An Account of Amoeba discoides; its Culture and Life History

Some years ago Schaeffer (1916) pointed out that there was great confusion concerning the description of Amoeba proteus, the largest of the fresh-water amoebae. He then proceeded to show that there were no less than three species of amoebae indiscriminately referred to as Amoeba proteus. There is no need to give here the details of Schaeffer’s investigation and the reasons for his conclusions. It suffices to say that he gave the names Amoeba proteus, Amoeba dubia, and Amoeba discoides to the three large amoebae.

Dr. Lucy Carter, who, in 1910, at the suggestion of Professor Graham Kerr, had undertaken to investigate’the life-history of Amoeba proteus, was confronted at the outset of her work with that confusion of nomenclature to which reference has already been made. She published the results of her experience in a paper entitled ‘Some Observations on Amoeba proteus’ (Carter, 1919). It seems quite clear from a scrutiny of the work of these two authors that Carter’s Amoeba proteus X corresponds to Schaeffer’s Amoeba dubia. Later Schaeffer (1926) resuscitating the Linnaean genus Chaos gave the new name Chaos diffluens to Amoeba proteus Pallas (Leidy) (== Y, Carter). Amoeba dubia (= Amoeba proteus, Penard = Amoeba proteus X, Carter) he named Polychaos dubia, and Amoeba discoides he named Metachaos discoides. In spite of Schaeffer’s suggested changes of nomenclature I shall use the old terms Amoeba proteus, Amoeba dubia, Amoeba discoides in this paper.

Although I have long worked on Amoeba proteus (= Chaos diffluens) and collected material in the district around Glasgow, I have never come across Amoeba discoides. Indeed, so far as I have been able to ascertain, it has never been recorded in Great Britain and seems to be absent from the fauna.

The material was kindly handed over to me by Mr. Watkinson and Mr. Sutcliffe, and to both these gentlemen I wish to express my very sincere thanks.¹

I also take this opportunity of expressing my best thanks to Sister Monica for having given me the material, and for having placed at my disposal throughout the progress of the work her varied knowledge and experience in the cultivation of microorganisms.

The amount of material at my disposal was limited. Its source was precarious, as any slight change in the conditions of the tropical fish tanks might kill off all the amoebae. Hence, it seemed advisable to establish good, strong, laboratory cultures before proceeding to fix large quantities preparatory to working out the cytology. The growth of all large free-living amoebae is slow, and it takes years to accumulate abundant material. Schaeffer (1916) said that Amoeba discoides was a slower grower than Amoeba proteus. As subsequent readings from my field book observations will show, I cannot endorse this statement unreservedly.

The first stock of amoebae arrived in a 250 c.c. capacity bottle. This was allowed to stand over night so that the debris containing the amoebae had time to settle on the bottom. Then into a glass trough (diameter = 4 inches, height = 2| inches) was poured Glasgow tap-water to a height of about J inch to which some of the supernatant fluid from the bottle containing the amoebae and eight wheat-grains boiled for five minutes to kill the embryo were added. To this were added amoebae from the surface of what remained of the original debris at the bottom of the bottle, great care being taken to avoid the silver sand which lay underneath and to take only the rich mud from its surface. The amoebae were added in groups at intervals of a day for several days.

The culture thus started on November 14,1934 (called culture A), was successful and has now (July 1937) been sub-cultured. At intervals of three months five or six wheat-grains and a little water have been added to it.

The bottle in which the amoebae arrived with its remaining debris (chiefly silver sand) was kept and filled up with Glasgow tap-water. This was left undisturbed to be used for future sub-cultures, and also to replenish the liquid in culture A at intervals. Each time that water was taken from this bottle it was replaced by fresh tap-water so that there was always a supply of water which had stood for some time over what remained of the original debris. After the culture had been going for about a year, water straight from the tap was used to replenish it, for as Glasgow tap-water is so admirably adapted to Amoeba proteus culture it was deemed safe for this particular amoeba.

