as a distinct amoeboid body when separated from its neighbours and introduced into a suitable medium, but which may also melt together to reform a common plasmodium when the sclerotium, as a whole, is exposed to conditions favouring its activity. As the whole of the body-substance of the Amœbæ is not expended in the formation of the sporoid cells, a portion remaining in the form of a common gelatinous investment, and as the latter, certainly in some cases, retains a certain degree of contractility and capacity of altering its form for some time after the formation of the reproductive cells has begun, I am inclined to regard the latter explanation of the phenomena in these exceptional cases as the true one.

The process of spore formation is not preceded by any true encystment; the parent body merely loses its active progressive movements and form-changes, and a very delicate surface layer becomes, in some degree, differentiated upon. it. The content substance within this next begins to show a constriction, dividing it into two lobes, and a gradual extension of the process ends in the separation of these as independent masses (Fig. 7). The entirety of the material of the parent does not, however, as before mentioned, seem to be expended thus; but a varying amount remains as a gelatinous intercellular matrix, which blends with the surface layer (Pl. XVIII, fig. 20). Under favorable circumstances, each of the daughter bodies originally formed, as described above, in its turn divides into two, and in this way groups of sporoids, containing large numbers of individual cells, may be actually observed to arise in the course of forty-eight to seventy-two hours (Fig. 8). Each of the bodies thus formed by division acquires a delicate investing layer, but the process of division may undergo arrest at almost any stage, and fusion of the partially differentiated bodies may then take place. The extreme variation in the size of the sporoids in different excreta thus finds an easy explanation in the processes by which they are formed. Where the processes of formation go on undisturbed in the medium, as, for instance, where the development occurs in specimens beneath a cover glass, the sporoids remain aggregated in groups embedded in their gelatinous bases, and exactly resembling those described and figured by Hallier. Due, however, to the tenuity of the matrix, they are readily detached from one another, and scattered under the influence of slight mechanical disturbances, and hence in the excreta in their nutural condition it is rare to encounter any save isolated individuals.

The sporoid cells thus arising by processes of division within the Amœbæ, we have next to inquire into their subsequent history. In cultivations of excreta, in which their development has been followed thus far, no further vital change appears to take place within them. The medium, sooner or later, seems to become unsuited to them, and they disintegrate and disappear. It is different, however, in the case of spores which have been developed within the intestinal canal of the host, for many of these, when exposed to favorable influences, appear to give origin to zoospores. The phenomenon of the origin of the latter may frequently be observed in specimens of fresh excreta, which have been treated with nutritive fluid. Under such circumstances media, which at first contained an abundance of sporoid cells and no zoospores, may within a few hours be found to contain hardly any of the former and numbers of the latter; the proportion of zoospores present at the close of the observation being in direct relation to the numbers of sporoids originally present and the proportion in which they have subsequently disappeared. It is difficult precisely to follow the stages in the process, as it only takes place in the thicker portions of the preparation—the sporoid cells, as before said, being rapidly destroyed when washed out into the surrounding fluid—but the cell wall of the sporoid seems to become gradually softened and absorbed, and does not remain behind as distinct cyst. It has unfortunately never been possible continuously to follow out the transition of any individual Amœbæ into a mass of sporoid cells, and the resolution of the latter into zoospores. Amœbæ have been seen to give rise to sporoids in some cases, and the origin of zoospores from bodies apparently in every respect indentical with these has been observed in others, but a link is still wanting in order to render the observation quite complete. In spite of this, however, there can, I believe, be little doubt that the Amœbæ, sporoid cells, and zoospores really constitute stages in one cycle of development, more especially when certain observations, an account of which will be met with farther on, are taken into account. Any direct origin of Amœbæ, from characteristic sporoid cells in human excreta, does not seem to take place.

Allowing, then, in the meantime, that the characteristic zoospores, Amœbæ, and sporoid cells occurring as parasites within the human digestive canal are all members of the developmental cycle of one specific organism, how can we account for the extreme frequency with which they are present? Taking the very great susceptibility of the organisms to the influence of external conditions, and the fact that the media in which they escape from the body seem, as a rule, to undergo changes certainly fatal to them, it appears at first sight very difficult to do so satisfactorily. We might, indeed, take refuge in the supposition that, after the germs have once obtained an entrance, they remain persistently within the body, giving rise to constantly recurring generations of the parasite, but such an hypothesis is hardly consistent with the fact that in the case of a given individual they may appear suddenly after considerable intervals of apparently entire absence, and after persisting for varying periods may again vanish, only to reappear as before at a later period. We should, therefore, be compelled farther to assume that periodical retentions, either of the germs or of the developed organisms, take place somewhere within the body, and alternate with uncertain periods of discharge, or that their appearance and disappearance from the excreta is determined solely by conditions in the latter allowing or preventing their persistence in the contents of the lower portion of the digestive canal.

While allowing the possibility of such explanations, I do not regard them as correct, but believe that the appearance and disappearance of the parasitic forms are due to the successive introduction of extraneous elements and the subsequent discharge of the result of their development. It is as difficult to give a definite opinion as to the precise source of these organisms as it is to state whence the oidial and bacterial elements of the intestinal contents are derived. They are, as has already been pointed out, almost constantly present in varying numbers in the intestinal canal, and are in all probability introduced with ingesta of various kinds.

It is now more than five years since, whilst studying the development of Pilobolus crystallinus, I first encountered what it appears may be regarded as the perfect fructifying or reproductive bodies of these intestinal organisms. In a specimen of recent cow dung, which had been reserved in a moist chamber for twenty-four hours, the surface was found to be studded with a multitude of minute glistening white spherules, adhering to projecting points of the basis (Pl. XVIII, fig. 1). At first sight these were regarded as basal dilatations of Pilobolus, in which an abnormal suppression of colouring had occurred, bnt on submitting them to microscopic examination this was found not to be the case. They were entirely unconnected with the mycelial tubes of the fungus which subsequently produced an abundant crop of normal fructification, and did not resemble the basal dilatations in structure, consisting of a membraneous sac crowded with spore-like bodies. These were circular, flattened, and biconcave, closely resembling red blood-corpuscles in general appearance. On being introduced into a solution of cow dung they rapidly became spherical, a contractile vesicle appeared within them and began to pulsate, and they sooner or later, as a rule, gave exit to minute amoebal bodies, which crawled off freely in the fluid, generally leaving a delicate cyst behind them in doing so. In other cases, however, in place of being resolved into Amoebae, they appeared to give origin to flagellate zoosporic bodies. Similar phenomena were observed at various subsequent periods, and the sporangia bodies being not unnaturally regarded as representing some low Myxomycete form, a repeated but futile search was made for the presence of plasmodia corresponding with them. Subsequently the appearance of these sporangic bodies came to be recognised as a normal and almost invariable event in specimens of cow dung reserved for the study of stercoreous fungi. The essential condition ensuring their appearance seemed to be that the basis should have been secured and set for cultivation whilst still quite recent, older samples almost invariably failing to produce a crop, or only producing a very scanty one. As the result of numerous experiments, it was ascertained that the appearance of these sporangia preceded that of any other form of fungal fructification, occurring, as a rule, within twenty-four or forty-eight hours from the commencement of a cultivation of perfectly fresh material, and being succeeded by that of various fungi in the following order:—PUdbolus crystallinus, Ascobolus sp., various species of Gymnoasci, Peziza sp., Coprinus sp. These fungi may be regarded as the regular and almost invariable results of the cultivation of fresh cow dung in this part of India, while occasionally other forms are interpolated in the series, as, for instance, a Syncephalis, which sometimes attacks the Pilobolus. Taking the normal series of developments, the sequence of events is shown in the following table:

Further investigations showed that the occurrence of these sporangia was not limited to cow dung, but also frequently occurred on horse dung too, where, as on the other medium, it preceded that of Pilobolus. or other forms of mucorine fungi.

