An Account of Amoeba discoides; its Cultureand Life History.

By

Catherine Hayes, S.N.D., B.Sc,

From the Zoology Department, University of Glasgow, and theLaboratory of Notre Dame, Dowanhill, Glasgow.

With Plates 31 and 32 and 6 Text-figures.

INTRODUCTION.

SOME years ago Sehaeffer (1916) pointed out that there wasgreat confusion concerning the description of Amoebapro teus , the largest of the fresh-water amoebae. He thenproceeded to show that there were no less than three species ofamoebae indiscriminately referred to as Amoeba p ro teus .There is no need to give here the details of Schaeffer's investiga-tion and the reasons for his conclusions. It suffices to say thathe gave the names Amoeba pro teus , Amoeba dubia,and Amoeba discoides to the three large amoebae.

Dr. Lucy Carter, who, in 1910, at the suggestion of ProfessorGraham Kerr, had undertaken to investigateHhe life-history ofAmoeba pro teus , was confronted at the outset of her workwith that confusion of nomenclature to which reference hasalready been made. She published the results of her experiencein a paper entitled' Some Observations on Amoeba pro teus '(Carter, 1919). It seems quite clear from a scrutiny of the workof these two authors that Carter's Amoeba proteus Xcorresponds to Schaeffer's Amoeba dubia. Later Sehaeffer(1926) resuscitating the Linnaean genus Chaos gave the new nameChaos diffluens to Amoeba proteus Pallas (Leidy)(== Y, Carter). Amoeba dubia (= Amoeba pro teus ,Penard = Amoeba proteus X, Carter) he named Poly-chaos dubia, and Amoeba discoides he namedMetachaos discoides. In spite of Schaeffer's suggestedchanges of nomenclature I shall use the old terms Amoeba

460 CATHERINE HAYES

p r o t e u s , Amoeba dub ia , Amoeba d i sco ides in thispaper.

Although I have long worked on Amoeba p r o t e u s(= Chaos diffluens) and collected material in the districtaround Glasgow, I have never come across Amoeba dis-co ides . Indeed, so far as I have been able to ascertain, it hasnever been recorded in Great Britain and seems to be absentfrom the fauna.

SOURCE OP THE MATERIAL.

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

I also take this opportunity of expressing my best thanks toSister Monica for having given me the material, and for havingplaced at my disposal throughout the progress of the work hervaried knowledge and experience in the cultivation of micro-organisms.

CULTURE OF THE MATERIAL CONTAINING AMOEBA DISCOIDES.

The amount of material at my disposal was limited. Itssource was precarious, as any slight change in the conditionsof the tropical fish tanks might kill off all the amoebae. Hence,it seemed advisable to establish good, strong, laboratory cul-tures before proceeding to fix large quantities preparatory toworking out the cytology. The growth of all large free-livingamoebae is slow, and it takes years to accumulate abundantmaterial. Schaeffer (1916) said that Amoeba d isco ides

1 For many years Mr. Harry Watkinson of Grimsby and Sister Monicahave exchanged material and notes on many problems connected withpond life and micro-aquaria. Three years ago Mr. Watkinson becameinterested in the organisms which he found in the fresh-water aquaria forrearing tropical fish, owned by the well-known aquarist Mr. Albert Sutcliffeof Grimsby, and endeavoured to make an ecological survey of each tank.While engaged on this work he came across a large free-living amoebawith which he was not acquainted. He had long possessed sub-culturesof Sister Monica's Amoeba p r o t e u s and was familiar with Amoebadub ia . He sent this unknown amoeba to Sister Monica for her inspection,who, suspecting that it was Amoeba d isco ides , asked Mr. Watkinsonfor more material which is that here described.

AMOEBA DISCOIDBS 461

was a slower grower than Amoeba p r o t e u s . As subsequentreadings from my field book observations will show, I cannotendorse this statement unreservedly.