A second consignment of 350 c.c. of material arrived from Mr. Watkinson (November 22,1934), and was found to contain many of the amoebae. The material was not removed from the bottle, but an attempt was made to cultivate the amoebae by adding occasionally very minute quantities of ‘Spratt’s Tropical Fish Food’, on which Mr. Sutcliffe fed his tropical fish and of which he had very kindly sent me a box. This experiment was a failure. The amoebae, plentiful at first, gradually disappeared, nor did they reappear as in the successful culture A. The entire disappearance of the amoebae may be due to the pabulum or to the fact of their having been kept in the bottle and not put out into flat vessels.

The amoebae evidently have died out in Mr. Sutcliffe’s tanks as no further stock has been received.

During the time that I have been caring for and examining the cultures of Amoeba discoides I have inclined to the opinion that the most successful were those reared in Petri dishes. During the Session 1936-7 I have had cultures in glass troughs 6 inches diameter and 4 inches deep, and others in Petri dishes 4 inches diameter and $34$ inch deep. The culture water in the troughs was about 3 inches deep. Though these respective cultures were obtained by subdividing the same parent culture, and though they were submitted to the same technique the results are outstandingly different. The cultures in the troughs have gradually grown poorer until now (July 1937) they seem to contain no amoebae at all, while the Petri dish cultures are most luxuriant, containing large numbers of beautiful amoebae.

The pH of the water in which Amoeba discoides lives appears to have no great significance in the cultivation of this rhizopod, if we except the fact that it is always lower than pH 7. Several good cultures vary between pH 6-5 and pH 6-8, but I have also a luxuriant culture whose present pH (Aug. 1937) is 4-5.

I have not been able, however, to perform any microinjections to test the pH of the cytoplasm.

As will be explained more fully later in this paper, the culture undergoes ‘depression’ periods and ‘optimum’ periods. A ‘depression’ period is one during which the culture contains no adult amoebae. Conversely during an ‘optimum’ period it contains large numbers of adult amoebae. Definition of these two terms is necessary at this point so that it may be clearly understood what I mean by ‘young’ adult amoebae and ‘old’ adult amoebae. When the full grown amoebae first appear in the culture after a ‘depression’ period I call them young adults. These live for from three to six or even seven months, during which time they increase by fission. Now comes a time when they begin to disappear gradually from the culture and when they exhibit features not seen three months earlier. At this stage I call the amoebae ‘old’ adults.

If a pipette full of the culture containing young adult amoebae be transferred to a glass cell and examined at once over a black background under the low power of a Zeiss Greenough binocular an observer at first experiences difficulty in recognizing the amoebae. A little patience brings its reward for these fantastically shaped and exceedingly translucent creatures make a beautiful picture as they lie emmeshed in the green algae. They possess numerous pseudopodia stretching outwards in all directions. When these same amoebae are placed on a slide in a drop of culture water under a cover slip, given time to grip the substratum and are then examined under a microscope with transmitted light, the shape is found to have changed completely (fig. 1, Pl. 31). They are no longer radial but long and flat, the pseudopodia being fewer in number and in one plane (Text-fig. 1, a, b, c). The cytoplasm of the healthy amoeba spreads out over a large surface area and consequently has but little depth or thickness. This makes the examination of the living nucleus and the cytoplasmic contents easy. The average length I found to be 420μ (Schaeffer (1916) gives 400μ as the average length): when the amoeba is stretched out to 420μ its width is about 140μ. The cytoplasm is very finely granular, exceedingly mobile and flows with great rapidity. The flow or movement is always in the centre both in the main bulk of the creature and along the pseudopodia. No forward movement of the cytoplasm at the sides can be detected. When the amoebae are fully stretched out there are no folds in the cytoplasm. The ectoplasm is exceedingly sparse, forming only a thin skin round the endoplasm. Even at the tips of the pseudopodia ectoplasm is only rarely seen. It is most conspicuous at the base of the pseudopodia, that is, in the angle between the main body and a pseudopodium or between two pseudopodia.