Even after the study of these sporangioid bodies was specially undertaken, it was not until after many months of continuous investigation that their true nature and mode of origin were satisfactorily determined, and that their relation to organisms, seemingly identical with the parasitic zoospores and Amœbæ of the human excreta, was ascertained. In studying the developments normally occurring in reserved specimens of cow dung as compared with human excreta, one of the most conspicuous differences presented by the media is that the former has no tendency to pass through the acid fermentation so constantly affecting the latter medium. Perfectly fresh specimens are either neutral or faintly alkaline in reaction, and when kept under observation exhibit a constant progressive development of alkalinity, so as to become strongly alkaline within twenty-four or forty-eight hours—a condition which they retain for indefinite subsequent periods. Another point distinguishing vaccine from human excreta lies in the relative amount of bacterial elements originally present in them; for while, as we have already seen, a very large proportion of the mass of the latter medium is formed of these elements, the proportion of them present in a developed form in the former appears normally to be very small. Farther when the medium follows a normal course in reference to the organic developments occurring in it, there is at no period that excessive multiplication of bacterial elements so characteristic of the later stages of decomposition in human excreta, the numbers and succession of fungal organisms appearing to a great extent to exhaust the nutritive properties of the basis. The two commonest forms of bacteria occurring in cultivations of cow dung are shown in the accompanying figures (Figs. 9,10).

The evidences of exhaustion of the basis, in so far as concerns certain of the organic developments which have occurred in any abundance of it, is unequivocal, each of them appearing in its turn and then absolutely and permanently dying out. The phenomenon is specially marked in reference to the sporangioid bodies and Pilobolus, which, as we have already seen, are the first forms which make their appearance. Crops of sporangia are rarely produced for more than two or, at utmost, three days, and crops of Pilobolus for more than six or seven, and in this case it is only for two or three that the development is abundant. We have an enormous primary production of reproductive bodies and an absolute, or almost absolute, failure of any further development due to these. Where the primary crop has been abundant, it is very questionable whether the reproductive elements then produced ever germinate in the same medium in which they were produced; for although, as before said, successive crops of fructification appear for two or three days, these apparently all belong to one generation. This certainly is the case in so far as Pilobolus is concerned; there is no evidence of successive generations of mycelia, and the successive crops of fruit are in many cases visibly the result of unequal rapidity in the development occurring in basal dilutations contemporaneously developed. The phenomenon is clearly one of exhaustion of the nutritive basis, and not due to defective germinal energy or the reproductive elements; for we have only to transfer some of them to a suitable fresh basis to secure their immediate development. While in the primary basis we find the surface covered with masses of reproductive elements totally incapable of farther development, so long as they remain there, we have only to transfer a few of them to an unexhausted basis in order to secure an abundant production of a fresh generation. This is of course merely the parallel of what we find occurring in regard to the Oidium and bacteria of human excreta, where a solitary and excessive development of the organisms occurs, terminating in the production of innumerable reproductive elements incapable of germination until transferred to a fresh medium. In the case of cow dung, we certainly cannot ascribe the failure of excessive bacterial development to exhaustion by organisms of the same nature; but the variety and succession of other organisms which are developed may, perhaps, practically produce a similar result.

While fresh cow dung is relatively deficient in bacteria, it is by no means devoid of distinct organisms generally. On the contrary, we find it almost invariably containing a very large number of zoosporoid bodies (Pl. XVIII, fig. 17), and sometimes smaller numbers of other infusorial forms of various kinds. After prolonged study of the zoospores under various circumstances, I am unable to indicate any constant differences to distinguish them from those of human excreta. Like the latter, they exhibit numerous varieties in form and in character of movements, but none of these are peculiar to them. They seem also to be similarly affected by reagents artificially added to the basis, or by spontaneous changes taking place in it, and altogether there can, I believe, be no doubt of the identity of the organisms in both media. Although so constantly present, they may, like those of the human excreta, readily escape detection, unless suitable precautions are taken in preparing the specimens for examination. A dilution of the basis with pure water is frequently so rapidly fatal to them as almost entirely to conceal their presence, and even other solutions more favorable to them must be added with caution so as to avoid too abrupt a change of conditions. The best method of treating preparations, with a view to their detection, is to spread out a minute portion of the basis in a thin layer on a slide, then apply a cover glass, and (having first focussed a field containing a view of a portion of the margin of the layer) to introduce some strong solution of the same dung from which the specimen was procured, and which has been previously filtered, boiled, and allowed to cool. On doing this, the organisms may be observed emerging from the margin of the basis, and, after swimming actively in the fluid for a short time, gradually passing on into the series of changes described as occurring in those of human excreta under similar circumstances. Here, too, we find that those which, in place of emerging into the peripheral fluid, enter some of the interstitial spaces existing between the solids of the basis, retain their vitality much longer than their neighbours.

The number of zoospores which may be detected in this way is in many cases very remarkable. The size of individual specimens varies much, which is no doubt greatly dependent on the frequency with which processes of division recur in them. Very often they measure about 10 μ in length by 5 or 6 μ in breadth. The number of flagella with which they are provided also varies from one to three or four; whilst in full activity, neither nucleus nor contractile vesicle can be detected as a rule, and after treatment with Liquor lodi they may or may not exhibit a nucleoloid particle. The latter reagent generally induces a peculiar series of phenomena. The body gradually loses its natural fusiform, or pear-shaped outline, and becomes circular and motionless. Shortly after it has ceased to move, a large vesicular protrusion is emitted at one or other point from the somewhat granular body, and general disintegration soon sets in. There is no evidence of any differentiated surface layer, and the flagella appear to be merely transitory and changeable protrusions of the protoplasm. The point opposite to the flagellar site seems to be that through which nutritive materials are absorbed, the body becoming attached to foreign particles by it, and being sometimes drawn out in consequence into a caudal process or filament of variable magnitude. AU the characters which the zoospores here present are, in short, identical with those occurring in the human parasite. The processes of multiplication are also similar, consisting in transverse division preceded by diminution or temporary arrest in activity, and the phenomena attending diminished vitality and disintegrative disappearance follow the same course.

While, however, the human and vaccine parasites appear to be identical in nature, their presence in the excreta is followed by different results. In the case of the human parasites we have already seen that a rapid and complete process of destruction sets in coincident with the changes normally occurring in their medium after its exit from the body, but this does not hold good in the case of the vaccine parasite. That it does not is probably due to the absence of any fermentive change in the medium corresponding with the acid development coincident with the appearance of Oidium. In place of disappearing from the medium the organisms in the cow dung, after continuing to multiply by division for some time, seem to pass on to further stages of development through which they are enabled to give origin to reproductive bodies providing for the perpetuation and diffusion of the species, or where conditions are unsuited to this, to resting forms capable of renewed activity on again encountering favorable conditions. The fully developed reproductive bodies consist of the sporangia, which have been already mentioned, the resting conditions are represented by encysted zoospores or much more frequently by encysted amoeboid bodies.

The following notes recorded in reference to the phenomena observed in a specimen of cow dung are generally typical of those in numerous other experiments of a similar nature. Some perfectly fresh cow dung was procured and set in a moist chamber at 10.30 a.m. The material was moist, faintly alkaline in reaction, and swarmed with large active zoospores. Five hours later a second preparation was taken from the specimen. In this an even greater number of active zoospores was present than in the first, and a certain number of still ones was also recognisable. A third preparation, procured seven hours later, showed no active zoospores but an abundance of still ones of oval and rounded form. After another interval of six hours a fourth specimen was taken and found to resemble the previous one in character save that a distinct contractile vesicle was visible in many of the cells. At this time (dawn) no signs of sporangia were visible, but a few hours subsequently they appeared in great abundance, while preparations of the basis showed an abundance of active amœboid bodies of various sizes, ranging from that of the still zoospores upwards.

Similar phenomena repeat themselves with monotonous uniformity in successive experiments. Again and again we find a basis abounding with zoospores increase in the numbers of these bodies for some time; a cessation in their activity; the appearance of multitudes of bodies agreeing in size and form with the inactive zoospores, but characterised by the presence of a contractile vesicle; the emergence and growth of these as active amœboid bodies and the appearance of sporangia. That the latter are certainly the product of the union of the amœboid bodies is clear from the result of other observations, but, as necessarily is the case in all massive cultivations, the evidence connecting the zoospores originally present with the amœboid bodies subsequently appearing remains imperfect. That the relation is one of identity is, no doubt, rendered probable by the fact that the excreta in the fresh condition never show any proportion of amœboid bodies or of still cellules capable of accounting for the enormous numbers subsequently present, unless an excessively rapid multiplicative division were assumed to take place from the scanty supply originally present. An assumption of this nature is, however, entirely devoid of any support from observation, as any division of the amœboid bodies previous to sporangic formation never appears to occur. On the other hand, we have the zoospores present in abundance from the outset, and capable of very rapid multiplication by division;¹ we find that it is impossible to distinguish between resting zoospores and bodies which pass on into an amoeboid state; and we know that the zoospores merely differ from Amœbæ in the character of the protrusions which they emit, and therefore in the nature of their movements. Allowing the identity of the two forms, we have a ready means of accounting for the regularity and abundance of the crops of sporangia and the general ratio of these to the numbers of zoospores, while rejecting it we have no explanation to give of the appearance of the multitudinous development of amoeboid bodies.

In the endeavour to obtain more positive evidence on this point, hundreds of cultivations on a small scale were carried out with more or less satisfactory results. In some cases there appeared to be no doubt that the zoospores originally present became converted into Amœbæ at a later period, but the difficulty of attaining to absolutely certain results appears to be almost insurmountable. In the first place, in order to render any such cultivation susceptible of continuous observation, it is necessary to introduce conditions which we have already found to exert a most prejudicial effect on the vitality of the zoospores, for the basis must necessarily be diluted with some fluid in order to render the organisms visible. A condition of fluidity of the basis, too, independent of any actually destructive effects, certainly influences the occurrence of developmental processes in other ways. The persistence of zoospore forms may be prolonged, and the appearance of amoeboid ones be delayed by an excess of moisture as may readily be proved by experiment. So again an excess of fluidity in the medium seems to be one of the agencies capable of causing amoeboid bodies present in it to assume the encysted condition in place of passing on to the normal sporangic development. Another great obstacle to the satisfactory decision of this question lies in the excessive and constant movement of the zoospores, which renders it impossible to secure any individual specimen for continuous observation over a prolonged period.

All that can be positively affirmed is that the amoeboid bodies which replace the zoospores primarily present appear to be directly derived from the latter, and that the two forms seem merely to represent different developmental stages of one and the same organism, connected with one another by the intervention of an inactive stage.

The Amœbæ, when they first appear, are of very minute size, ranging from 5 μto 7 μ. and upwards in diameter when in a spherical condition (Pl. XVIII, fig. 1). In some cases a contractile vesicle is at once distinctly visible within them; in others such a structure can only be detected with extreme difficulty, and in still others it appears to be absent. The same inconstancy seems to prevail in regard to the presence of a nucleus and nucleolus. Generally, I believe, a clear nuclear area is visible at a comparatively early stage, but a differentiated nucleolus often does not appear until much later; when it does appear it is as a flattened circular disc in the nuclear area. The rate of growth in the Amœbæ and the size ultimately attained ere the occurrence, of sporangic formation varies very much, apparently in accordance with the nature of the medium. In favorable cases it is wonderfully rapid, and where the growth is considerable, it is usually associated with further development of the nucleus (Pl. XVIII, fig. 10). A division of the nucleolus occurs, and the resultant bodies move somewhat apart, so that a pair of greenish discs replace the originally solitary one. Farther than this the nuclear area seems to become differentiated from the rest of the body-substance by a boundary layer, and a cross partition of similar nature passes inwards to separate the nucleoli (Pl. XVIII, fig. 15).

The characters of the movement also vary greatly in different specimens, and in one and the same specimen at different times. Sometimes it is of a free flowing character, the organism moving rapidly forward by means of successive protrusions of its substance. In other cases we find such movement alternating with a more sluggish action, in which the body presents an irregularly lobed or cornuate form, and only gives origin to limited extensions (Fig. 11). This condition frequently seems to coincide with defective nutrition, as the addition of fresh nutritive matter will often cause it to be exchanged for free progression. In still other cases again the body assumes a peculiar flattened scale-like condition, adhering by one surface to the glass of the slide, and moving forward with a slow gliding motion, accompanied with comparatively little change of form (Fig. 12). In such cases the free surface often shows curious linear markings due to the presence of longitudinal thickened ridges. Three distinct areas appear to be present in the body in this state; we have first a dense granular central portion constituting the sub-stance of the ridges and the thicker portions; beyond this is a highly refractive area devoid of granules, and external to this again is a delicate tenuous layer of the protoplasm, often only distinguishable with difficulty from the surrounding medium. All the vital processes seem to be carried on in such cases with extreme slowness. The contractile vesicle dilates very gradually and often only undergoes imperfect obliteration on contraction, or it may remain absent for prolonged and uncertain intervals. In other cases it seems to be rigidly fixed in full dilatation. In appearing it sometimes is developed from a single centre, in other cases several minor vesicles appear and fuse into one as they increase in size. Granules of nutritive matter are ingested and frequently accumulate in spherical masses within vacuolar spaces filled with fluid. In other cases, however, they are irregularly diffused. When in full activity, a constant succession of fluctuating vacuoles is present in the body-substance. In some cases, and apparently connected with or preparatory to the resolution of the body into a collection of sporoid reproductive bodies—as it only occurs where they have ceased to move and have become aggregated in sporangoid masses—the large Amœbæ in place of showing at utmost two large nucleolar bodies as they normally do, contain from three to eight of smaller size (Pl XVIII, fig. 11). From the appearance and arrangement of these in different cases, there can be no doubt that the increased numbers are due to repeated binary division of the nucleoli originally present.

The size of the Amœbæ and nuclei varies so extremely in different cases and at different times that it is impossible to give any useful average measurements. They very frequently, when in an irregularly rounded condition, measure from 15 to 25 μ in diameter, with nucleoli, which, when paired, have a diameter of 3·5 μ₃ and when in larger numbers measure only half as much or under.

After continuing to progress through the medium for variable periods, the Amœbæ either cease to move, and remaining more or less isolated, become encysted, or becoming aggregated into masses give origin to sporangia. Where they are present in abundance, and where the conditions of the medium are unfavorable to sporangic development, the accumulation of encysted bodies on the surface often covers the medium with a fine greyish bloom, which, unless closely examined, may readily be ascribed to the presence of mycelial elements. The encysted bodies are either quite free or are associated in little groups and knots. As a rule, no further change appears to occur within them, and they remain unchanged for indefinite periods, ready to resume activity when favorable conditions again present themselves. In place of becoming encysted, however, we normally find thea Amœbæ, after some time, becoming more sluggish in their movements, and adhering to one another in pairs or groups of various sizes, the union becoming very intimate, and in some cases proceeding to such a degree of apparent fusion that we are only able to estimate the number of individual elements entering into the formation of a group by the number of nuclei or of rigidly dilated contractile vesicles which may persist (Fig. 13). This phenomenon is so far parallel to that occurring in the case of various Rhizopodous organisms, such as Aciinophrys sol, &c., but the results following adhesion and union rather resemble those following the formation of Plasmodia in Myxomycetes, as the formation of the compound body is distinctly the antecedent to spore formation, the protoplasmic material becoming in greater part resolved into a mass of spores or reproductive cells, while a certain amount of it remains as an investing and intercellular substance (Fig. 14). All the processes which have been just described can sometimes be observed to occur in slide cultivations beneath cover glasses, and capable of continuous observation; but under such circumstances they naturally never attain the magnitude and perfection exhibited under natural conditions where the sporangia are developed on an exposed basis of large bulk.

In such cases we may trace all stages of the formation of perfect sporangia from that in which we have mere irregular aggregations of closely adherent Amœbæ (Fig. 15), which on being detached and introduced into a new nutritive medium become resolved into their constituent Amœbæ (Fig. 16) by resumed activity of the latter, to that in which we have perfectly developed sporangia, with a distinct investing membrane, and even, in certain cases, an internal meshwork representing what may be regarded as a rudimentary capillitium. The degree to which an actual fusion of the constituents of the sporangic mass takes place varies greatly in different cases and in different portions of one and the same specimen. Complete fusion often occurs in the basal portions of pedicillate sporangia, while the process is only partial higher up. In such cases while we find the stem consisting of a seemingly homogeneous mass, the head is generally more or less distinctly marked out into a series of irregular areas, which are in some cases so defined as to give the surface when viewed under a low microscopic power a somewhat granular or faintly nodulated aspect.

The appearances presented by the sporangic masses, where the constituent Amœbæ are yet distinctly recognisable, are very curious. The surface presents a strangely epithelioid appearance, due to the dense aggregation of irregular cells closely adapted to one another, so as to form a continuous layer, while on deeper focus we encounter sectional views of the interior, consisting of a dense mass of similar bodies (Pl. XVIII, fig. 3). We have a regular tissue formation due to aggregation and union of originally independent elements.