Embedded in the cytoplasm is a number of crystals and many minute particles which may be nascent crystals. These crystals resemble the characteristic bipyramidal ones found in Amoeba proteus but they are much less truncated than those of Amoeba proteus, also the angles or edges are less sharp and the whole crystal has a more oval appearance. I cannot agree with Schaeffer that this amoeba is more stuffed with crystals than Amoeba proteus. During the many years that I have worked in the Notre Dame Laboratory I have examined innumerable Amoeba proteus, and it is my experience that under certain conditions of culture and age the crystals are far larger and more numerous in Amoeba proteus than in Amoeba discoides. On the other hand, Sister Monica Taylor has often shown me specimens of Amoeba proteus containing very few and sometimes no crystals at all. A few years ago I carried out some experiments on the crystals of Amoeba proteus, but I have arrived at no satisfactory conclusion about their formation or function. I think it certain that they grow in size and number as the amoeba grows in age. Schaeffer (1916) states that he has ‘never with certainty been able to find any other form of crystal i.e. the dipyramidal ‘in this amoeba’. I have seen small cubic crystals in several specimens, but these cubic crystals are never as large as those seen in Amoeba proteus and their occurrence is much rarer. I have never seen in Amoeba discoides the large square plate-like crystals which occur in Amoeba proteus. Also in the cytoplasm of the young adult is a number of nutritive spheres. These are small (2 to 3/i), and of a definite greenish appearance. At this stage the nutritive spheres are quite structureless, and are not stained by Ehrlich’s haematoxylin.

I have been able to confirm all Schaeffer’s published observations on the living nucleus. It is normally single, disk-shaped with rounded edges, and with an average diameter of about 40μ by 15μto 18μ. thick. One nucleus was 56μ in diameter, though why it should be so much above the average size I am unable to say. The two larger surfaces are generally concave (Text-fig. 2 a). The surface is smooth, without folds. The nucleus is carried along in the flowing endoplasm, but generally holds a position about one-third the length of the amoeba from the posterior end. If and when it is carried nearer the anterior end it remains stationary while the cytoplasm flows over and round it, and thus it regains its normal position. While being carried along the nucleus is all the time rolling over and over. It is my opinion, however, that the nucleus of Amoeba discoides rolls over more quickly and consequently more often, and so one obtains an edge view or ‘elevation’ view much more frequently, than in Amoeba proteus. In rolling over the nucleus sometimes bends on itself and may remain so for some time, having thus the shape of a kidney-bean (Text-fig. 2 &), as observed by Schaeffer (1916). As the cytoplasm of Amoeba discoides is much less voluminous than that of Amoeba proteus, and as it spreads this smaller volume over a large surface area, the living nucleus is always visible, and the chromatin masses under the nuclear membrane and in the karyosome are easily seen. The nucleus has a coarse mottled appearance. There appears to be a great deal of fluid of a mobile nature within the nucleus, and in this the karyosome changes position so that sometimes it is central and at other times lies to one side. Indeed one gets the impression that the karyosome itself contains mobile liquid readily changing position from one part of it to another as the nucleus itself is being rolled about in the endoplasm. This hypothesis is confirmed by the variety of appearances which one comes across when studying a large number of fixed specimens. In many of the young adults the nucleus lies in clear cytoplasm, yet I am not prepared to call this clear space a vacuole. There does not seem to be any definite boundary line round it as there is, for instance, round the contractile vacuole. It seems to me to be a region of very clear and very mobile cytoplasm free from cytoplasmic inclusions, which gradually merges into the ordinary cytoplasm containing crystals, nutritive spheres, &c.

In most specimens there is a single contractile vacuole which reaches a diameter of 30 μ. It is however quite a common thing to find two contractile vacuoles, a primary one at its maximum size and a secondary one beginning to grow. As the primary one bursts the secondary takes its place, while another secondary is formed at once. The contractile vacuole is generally formed near the nucleus, and moves along with it until it is ready to burst when it lags behind the nucleus and takes up a position near the posterior end of the amoeba. In this position it bursts very slowly and very gently without causing any apparent disturbance within the cytoplasm or in the surrounding fluid. The rate of growth of the contractile vacuole is not regular, but I have observed it to be more rapid and more regular in young specimens. The irregularity may, of course, be due to the imprisoned conditions in which it is necessary to examine the amoebae.

Amoeba discoides feeds on Flagellates of various sizes, on green unicellular plants, and on Rotifers. When the latter are plentiful in the culture they generally form the staple food of the amoebae and it is quite a regular occurrence to find as many as nine while occasionally twelve Rotifers may be seen ingested by one individual amoeba. The variety and the amount of food which an individual amoeba can contain at the same time in its cytoplasm is truly amazing.

I have a culture at present (July 1937) in which, though it contains a plentiful supply of the above-named organisms, the amoebae have taken to ingesting long pieces of filamentous algae. Whether these latter are wholly digested or ejected undigested I am as yet unable to say.

Viewed by reflected light the amoebae at this stage (i.e. old adults) are white and opaque. In transmitted fight the crystals are seen to be large and very numerous. The nutritive spheres which at the beginning of the ‘optimum’ period were small and inconspicuous, are now large (5μ to 6μ) and exceedingly numerous, filling up much of the endoplasm. Their definite green appearance tints the whole Amoeba. The spheres tend to collect together into groups of from twenty to thirty, and such a group is often seen at the tip of a pseudopodium to the exclusion of crystals and other cytoplasmic inclusions (Textfig. 3). Schaeffer (1916) was of the opinion that the number of these spheres which he designated ‘so-called excretion spheres’ depended on the amount of food digested by the amoeba. I confirmed that observation in 1924 on Amoeba proteus (see Taylor, 1924). Now, however, in the light of much more experience and intensive observation, I think that the amount of food eaten by the amoeba is not the only factor at work. To give but one out of many instances, in the early part of 1936, owing to stress of other work, my culture of Amoeba discoides was neglected. Whilst not actually starved, it was decidedly underfed, yet when I resumed work on it in July I found that all the amoebae were stuffed with very large nutritive spheres. These amoebae were then about six months old, and from that time onwards they began to disappear gradually from the culture.

The nutritive spheres grow in number and in size as the amoebae grow in age. When a culture is healthy and undergoing its normal cycles the amoebae are always found to contain many large nutritive spheres just before the so-called ‘depression’ periods. It has been shown for Amoeba proteus (Taylor, 1924) that the nutritive spheres are intimately connected with the formation of encysted young amoebae. As will be shown later, I have had evidence of this being true also for Amoeba discoides.

In April 1937 a culture of Amoeba discoides reared in a Petri dish was in an especially flourishing condition, containing a very large number of adults. The food organisms however in this culture were sparse, and it was evident that in a short time starvation conditions would prevail. Consequently a subculture was made by transferring a portion of the material (about one-tenth of the whole) to a Petri dish containing an excellent culture of food organisms, Rotifers, Flagellates, &c. Some food organisms were also added to the parent culture. On examining both cultures three months later (July 1937) their conditions were found to differ greatly. The parent culture contained no food organisms. The amoebae, though still plentiful, had become senescent—being black by transmitted light—full of crystals and large nutritive spheres—sluggish—refusing to grip the substratum and flow like healthy individuals. In the subculture which still contained a good supply of food organisms, the amoebae had multiplied to an extraordinary degree. They were in beautiful condition, and as soon as placed under the cover-slip they gripped the substratum and flowed actively. The crystals and nutritive spheres were small. The lack of food in the one case had brought on senescence. The plentiful supply of it in the other had warded off this condition, the amoebae being kept in good condition by repeated fission. However, this fission cycle will not continue indefinitely no matter how plentiful the food supply, and there is evidently still much work to be done on the nutrition of the amoebae.

For the purpose of examining the fixed and stained nucleus three types of preparations were made:

1. By means of a fine pipette a drop of the culture water containing the amoebae was put on to a glass slide and a coverslip placed over it. The slide was then left to stand for a time, in a damp chamber, sometimes overnight, so as to give the amoebae time to settle down, grip the slide, assume the flowing state and stretch out to their full length. Aceto-carmine was then run under the cover-slip and the preparation thus made examined at once. Very many specimens fixed and stained by this method were examined.

2. The amoebae were put on to the slides and given time to settle, but instead of aceto-carmine Bonin’s fluid was used to fix them. After fixation in Bouin the amoebae were washed, stained in Ehrlich’s haematoxylin, dehydrated, cleared in xylol and made permanent with Canada balsam, all by the irrigation method. Many beautiful preparations have been obtained by this method.