The sporangia vary greatly in size as well as in the extent to which a distinction between a stem and head is present. In many cases no stem formation takes place, and the spherical sporangium is merely attached at one or other point of its circumference, in others a pedicle of considerable length is present (Pl. XVIII, fig. 1). The heads may attain a diameter of 0·37 of a millimeter, and the pedicles a length of 0·25. When the pedicle is of any length it is usually dilated basally into a disc or into several root-like expansions, which embrace the body to which it is adherent. The sporangia are almost invariably situated on prominent projecting points of the basis, such as minute fragments of vegetable tissue, &c. When developing, they first appear as minute hyaline prominences or rods projecting on the surface of the basis. As their development advances they become dilated—the dilatation in the rod forms occurring terminally, and causing them to assume a capitate aspect—and at the same time an opalescence appears in the previously hyaline material. This increases in intensity and passes on to opacity, and the fresh mature sporangia are of a bright glistening whiteness, passing into various stages of yellow, buff, and amber, as drying sets in.

On examining many sporangia, even when the constituent Amœbæ are yet recognisable through more or less of their substance, the presence of a distinct investing membrane may be made out, and in all mature sporangia such a structure is invariably present. Owing, however, to variations in its structure under different circumstances, it is much more readily recognisable in some cases than in others, and may, indeed, sometimes readily escape detection in developing or recently formed sporangia, which have been kept in a very moist atmosphere; it is very soft, and quickly dissolves and disappears in water, although readily visible ere the addition of fluid, and especially so long as no pressure has been applied. In such cases, too, certain reagents, such as Liquor lodi, readily demonstrate its existence. In older sporangia, which have undergone a certain amount of drying, it appears as a distinct, somewhat resistant, and very elastic membrane, of a yellowish colour. In structure it is finely molecular, and the external surface is covered with projecting organic corpuscles (PL XVIII, fig. 6). In the course of thorough desiccation again, it appears gradually to disintegrate and more or less completely disappear, leaving its contents exposed, and only adherent to one another by intercellular material. Its inner surface is sometimes distinctly mapped out by a series of prominent thickened ridges into polygonal areas corresponding with the formative Amœbæ (Fig. 17).

After the sporangia have been, as it were, planned out by the aggregation and more or less intimate union of the Amœbæ, and the formation of an investing membrane, the process of spore formation normally sets in. When this is regularly carried out the bodies of the Amœbæ become resolved into masses of spherical spores, measuring from 5 to 9 μ in diameter. In cases where the fusion of the parent bodies seems to have been complete these are indiscriminately massed in the cavity of the sporangium embedded in an intercellular basis; where, on the other hand, the process has not gone so far they tend to adhere in groups of various sizes corresponding to individual Amœbæ, or to small groups of these. The intercellular material in recently developed sporangia is soft and seemingly more or less fluid, resembling the intercellular matter within Mucor sporangia. Like the material of the sporangial wall, however, it concretes or sets in drying, so as to appear in many preparations of partially dried sporangia in the form of a network, in the interspaces of which the spores are situated. The character and definition of this vary considerably in different instances, and in some cases it may be distinctly resolved into two series of meshes—a larger one, seemingly corresponding with the parent Amœbæ, and a smaller one with the individual spores (Figs. 18. 19). Both the sporangial wall and the intrasporangic network are, when fully developed, stained of a deep red brown by solutions of iodine, whilst the spores merely acquire a yellow tint. No blue colouration follows treatment with iodine and sulphuric acid, nor does any effervescence occur under the influence of acids.

The spores, as before mentioned, are, when first formed, of a spherical outline, or are, at all events, spherical when free, for, due to mutual pressure, they are frequently more or less polygonal while within the sporangium. The process of spore formation seems to be preceded by a disappearance of the nucleoli of the parent bodies, resulting, apparently, as some cases seem to show, from a process of repeated binary division (Pl. XVIII, fig. 11). As the sporangia mature and dry the spores lose their spherical form, a condensation of their substance seems to take place, and they become biconcave; when in this condition they closely resemble mammalian red blood-corpuscles, and, indeed, in many cases can hardly be distinguished when mingled with human blood. When in this state the margins measure about 2·7 μ and the central portions about 2·3 μ in thickness, the margin being of a faint greenish tint, and the centre almost colourless (Pl. XVIII, fig. 7). No evidence of the presence of a nucleus is, as a rule, present in such spores.

When a mature sporangium containing such biconcave spores is introduced into a suitable medium, the former very rapidly swell out and become spherical, and by their increased bulk exert a constantly increasing tension on the sporangial wall. The capsule ultimately ruptures at one or more points and contracts, forcing the spores out in streams and masses into the fluid (Pl. XVIII, fig. 2). Where the capsule has disintegrated and disappeared the process of swelling up is accompanied by curious writhing movements of the spore masses. A contractile vesicle soon makes its appearance in the spherical spores, which now, as a rule, show a clear central nuclear area surrounded by finelyclouded substance, and sometimes apparently containing a nucleolar particle of a greenish colour (Pl. XVIII, fig. 8,a). After the contractile vesicle has continued to pulsate for a short time the body begins to emit a delicate protrusion, and rapidly unfolds into a minute Amæbula, which crawls freely in the medium (Pl. XVIII, fig. 8,b, c, fig. 9). In many cases no evidence of any cyst is left behind, but in others, specially where the spores are derived from sporangia which have been subjected to prolonged desiccation, such a structure is present, appearing in the form of a delicate ring after the inmate has escaped. The spores then, as a rule, give origin to minute Amœbæ, but, in certain cases, in place of doing so, they appear to be resolved into flagellate zoospores,,which, swim off actively in the fluid.

The assumption of activity by the sporoids is manifestly influenced, both by the nature of the fluids into which they are introduced, and by the conditions to which they have previously been exposed. When introduced into ordinary pure or distilled water they remain unaltered for prolonged periods, and either fail entirely or in greater part to become active. A momentary exposure to the influence of boiling water, as by dropping the fluid on sporangia situated on a slide, does not prevent the sub-sequent development of the spores. The assumption of activity is retarded, but a certain number of the spores survive and subsequently give vent to Amœbæ. Prolonged boiling, however, is certainly fatal to them. They are capable of surviving a twenty-four hours’ immersion in Liquor lodi, remaining seemingly unaltered, and becoming active on the substitution of the reagent by a nutritive fluid. They can also survive immersion for several hours in 1 per cent, solutions of rectified spirit and of the pharmaceutical acetic acid. Mineral acids, even in very small proportions, appear to be fatal to them, and hydrochloric acid also immediately reduces any which are spherical to the biconcave form. The capacity for resisting various external influences is also regulated in some degree by the condition of the body. It is only the condensed biconcave spores which are capable of any decided resistance, those which are in the dilated spherical condition being much more susceptible to detriment.

Prolonged desiccation appears to influence the rate at which activity is developed, but certainly is not fatal. A careful series of experiments on this point showed that whilst Amœbulæ began to emerge from (perfectly fresh spores within periods ranging from fifteen to twenty-five minutes, a gradual retardation of the process corresponding with different periods of desiccation manifested itself, so that, after a period of eighty-two days, emergence did not occur until within between five to twenty hours’ exposure to favorable conditions.

When sporangia are introduced into preparations of freshboiled cow dung they rapidly disappear, and the cultivation within twenty-four hours, in favorable cases, shows an abundant new crop of sporangia. This process may be repeated again and again indefinitely so long as a fresh medium is supplied for each experiment; for, as in the case of the natural development, the soil appears to be exhausted in the process of producing a single crop. As a rule, in these cultivations we do not find a zoosporic stage represented, the spores at once giving origin to amœboid bodies, which, after having increased in size, become associated to form new sporangia. The crops of ‘sporangia thus produced are, as a rule, peculiarly abundant and well developed as compared with natural ones, due, no doubt, to the comparative freedom which the organisms here enjoy from a struggle for existence. In some cases, however, in these artificial cultures we find a failure of development or a failure of sporangial formation, the surface becoming covered with a bloom of encysted Amœbæ.

Both in natural and artificial cultivations there is a distinct tendency to periodicity in reference to sporangial formation. In the notes regarding one case which were previously given, it is recorded that while at dawn the cultivation showed no traces of the presence of sporangia, an abundant crop of such bodies appeared within the course of the next few hours. This is merely an illustration of the fact that the development is regularly limited to the period between dawn and noon or at latest 1 p.m. If sporangia have not appeared by the latter hour they will not appear until the following morning. At first sight it appeared not improbable that light-conditions were the determinent of this phenomenon, but experiments proved that this was not so, for the development followed the same course even where all light was carefully and absolutely excluded.