3. At times when the amoebae were very plentiful in the culture large numbers of them were put into a Petri dish. Then working under the low power of the binocular with the help of needles and a fine pipette the amoebae were freed as much as possible from the debris of the culture. This latter was as far as possible removed, as was also much of the supernatant water, leaving only just enough water to keep the amoebae ‘happy’. The Petri dish was then flooded with Bouin’s solution. The material thus fixed was pipetted into centrifuge tubes where the washing, staining, dehydration, and clearing was carried on. Finally, the amoebae were permanently mounted on slides in Canada balsam. Bouin’s fluid proved an excellent fixative, and Ehrlich’s haematoxylin as a stain leaves nothing to be desired.

The fixed and stained nucleus varies in diameter from 21 μ. to 45μ, with an average diameter of about 36μ. It is surrounded by two membranes. Of these, the outer is very definite and sharply differentiated, looking like a distinct blue skin in the stained preparations. The inner membrane is much less definite. There is a clear space between the two membranes (figs. 3, 4, 7, 8, 11, Pl. 32). Immediately within the inner membrane, in fact close against it, lie the large chromatin blocks (figs. 2-12, Pl. 32). In optical sections of the nuclei these blocks appear very regularly arranged and equally spaced. There is generally a second layer of chromatin blocks within the outer one, occasionally there is a third such layer, but the blocks of these inner layers are not so regularly arranged as those of the outer. The chromatin blocks stain easily in Ehrlich’s haematoxylin—each block standing out clear cut and richly coloured like fully differentiated chromatin, reminding the observer of a chromosome of a metazoan nucleus. The karyosome which carries masses of chromatin lies within the chromatin blocks region. As a rule its diameter almost equals that of the nucleus. Sometimes, however, when the latter is seen in ‘plan’, the karyosome cannot be distinguished from the rest of the nucleus, which means that it extends right out to the inner nuclear membrane (fig. 6, Pl. 32). As has already been said in describing the living nucleus, the karyosome takes up various positions in the nucleus, and the stained preparations show that it often is much bent on itself. That the nucleus, and even the karyosome, contain much fluid also becomes evident from an examination of the stained preparations (figs. 3, 5, 6, 7, 11, Pl. 32). I think there can be little doubt that there is also a layer of fluid between the inner and outer nuclear membrane.

I have fixed, stained, mounted, and examined a very great number of Amoeba discoides at different times in the life of the culture. The beautifully expanded condition of the amoebae, and the details of the structure which can be seen in the organisms present in the food vacuoles are evidence that the fixation is absolutely satisfactory. Many of the nuclei examined are dividing—some just beginning to divide (figs. 5 and 6, Pl. 32), others further advanced in the process of division (figs. 8,9,10,11, Pl. 32), but in no case is there any sign whatever of mitotic division. The division of the nucleus as a whole is amitotic, and so far as I can see a very simple, straightforward case of amitosis. The cleavage divides the nucleus as a whole as well as the karyosome into two parts. Each of these parts forms a new daughter nucleus. In one nucleus which had so divided, the two daughter parts had not separated from each other yet there was evidence of a second division taking place at right angles to the first. This observation was made on a temporary preparation in aceto-carmine, so I can only represent diagrammatically what I saw (Text-fig. 4). In Amoeba proteus it is possible to find four or even eight nuclei in individual amoebae. In the present investigation I found only one specimen in which one of the newly formed daughter nuclei had already divided into two before the cytoplasm showed any signs of division (fig. 11, Pl. 32).

So as to make quite clear what has to follow in this section I think it advisable to give at this point some readings from the record of the culture A.

14/11/34. The culture was started in the manner explained at the beginning of this paper (p. 461).

12/12/34. No adult amoebae could be found when the culture was examined with the low power of the Greenough binocular. An examination by the ordinary microscope was not made.

11/3/35. The culture contained numerous adult amoebae some of which were yellowish in tint. A sub-culture made at this date did not for some unknown reason succeed.