The sporangia and spores described above are such as occur by far most regularly and may be regarded as the typical form of reproductive bodies in the organism, but certain other sporangial bodies occasionally accompany or replace them, which although differing in various particulars are, I believe, mere aberrant varieties determined by the coincidence of special conditions. In the first place, in place of those containing normal spores, only varying within the limits as to size and form ordinarily encountered, we also meet with sporangia which, in addition to normal spores, contain a greater or less proportion of irregular, unformed looking bodies (Fig. 20).

These, as a rule, are of larger size than the others, but are connected with them by a series of intermediate forms, and exhibit a precisely similar series of developmental changes in passing into a state of activity. Sporangia in which such bodies abound are frequently of an irregular form, and in some cases may assume a dendritic character, appearing in branched tufts which may attain a height of 1·5 mm. and a breadth of 2 mm. (Pl. XVIII, figs. 4, 5); in other cases either in association with these ill-formed spores or in normal sporangia, isolated encysted Amœbæ may be present, or bodies which resemble the framework of an Amœba containing sporoid bodies.

There is, however, a much more remarkable form of sporangium which appears to be interchangeable with the common one, sometimes almost entirely replacing it, sometimes occurring in various proportions along with it, and sometimes appearing in curious intermediate forms which combine the characters of both varieties in one and the same individual. The first occasion on which they were observed was in an artificial cultivation, consisting of a portion of recent, freshly boiled cow dung into which a normal sporangium from a previous cultivation had been introduced. Forty-eight hours after the cultivation had been set, the surface was found to be covered by a sprinkling of very minute hyaline sporangoid bodies situated on the projecting points of the medium. These on microscopic examination were found to consist of aggregations of large Amœbæ which in general were still readily separable and capable of resuming independent activity in the nutritive fluid into which they were introduced. On the following day the sporangia had increased in size and numbers, some of them being of a pearly-wliite colour, others pale yellow, and others of a bright warm Indian yellow. The white ones consisted of amœbal aggregates like those observed on the previous day; the pale yellow ones contained similar bodies, and a certain proportion of masses of minute oval or broadly fusiform cellules (Pl. XVIII, fig. 14); the Indian yellow sporangia contained enormous accumulations of such cellules and a few large Amœbæ. The sporangial membrane was very distinctly defined in some cases, and on its rupture masses of the cellules (Pl. XVIII, fig. 12) and large distinct Amœbæ were forced out into the fluid of the preparation. The cellules were, as before mentioned, broadly fusiform or oval in outline (Pl. XVIII, fig. 13,b). They were flattened, colourless, and contained a large refractive and apparently oily nucleolus of greenish yellow colour with a brilliant shining nucleolus within it. The cells measured on an average about 6’2 × 3’7 p, and their oily nuclei 1’8 × 0’9 p. In most cases when they first escaped from the sporangia they were aggregated into small lumps or groups by means of a gelatinous and very faintly molecular basis-substance which soon dissolved and disappeared in the nutritive fluid (Pl. XVIII, fig. 13 a)..

The presence of sporangia containing similar cellules was recognised on several subsequent occasions. The sporangia in these cases varied in colour from clear pale yellow to full bright vermilion, a phenomenon dependent partly on the proportion of cellules present in them in relation to Amœbæ or normal spores, and partly on the proportion of oily matter around the nucleoli. In some cases this was hardly represented, in others it formed a large full-coloured globule, and in these the colour of the sporange was always highly developed. In some cases curious particoloured or piebald sporangia were present in which localised portions of the contents consisted respectively of cellules and of normal spores. The size of the cellules varied considerably in different instances, ranging from that previously given downwards to specimens measuring only 3 or 4×2·7μ on other occasions in which there was no microscopic evidence of their presence, isolated masses of them were encountered among the Amœbæ and sporoids within normal sporangia or in those in which only an imperfect spore-formation had taken place.

When the masses of cellules are allowed to remain in a suitable nutritive fluid, the gelatinous investing substance in which they are embedded gradually dissolves and disappears leaving the individual cells free. These now show a slight gradual increase in size, the oily matter of the nucleus gradually disappears leaving the greenish nucleolar particle very conspicuous, and a minute contractile vesicle generally appears. The outline of the cell also becomes somewhat modified, for while one extremity retains its original pointed character, the other becomes somewhat rounded. Subsequently one or more flagellar filaments are protruded from the latter, and the body swims off as an active pear-shaped nucleated zoospore of minute size. The zoospores after continuing their active movements for some time, and in doing so frequently exhibiting very extensive amœboid changes of form, gradually cease to move, becoming at the same time more or less rounded, and finally creep off as minute Amœbæ (Pl. XVIII, fig. 16 a, b, e, e, /). Both zoospores and Amœbæ generally show the nucleolar particle originally present very distinctly. In other cases the flagellated zoosporic stage seems to be omitted and the cells, after undergoing a certain amount of increase in size, pass off at once as nucleated Amœbæ.

In one or two instances I have met with large active Amœbæ containing varying numbers of these cells within them, but whether this were a case of ingestion or of commencing development, could not be ascertained (Fig. 21). These cells certainly appear to resemble very closely, if not to be identical with those described by Cienkowski under the name of Diplophrys stercorea, as forming sporangioid aggregations on specimens of moist horse dung.¹ According to him, however, the sporangia were devoid of any investing membrane or matrix, both of which are unequivocally present in the present instance. The characters of the movements in the cells when in the active state is moreover different, and there does not seem to be any tendency to the formation of compound groups by adhesion of active cells as described by Cienkowski. Certainly, too, the cells here are unprovided with anything of the nature of a shell or differentiated external investing coat, which is regarded as probably present in Diplophrys.

Taking the facts that they are so closely related to the ordinary Amœbæ and spores of the media in which they occur; that they are included in many cases within the same sporangia with these bodies; that they appear to be developed in groups such as would naturally result from processes of division in Amœbæ, and that in the active state they present characters so similar to those of the zoospores and Amœbulæ developed from the spores of the common form of sporangia, I am inclined to regard these cells as merely a variety of reproductive bodies belonging to the same organism, and not as the representatives of a distinct species.

While in the excreta of cows and horses we find media which permit of the continued vitality and further development of the parasitic organisms which they contain whilst still within the body, it must not be supposed that they are peculiar in doing so. The excreta form the normal site for the reproduction of continuous series of generations external to the body, but other animal fluids apparently may more or less replace them in this respect. With regard to one at all events—blood—there can be no doubt. In comparing the appearances presented by the biconcave spores of normal sporangia with those of blood-corpuscles, it was accidentally ascertained that the spores in place of being destroyed by their transfer to the abnormal medium, appeared to find in it the conditions for further development. A series of special cultivations in isolated wax-cells was therefore carried out with the following results. When a drop of normal blood suspended from a cover-glass is sealed in a wax-cell, coagulation rapidly sets in, and with the contraction, of the clot a clear peripheral ring of serum is generally formed into which white corpuscles emerge in varying numbers, retaining their activity for various periods up to twenty-four hours. In about four days the rouleaux have entirely broken up, leaving the corpuscles loose in the fluid. At the close of a period of a week the serous ring begins to become stained by the solution of the haemoglobin, and shortly afterwards the colour of the central portion of the preparation begins to lose its brilliant scarlet and to acquire a carbuncle red hue. This change in colour becomes more pronounced, and as the staining of the peripheral portion advances, the entire drop assumes a uniform deep ruby colour. With the solution of the coagulum the white corpuscles which have been entangled in it, as well as those in the peripheral area which have not disintegrated, come conspicuously into view, appearing as shining, white, oily-looking globules among the surrounding deep red fluid; after this no further change occurs, and the preparation remains seemingly unaltered for months.