23/7/35. Only one opaque adult was found after a careful search, but there were many very beautiful young amoebae. When these were put on to a slide in a drop of the culture water for examination they spread out quite readily and formed long pseudopodia (Free-hand outline sketches of these young amoebae are shown in Text-fig. 6, a and V). These young amoebae were feeding heavily on unicellular algae, this being the only available food at the time.

11/10/35. Many large adults were present. These when examined with the microscope were found to be in an active healthy condition.

30/11/35. Amoebae were not nearly so plentiful as when last examined. It was evident that the culture was approaching a ‘depression’ period.

4/1/36. Very few adults could be found. Many young amoebae present, these were healthy looking and feeding heavily.

13/4/36. Adult amoebae were plentiful.

6/7/36. Adult amoebae plentiful, but very opaque when seen under the binocular with reflected light over a black background. When examined with the microscope the amoebae were found to be stuffed with crystals and very large nutritive spheres. As these amoebae were about six months old, the culture was carefully watched during the next few weeks. The number of adult amoebae grew less and less, but before they had all finally disappeared the culture contained numbers of micro-amoebae. These microamoebae will be described later.

(Note.—The term micro-amoeba is used for the small amoebae recently emerged from the cyst and undergoing development.)

From a study of this record it is evident that, as has already been said, there are times when the culture contains no adult amoebae but such times are only so-called ¹ depression’ periods for during them numbers of micro-amoebae can be found. These young amoebae grow up and in their turn increase in number by fission.

The question now to be considered is the formation of these young amoebae, and though I have not yet seen all the stages described for Amoeba proteus (see Taylor, 1924), those I have seen are, I think, sufficient to prove that agamontogony in Amoeba discoides is similar to that in Amoeba proteus.

The ‘old’ adult with its large nutritive spheres is the agamont. When an agamont has been fixed and stained in Ehrlich’s haematoxylin it becomes evident that there are two types of spheres in its cytoplasm—the ordinary large deeply stained nutritive spheres and other still larger spheres which are definitely differentiated into palely stained and deeply stained regions. These larger spheres are the developing agametes. In the process of differentiation they use up the nutrient material of the nutritive spheres which latter in consequence lose their staining capacity. In a fully formed agamete it is possible to distinguish the new cytoplasm which is pale-staining from the chromatin which stains more deeply (fig. 13, Pl. 32). Dr. Monica Taylor (1924) has shown how in Amoeba proteus the chromatin blocks escape from the nucleus and become associated with the nutritive spheres to form the rudiment of the agametes.

I have not seen the chromatin blocks actually escaping from the nucleus of Amoeba discoides. I have, however, found specimens, containing fully differentiated agametes, in which the nucleus had no visible membrane (fig. 12, Pl. 32). Now ordinarily the nuclear membrane is a conspicuous structure which, stains very readily. It seems reasonable then to conclude that in these specimens the nuclear membrane has dissolved in order to set the chromatin blocks free into the surrounding cytoplasm. Here these masses of chromatin uniting with the nutritive spheres differentiate round themselves a certain amount of cytoplasm and become fully formed agametes (fig. 13, Pl. 32). When the agamont disintegrates the agametes are set free into the culture water where they remain in an inactive, encysted condition for a varying period of time (fig. 14 a-g, Pl. 32). Finally, the little amoebae escape from the cysts. At first these micro-amoebae are circular in general outline, from 20μ to 25μ. in diameter, with a great number of very short blunt pseudopodia radiating in all directions (Text-fig. 5, a and b). The cytoplasm is clear and streams very slowly, so that there is little change in the creature’s position on the slide. The nucleus is very conspicuous, the pale green-looking karyosome standing out clearly, the rest of the nucleus forming a clear zone round it. At this stage the young nucleus of the microamoeba does not roll over in the cytoplasm, it is always seen in ‘plan’. The contractile vacuole which lies beside the nucleus is also a conspicuous feature. It grows rapidly, bursts regularly and very gently. Many of those micro-amoebae contain no food vacuoles while others are stuffed with them, the former, I conclude, being those most recently emerged from the cysts.