The phenomena in cases where a sporangium or spores have been introduced into the drop are very different. The following are the notes recorded regarding one set of experiments:—A drop of blood was inoculated with a couple of normal sporangia from a cultivation of cow dung, and sealed in a wax-cell. For some hours the specimen exhibited similar appearances to those of a pure blood specimen set at the same time for comparison; clear serum being freely expressed to form a peripheral zone into which white corpuscles emerged, and the colour of the clot remaining bright scarlet. Subsequently, however, a dark zone appeared around each of the sporangia, indicative of local deoxidation, a phenomenon frequently observed in ordinary slide preparations of sporangia in blood. On the following morning, twenty hours after the commencement of the experiment, the clot was of a dirty brownish colour and the serum was deeply stained. It contained an abundance of active, freely crawling amoeboid bodies which, had it not been for the altered condition of the fluid and the fact of the sporangial inoculation, might have readily been taken for persistently active white blood-corpuscles, as in size, general appearance, and character of movement they were indistinguishable from such bodies. On the next day the clot and serum were somewhat darker coloured. The latter was full of active and still amoeboid bodies of various sizes; some when spherical measuring from 12 to 15 μ in diameter. As a rule, they showed a single well-defined nucleolar particle, and some contained a pair of such bodies. They showed no signs of possessing a contractile vesicle. Some of them, in addition to the ordinary scattered granules, contained a more or less altered red blood-corpuscle in their interior. An abundance of still molecular matter was also present in the serum, but no active bacteria could be detected. On the next day the serum was full of circular cells, either motionless or still exhibiting slow form-change, while a few continued to progress slowly. The average diameter of the circular cells was about 15 μ, some of them contained three nucleoli. They appeared as bright, white, shining bodies in the yellow-stained serum. Their protoplasmic contents in some cases were aggregated into a central or lateral granular mass, leaving the rest of the body apparently occupied by a homogeneous fluid; a few much larger bodies were present, attaining in some cases a diameter of 45μ, and of an even molecular substance. The still, amœbal bodies completely filled the field in many parts of the preparation, especially an the margins of the clot, were they formed continuous sheets and masses. A fresh drop of blood was now taken, inoculated from the previous one and sealed like it in a wax-cell. The initial phenomena in this were just those characteristic of normal blood. Twenty-four hours later, however, the clot had become of a dark red-brown colour, and the serum contained much molecular matter and numerous slowly moving amoeboid bodies. On the subsequent day, the latter were again observed, the activity of movement being now more decided. They contained a dim nucleolus within a clear nuclear area, but were devoid of any contractile vesicle. Twenty-four hours later the serum was full of small Amœbæ, measuring about 10μ in diameter when circular, and provided with a distinct nucleolus. After another interval of twenty-four hours the clot was found to be almost everywhere surrounded by a bank of circular and slowly moving binucleolate Amœbæ, measuring when at rest about 15μ in diameter, and appearing as bright punched-out areas in the brownish serum.

A second transfer was now made as before, inoculation being effected by means of a needle which had, as formerly, been heated to redness, and allowed to cool immediately previous to the operation. In this case the results were of a similar nature to those in the previous experiment, only the appearance of the Amœbæ was somewhat retarded. Ultimately an enormous accumulation of still amoeboid cells was formed as before. A third transfer was next carried out, but was followed by no development of Amœbæ, the basis merely rapidly breaking up and becoming full of molecular matter. The experiments as they stand, however, clearly show that the spores of the organism, although finding their natural medium in excretal matters, are perfectly capable of life and activity in other media. In some of the series of blood cultivations there certainly appeared to be a certain amount of spore formation, as subsequent to the appearance and cessation of activity in the Amœbæ, masses of much smaller spheres made their appearance among the amœbal aggregates, the individual cells of which measured from 4 to 6 μ in diameter. As no process of multiplication by division during activity was ever observed to take place, and as, at the same time, no evident diminution in the numbers of bodies developed in successive inoculations manifested itself, it seems, indeed, probable that the occurrence is a normal one. Cell cultivations in which boiled milk was substituted for blood failed to show any similar phenomena, the intense acidity developed in the medium subsequent to inoculation seeming to be fatal to the spores.

Numerous attempts were made to cultivate the sporangial spores and Amœbæ of cow dung in human excreta, but at first without any result. Like the similar bodies naturally present in the medium, they invariably appeared to be killed by the stage of acid fermentation. Even where the development of acidity was very limited, as in cases where the occurrence of Oidium was prevented by prolonged boiling, it was long before any positive results were obtained in dealing with fresh excretal matter, and the investigation had almost been given up, when, due to an accidental case of inoculation, it was ascertained that the case is very different when the medium has once entered on the alkaline stage of fermentation. Here, in place of being unfavorable to the vitality of the organism, the material appears rather to be specially adapted to it in some respects, although, at the same time, the normal cycle of developmental phenomena characterising it in its natural medium fails to occur with constancy. There is not the same strong tendency to the formation of regular sporangia, and the individual amœboid elements tend rather to retain an independent existence, attaining at the same time an abnormal magnitude; sporangial formation,however, is not always absent, although in most cases in which it occurs assuming an abnormal character.

The history of a case in which an imperfect development of sporangia occurred is as follows:—A portion of perfectly fresh normal human excreta was boiled for half an hour, and then set in a moist chamber. The material was almost neutral, and contained the usual microscopic constituents—débris of various sorts, an enormous accumulation of still bacterial matter, and a sprinkling of still circular Amœbæ possessing one or two distinct nucleolar particles. On the following day the material was unaltered in appearance. Its reaction was decidedly and permanently acid, and all the bacteria were still. Twenty-four hours later the acidity was less pronounced; at the close of forty-eight hours it had been replaced by strong alkalinity. The surface of the material was now covered with a creamy greyish-yellow layer of bacteria, which at once began to move actively in nutritive fluid. On the following day it was inoculated with one or two normal sporangia from a dried cultivation of cow dung, the condition of the sporangia being specially favorable to their ready transfer. Two days later the cultivation was again examined. The basis retained its highly alkaline reaction, but the bacterial rods had now been almost entirely reduced to series of spores (Tig. 3), so that the surface coating consisted of little save dense masses of brightly refractive granules. In the gelatinous matter of this coating numerous large Amœbæ were slowly crawling about. None of them at this time showed a contractile vesicle, but the majority possessed a distinct clear nuclear area containing two disc-shaped greenish nucleoli. In some cases the denser portion of the body was crowded with an amorphous mass of granules, and in others similar granules were aggregated into spheres contained in fluid vacuoles (Fig. 22). In appearance and measurements the granules within the Amœbæ were identical with the bacterial spores of the medium. The Amœbæ precisely resembled those frequently encountered in fresh human excreta, but μ size considerably exceeded those normally developed in cow dung cultivations.

On the following day the cultivation was found to be crowded with huge active Amœbæ, like those of the previous day. When first introduced into the nutritive fluid of the preparations they presented a peculiar tuberculate or irregularly copulate outline, but they rapidly unfolded and crawled freely about. Their nucleoli varied greatly in size; in some cases the discs attained a diameter of 5’5 μ. Specimens of Amœbæ which had been reserved beneath a cover-glass had passed into the condition of dilatation and rigidity normal under such circumstances. They were circular, entirely or almost entirely motionless, the granular matter which they contained gathered into hard lumpy masses, and with a large sharply-defined clear vacuole, apparently a rigid contractile vesicle. Some of them showed a very instructive phenomenon. In such specimens vacuolar cavities containing spherical masses of bacterial spores were still present. The remarkable thing in reference to these was that in many instances a development of a new generation of active bacteria had occurred within them, active rods darting hither and thither in the peripheral fluid of the vacuoles, and knocking and turning about the persisting granular mass (Fig. 23). Interpreted in accordance with current theories of disease, this phenomenon would indicate that the Amœbæ were dying, due to their infection with bacterial organisms; whereas, in fact, there can be no doubt that the presence of the latter was really the result of processes of development in the ingested spores occurring after the Amœbæ had been enfeebled or killed by the supervention of unfavorable conditions in the medium. The Amœbæ, as usual, were apparently slowly asphyxiated by insufficient access of air to the medium, and the bacterial elements within them now underwent development in place of digestion. They clearly had not invaded the amcebal body subsequent to its death, being confined solely to the digestive vacuoles of the interior, whilst the peripheral substance remained entirely free from them. Moreover, the Amœbæ, in certain instances, appeared to be enfeebled rather than actually dead, as faint changes in their form continued to manifest themselves with more or less distinctness.

On the following day the cultivation continued to swarm with huge Amœbæ. The body-substance was denser and less transparent than previously, and the nucleoli were very hard to distinguish, and in many cases indeed quite irrecognisable. In some cases they had begun to form masses of epithelioid tissue consisting of various numbers of more or less fused individuals. The figure on a former page (Fig. 13), illustrative of conjugate Amœbæ, was taken from this cultivation, and shows an example consisting of three individuals, each provided with a solitary large rigid vacuole. The Amœbæ in these masses were perfectly motionless. Twenty-four hours later the condition of the cultivation remained much as before. In still, dilated Amœbæ which had passed into a condition of rigor in a preparation of the previous day, the nucleus was in some instances very clearly defined. It was here seen to form a distinct bilocular capsule, which in the course of disintegration of the Amœbæ to which it belonged sometimes escaped entire into the fluid of the preparation (Pl. XVIII, fig. 15). Each of the cavities contained one sometimes two greenish discoid nucleoli of varying size. On the next day isolated and aggregate Amœbæ continued to be present in extreme abundance in the cultivation. They were now almost all characterised by the extreme indistinctness and very small size of their nucleoli, so that these bodies in many cases could not be detected even when specially sought for.

Two days later the aggregations of still Amœbæ had become so large in many cases as to form distinct irregular whitish masses visible to the unaided eye on the surface of the medium, and in many of these there were considerable numbers of smaller sporoid cells. Some of the large Amœbæ were in the flattened scale-like condition, and here the nucleus was almost or entirely invisible. A large, slowly acting contractile vesicle was, however, frequently present, which generally was formed by the fusion of several originally independent vacuoles which underwent fusion as they expanded. Some of the large adherent Amœbæ measured as much as 40 × 37·5 p and upwards. The sporangioid bodies continued to increase in numbers and size, and in the course of the next three days were entirely converted into masses of spores, hardly a single large Amœbæ remaining recognisable. The spores were somewhat larger, as a rule, than those ordinarily present in the normal sporangia developed on cow dung, but they varied considerably in size in individual instances, ranging from 5 to 10 μ in diameter. Shortly after the sporangial masses were introduced into nutritive fluid, zoospores began to emerge from them. Most of these were of large size corresponding with that of the spores. They swam about actively in the fluid, and in many instances exhibited very free amœboid changes of form while doing so. In all their characters they were quite indistinguishable from similar bodies as encountered in fresh human excreta, and like them were frequently observed to multiply by division. Due to this and to the constant emergence of new individuals, the preparation in the course of a few hours was swarming with active zoospores. At this time a certain number of small active amoeboid bodies was also present, which seemed in most cases to emerge directly from some of the larger spores. The preparation was reserved and again examined on the following day. The margins of the fluid still swarmed with active zoospores, some of them of very large size, but otherwise agreeing with their compeers in every respect. In many of them a contractile vesicle was clearly visible. Their movements varied greatly from time to time, free swimming being alternated with caudal adhesion, or with a crawling motion accomplished by means of amœboid protrusions of the body-substance.

The cultivation after this appeared to remain unchanged, no further development occurring, and the surface continuing covered with a thick layer of bacillar spores and of spore-cells derived from the Amœbæ. It remained throughout entirely free of any fungal mycelium.

In the majority of similar cultivations the results closely resembled those just described, but in one case, at all events, a development of well-formed normal sporangia took place, and in others various deviations in the form of arrested development occurred. The series, taken as a whole, appeared unequivocally to prove the identity of the organisms occurring in human and vaccine excreta, and also that the zoospores are merely a form which the reproductive bodies resulting from processes of division in the Amœbæ may assume, interchangeably with the common amœbal form directly developed in cultivations of sporangia in cow dung. In showing this they also afforded a ready explanation of the extreme frequency of the parasite in the human subject, for they indicated the presence of a constant source of readily transferable reproductive elements.

We have already seen that the vaccine excreta furnish the conditions for the continued existence and repeated reproductive multiplication of the parasites external to the host-body, crops of sporangia being apparently indefinitely produced so long as the spore-cells obtain access to the medium while in a recent state, if certain conditions of temperature and moisture be provided. Further, we have ascertained that the reproductive elements are capable of retaining their vitality for prolonged periods when in a dry state, and that they are then also capable of resisting influences which are fatal to them in activity, so that a constant supply is always at hand for introduction. These may of course obtain access to the body by various means. The transfer in the case of cattle probably occurs by means of fodder in which the sporangia are constantly liable to be present. In the case of the human subject it, no doubt, occurs in various ways, the great means of diffusion in all probability being the air. That the air is the chief agent by which the reproductive elements are diffused appears probable for several reasons. There can be no doubt as to the constant entrance of the reproductive bodies into the air, both as isolated spores and entire sporangia. The sporangia, when thoroughly dried are detached by the slightest contact from their points of attachment, and having been so, are so light as readily to be carried about by air-currents. One of the difficulties encountered in the study of dried sporangia is, in fact, dependent on their extreme lightness and the ease with which they are swept away by the air. This is not all, however; it is not only evident that a possibility for constantly recurring diffusion of the reproductive bodies by means of the air exists, but it also appears probable that when thus diffused they are more likely to undergo subsequent development than when diffused by the only other medium which can be supposed to play an influential part in the process—water. The more thoroughly dessicated the sporangia and spores are, the more are they capable of retaining their vitality under exposure to unfavorable conditions. When active, or when without being so, they have become softened and distended by immersion in passive fluids, they readily succumb to influences which, when dried, they are capable of resisting with impunity for considerable periods. Now, there appears to be little reason to doubt that in the acid gastric fluids we have such unfavorable media, likely to act prejudicially on the reproductive bodies entering the digestive canal unless specially protected. Active or softened elements will thus probably fail to reach a locality favouring their further development, while those in a desiccated condition will pass on unaffected to assume activity in the lower portions of the digestive tube.

In so far as the observations here recorded justify us in coming to a conclusion, the development of the parasite appears, as a rule, to follow a somewhat different course according as it takes place within or without a host-body. In media external to the body the spore-cells generally give direct origin to Amœbulæ, which in their turn produce a new generation of sporangia. Now, certainly, any true sporangial formation never occurs within the body, indeed, it is-scarcely possible that it should occur seeing that the constant movements of the medium must mechanically tend to prevent the initial aggregation of the formative units. It is not so easy to determine to what extent any new spore formation takes place at all, or how far the entering spores normally assume an amoeboid condition on emergence, or whether the zoosporic condition replaces the amœboid one as it does in certain cases external to the body. That they sometimes do give origin to Amœbæ, and that the latter, although failing to produce sporangia, may, in some cases, develop a new generation of reproductive elements, seems to be clear; but it remains undetermined how far this is a normal event. The question is, does an amœboid stage normally intervene between the entering spore and the zoosporic elements abounding in the lower portion of the intestinal tube? In other words, are the zoospores there the products of spores developed in Amœbæ derived from the extraneous reproductive elements, or are they directly derived from the latter ? is the flagellate zoospore the normal form assumed by the reproductive elements within the body as the Amoeba is external to it ? This is a question which cannot, in the meantime, be definitely answered. There seems to be no doubt that zoospores, in certain circumstances, are developed as the normal product of the intra-intestinal Amœbæ; but this of course does not exclude the possibility of their coincident development from extraneous spores also.

It now remains to consider the relation which the presence of the parasite bears to cholera and other morbid conditions with which it appears to be specially associated. It may be asked why any special association should occur if the reproductive elements of the organism are so generally diffused and so constantly liable to be introduced into the digestive tube as they appear to be. The answer to this question appears to be as follows:—The special prevalence of the parasite in the excreta in cholera and other intestinal disorders seems to be determined by the abnormal characters of the intestinal contents.

With regard to this point it may be sufficient to recall the fact that the alkaline choleraic fluids may be readily demonstrated to be an efficient nutritive medium—a medium much more favorable to the parasite than the material of normal excreta is. They have frequently been employed as such in the ‘study of the parasite as present in normal excreta, and again and again it has been observed that elements which in. their natural medium were in an inactive and seemingly dying condition were rapidly roused to activity and multiplication under their influence. Leaving the chances of excessive or repeated introduction of extraneous reproductive elements entirely out of account, the rapidity with which multiplication by division may occur, under favorable circumstances, appears to be amply sufficient to account even for the excessive multitudes of zoospores present in certain specimens of choleraic excreta. As a matter of observation, it is undoubted that processes of division may recur at a rate of two per hour in the same zoospore, and a calculation of the numbers which may thus be developed under favorable circumstances, even within comparatively brief periods, renders it evident that the numbers of parasitic elements present, even where most excessive, do not necessitate the conclusion that the parent bodies originally introduced must have been very numerous.