In the next stage the blunt pseudopodia have been withdrawn and replaced by a single large pseudopod so that the amoebae are no longer circular but oblong in outline, about 30μ. to 40μ. long (Text-fig. 5 d, e,f). A great part of this pseudopodium— the anterior portion of it—consists entirely of clear cytoplasm (Text-fig. 5 d, e, f). The cytoplasm flows rapidly so that the little creature travels along at a considerable rate. The nucleus which is very conspicuous, and still seen always in plan, seems to act as a dividing centre for the cytoplasm which flows round it in two streams, one on either side (Text-fig. 5 f). The contractile vacuole beside the nucleus behaves as in the earlier stage, while the food vacuoles are larger and more numerous than they were in that stage.

After this the amoebae begin to look much more like the adults (Text-fig. 6 a and b). The single pseudopodium which consisted mostly of clear cytoplasm being replaced by many pseudopodia in which the endoplasm is more granular. The tips of the pseudopodia are always, at every stage of development as well as in the fully grown adult, blunt, that is, rounded.

I wish to offer my sincerest thanks to Professor Graham Kerr under whom this work was begun, and who has continued from afar to watch over it with ever kindly interest and encouragement and who has read the paper in typescript.

My thanks are also extended to Professor Hindle, under whom the work was completed, for his kind advice and for reading the paper in typescript.

In conclusion I would like to express my appreciation of her skill and of the care and trouble bestowed by Miss Brown Kelly in the execution of the original drawing of fig. 1, Pl. 31.

EXPLANATION OF PLATES 31 AND 32

LETTERING.

N., nucleus; c.b., chromatin blocks of the nucleus; C.V., contractile vacuole; F. V., food vacuole (the organism being a large encysted Flagellate); F.r., food vacuole (small Flagellates); s.c., cubic crystal; c., dipyramidal or oval crystal; N.S., nutritive spheres; k., karyosome; n.m., nuclear membrane; n.sp., nuclear sap; n.s., nutritive sphere; ch., chromatin in the karyosome.

PLATE I.

Fig. 1.—Free-hand drawing (not to scale) made from a large, living adult Amoeba discoides.

PLATE II.

All the figures were drawn from specimens fixed in Bouin’s fluid and stained in Ehrlich’s haematoxylin. Camera lucida with a No. 5. compens.ocular and a Zeiss Apochromat. 2 mm. oil immersion objective.

Figs. 2, 3, and 4 represent resting nuclei; the karyosome of 2 is seen in ‘plan’; of 3 in ‘elevation’, and of 4 pushed to one side of the nucleus.

Figs. 5 and 6 early stages of division of the nucleus.

Fig. 7.—Nucleus dividing and bent into a figure of eight shape.

Fig. 8.—Division of the nucleus almost complete, half the karyosome passing into each daughter nucleus.

Figs. 9 and 10.—The nuclear division is completed, but the daughter nuclei have not separated.

Fig. II.—Three nuclei from one amoeba. Cytoplasmic division has not kept pace with nuclear division for one of the daughter nuclei of the first division has already divided.

Fig. 12.—A nucleus with no trace of a nuclear membrane.

Fig. 13.—Small portion of an amoeba—an agamont showing nucleus without nuclear membrane; a, agametes and n.s., ordinary nutritive spheres.

Fig. 14.—Encysted agametes, a-g, after disintegration of the agamont.

Fig. 15.—A young Amoeba discoides.

For many years Mr. Harry Watkinson of Grimsby and Sister Monica have exchanged material and notes on many problems connected with pond life and micro-aquaria. Three years ago Mr. Watkinson became interested in the organisms which he found in the fresh-water aquaria for rearing tropical fish, owned by the well-known aquarist Mr. Albert Sutcliffe of Grimsby, and endeavoured to make an ecological survey of each tank. While engaged on this work he came across a large free-living amoeba with which he was not acquainted. He had long possessed sub-cultures of Sister Monica’s Amoeba proteus and was familiar with Amoeba dubia. He sent this unknown amoeba to Sister Monica for her inspection, who, suspecting that it was Amoeba discoides, asked Mr. Watkinson for more material which is that here described.