Experiments on the artificial introduction of the sporangia into the bodies of healthy animals have never been followed by any special result. I have again and again caused a dog to swallow large numbers of sporangia in all stages of development and desiccation without the treatment producing the slightest appreciable effect, and on one occasion introduced a solution crowded with spores into the peritoneal cavity of a guinea pig with as little result. The presence of morbid conditions certainly determines the degree of development of the parasite, but the presence of the latter seems to be incapable of giving rise to disease. The result of these experiments is suggestive, inasmuch as it shows how closely parasitic organisms may be associated with disease without being causally related to it. In many cases in which experiments have been supposed to demonstrate the essential dependence of disease on parasitic organisms, the procedure has not, as in the present case, consisted in the introduction of these organisms per se, but in the introduction of morbid fluids or other materials containing them. For example, we find Lösch affirming the essential causation of certain dysenteric conditions to lie in the presence of his Amœba coli, because in one instance where he injected dysenteric excreta containing the parasite into the rectum of a dog, dysenteric lesions and a development of the parasite ensued. Now, there can be doubt that if in the present series of experiments morbid fluids containing the parasite had been employed in place of clean specimens of sporangia and spores, the results might have been very different. If a solution of choleraic or normal excreta containing the parasite had been substituted for the solution of the spores per se, in the experiment on the guinea pig, it may safely be affirmed that septicaemia leading to a fatal result would have followed; and it is very probable that had the parasitic elements in the excretal solution consisted of dried or encysted spores, we should have had a coincident development of the parasite parallel to that occurring in the blood cultivations previously described. Had this been so we should, following a line of argument similar that adopted in reference to the relation of Amœba coli to dysentery, have been led to conclude that the parasite was the cause of death.

The phenomena presented by the parasite whose life-history forms the subject of the foregoing pages, in the various stages of its development, render it somewhat difficult to determine to what group of organisms we ought properly to refer it. In any attempt at doing so, the question of its animal or vegetal nature need not occupy us, as it appears certainly to belong to that series of organisms which in the mean time, at all events, must be included in the Protista, the intermediate kingdom to which all doubtful organisms wanting in differentiated animal or vegetal characters are conveniently referred. There are two groups in this no-man’s-land to which it shows certain points of affinity, appearing in some respects, indeed, to occupy an intermediate position between them. These are the Monadinœ, as they are termed by Cienkowski, or the Protomonadinœ, as they have been subsequently named by Hæckel, and the Myxomycetes, which are by some still regarded as an order of fungi. It appears to be related to the Monadinœ in the absence of any definite plasmodial stage interposed between the zoosporic and the sporangial one, and in the fact that individual units developed from single spores appear occasionally to proceed to spore formation. On the other hand, the complex nature of the sporangia, which are developed as the result of the close association and more or less complete fusion of distinct zoosporic elements, points to a close affinity to the Myxomycetes. In some cases, indeed, the fusion of the formative elements advances so far as practically to be equivalent to plasmodial formation, but the occurrence of such a phenomenon cannot be regarded as normal, the spores, as a rule, being developed in groups corresponding to individual units, and the fusion in any case being immediately antecedent to spore formation. In characters, too, the sporangia closely resemble those in certain forms of Myxomycetes. The organic granules developed in the walls closely resemble those characterising some myxomycete sporangia, and the ridging or reticulation of the inner surface of the membrane and the rudimentary capillitium clearly correspond to myxomycete structures. Taking all its characters into consideration, the organism appears rather to represent a rudimentary form of the myxomycete group, and it may, therefore, be conveniently distinguished by the name of Protomyxomyees coprinarius.

It has already been pointed out that the different developmental forms of the parasite exhibit a high degree of variability under the influence of variations in the external conditions to which they are exposed. Various forms of the zoospores are thus encountered, replacing one another in different media and in the same medium at different times. In some cases the flagellate zoospores show a distinct contractile vesicle and nucleolar point; in others any differentiation of such structures seems to be wanting. The degree and character of movement, the consistence, size, and outline of the body are also extremely inconstant; and a similar variability, although perhaps to a somewhat slighter extent, prevails in the amoeboid stage. There is one very distinct form of the flagellate zoospores which in many respects is so unlike the common ones that it might readily be regarded as an indication of specific difference, were it not possible to observe its origin as a mere transition form. In this case the body is characterised by a peculiar spathulate flattened contour, and exhibits a peculiar type of movement, consisting in a hinge-like flexion of the posterior slender portion of the body on the anterior broader part. In some specimens of choleraic excreta, as was previously pointed out,¹ this variety almost entirely replaces the normal one, but its occurrence is not limited to such media, as it has more than once been observed to arise in cultivations of cow dung. Variation in the size of the spores in the same or in different sporangia is a phenomenon of constant occurrence, and one which runs through a wide range of development. As we have previously seen, moreover, there is some reason to believe that there are two distinct forms of spores, which may replace one another more or less completely under different circumstances, the commoner one being distinguished by its spherical or biconcave figure, the other by its smaller size, more or less fusiform outline, and well-marked nucleation.

So far as I have been able to ascertain, the occurrence within the digestive canal of the human subject in this part of India of zoospores or amœboid bodies belonging to any other developmental cycle than that which has been described above is very rare and quite exceptional. It is different, however, in the case of other animals in which the same parasitic forms occur. In many specimens of fresh vaccine excreta smaller numbers of various other organisms are also occasionally present. Some of these are unquestionably specifically distinct, and others, while not unequivocally so, still present certain characters requiring that they should in the meantime be kept apart. Of the former class of bodies one of the most frequently present is apparently a species of Chlamydophrys, Cien.,¹ while as representatives of the latter we have various zoosporic forms characterised by the possession of a differentiated cell-wall, and by the fact that in the process of multiplication the line of division is longitudinal and not transverse to the original long axis of the body, and starts from the point of emergence of the flagellum (í’ig. 24).

Another characteristic organism, occasionally present in considerable numbers, appears in the form of peculiar, somewhat crescentic, colourless cells, which closely resemble certain fungal conidia, and are frequently aggregated in linear series (Fig. 25).

In some cases, too, a peculiar form of sporangioid structures makes its appearance either on the same basis with the characteristic sporangia, or apparently replacing them. In colour they vary considerably, in some cases being pale buff, in others salmon-coloured, and in others orange or red. They are always of relatively small size, of irregular outline, and unprovided with a pedicle (Fig. 26).

Their texture is firm, and they have a more or less distinctly defined capsule. Within this, as a rule, we find a thin layer of granular matter surrounding a dense mass of minute circular sporoid bodies measuring about 1*8 μ in diameter. The development of these sporangia has not been followed out, so that their true nature remains uncertain.

The principal conclusions which seem to be warranted as the result of these investigations have been already stated in the course of the narrative, but in concluding, it may be well to bring them together into a continuous series. They are as follows:

1. Special parasitic forms may be specially associated with particular forms of disease without holding any causal relation to them.

2. The monadic, amœbal and sporoid bodies, so abundant in many choleraic excreta, are all developmental forms of one species of parasite which I propose to call Protomyxomyces coprinarius.

3. This parasite appears to be closely related to the organisms included within the Protist groups of Protomonadinæ and Myxomycètes, and in certain respects seems to represent a connecting link between them.

4. It is not confined to choleraic or even to human excreta as a basis, and only attains its full development external to the bodies of the animals within which it occurs.

5. Its immature forms occur parasitieally as normal inmates of the digestive canal in certain of the lower animals.

6. In the human subject, both in health and disease, they are very frequently present in varying numbers.

7. During health the number and activity are limited, due to repressive influences exerted by the normal intestinal contents as a medium.

8. Their excessive abundance in certain forms of disease is due to abnormal conditions of the intestinal contents, permitting of the occurrence of processes of rapid multiplication.

9. Normal human excreta do not form a medium in which any farther development of the parasitic elements outside the hostbody can occur.

10. On the contrary, the normal series of fermentative changes through which the excreta pass after exit from the body ensures the complete destruction of the parasitic elements.

11. No such destructive effect, however, is exerted by the changes occurring during the decomposition of the excreta in certain lower animals—specially cows and horses; and here the parasitic elements on their escape from the body undergo farther processes of development resulting in the production of reproductive bodies securing the continuance and diffusion of the species.

12. Such excretal matters, therefore, serve as a constant source whence parasitic elements may be transferred to the bodies of other animals.

13. Human excreta which have passed through the initial processes of decomposition, and which have thus become alkaline, allow of the continued existence and multiplication of elements of the parasite which may then obtain access to them, and may thus serve as a second centre of reproduction.

14. The introduction of the reproductive elements of the parasite into the human body is mainly effected through the medium of the air.

15. The introduction of the reproductive elements per se seems to be quite innocuous.

16. The special association of the parasite with intestinal disorders appears to be dependent on the abnormal condition of the intestinal contents allowing of the rapid multiplication of reproductive elements which may obtain access to them.