The first stock of amoebae arrived in a 250 c.c. capacitybottle. This was allowed to stand over night so that the debriscontaining the amoebae had time to settle on the bottom. Theninto a glass trough (diameter = 4 inches, height = 2J inches)was poured Glasgow tap-water to a height of about \ inchto which some of the supernatant fluid from the bottle con-taining the amoebae and eight wheat-grains boiled for fiveminutes to kill the embryo were added. To this were addedamoebae from the surface of what remained of the original debrisat the bottom of the bottle, great care being taken to avoid thesilver sand which lay underneath and to take only the richmud from its surface. The amoebae were added in groups atintervals of a day for several days.

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

The bottle in which the amoebae arrived with its remainingdebris (chiefly silver sand) was kept and filled up with Glasgowtap-water. This was left undisturbed to be used for futuresub-cultures, and also to replenish the liquid in culture A atintervals. Bach time that water was taken from this bottle itwas replaced by fresh tap-water so that there was always asupply of water which had stood for some time over whatremained of the original debris. After the culture had beengoing for about a year, water straight from the tap was used toreplenish it, for as Glasgow tap-water is so admirably adaptedto A m o e b a p r o t e u s culture it was deemed safe for thisparticular amoeba.

A second consignment of 350 c.c. of material arrived fromMr. Watkinson (November 22,1934), and was found to containmany of the amoebae. The material was not removed from thebottle, but an attempt was made to cultivate the amoebae byadding occasionally very minute quantities of' Spratt's TropicalFish Food', on which Mr. Sutcliffe fed his tropical fish and of

462 CATHERINE HAYES

which he had very kindly sent me a box. This experiment wasa failure. The amoebae, plentiful at first, gradually disappeared,nor did they reappear as in the successful culture A. The entiredisappearance of the amoebae may be due to the pabulum orto the fact of their having been kept in the bottle and not putout into flat vessels.

The amoebae evidently have died out in Mr. Sutcliffe's tanksas no further stock has been received.

During the time that I have been caring for and examiningthe cultures of Amoeba d i s c o i d e s I have inclined to theopinion that the most successful were those reared in Petridishes. During the Session 1936-7 I have had cultures in glasstroughs 6 inches diameter and 4 inches deep, and others inPetri dishes 4 inches diameter and f inch deep. The culturewater in the troughs was about 3 inches deep. Though theserespective cultures were obtained by subdividing the sameparent culture, and though they were submitted to the sametechnique the results are outstandingly different. The culturesin the troughs have gradually grown poorer until now (July1937) they seem to contain no amoebae at all, while the Petridish cultures are most luxuriant, containing large numbers ofbeautiful amoebae.

The pH of the water in which Amoeba d i s c o i d e s livesappears to have no great significance in the cultivation of thisrhizopod, if we except the fact that it is always lower thanpH 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 micro-injections to test the pH of the cytoplasm.

DESCRIPTION OP THE LIVING ADULT AMOEBA DISCOIDES

(ScHAEFFER.)

As will be explained more fully later in this paper, the cultureundergoes ' depression' periods and ' optimum' periods. A ' de-pression ' period is one during which the culture contains no adultamoebae. Conversely during an 'optimum' period it containslarge numbers of adult amoebae. Definition of these two terms

AMOEBA DISCOIDBS 463

is necessary at this point so that it may be clearly understoodwhat I mean by ' young' adult amoebae and' old' adult amoebae.When the full grown amoebae first appear in the culture aftera ' depression' period I call them young adults. These live forfrom three to six or even seven months, during which time theyincrease by fission. Now comes a time when they begin todisappear gradually from the culture and when they exhibitfeatures not seen three months earlier. At this stage I call theamoebae 'old' adults.

If a pipette full of the culture containing young adult amoebaebe transferred to a glass cell and examined at once over a blackbackground under the low power of a Zeiss Greenough binocularan observer at first experiences difficulty in recognizing theamoebae. A little patience brings its reward for these fantasti-cally shaped and exceedingly translucent creatures make abeautiful picture as they lie emmeshed in the green algae.They possess numerous pseudopodia stretching outwards in alldirections. When these same amoebae are placed on a slidein a drop of culture water under a cover sup, given time to gripthe substratum and are then examined under a microscope withtransmitted light, the shape is found to have changed completely(fig. 1, PL 31). They are no longer radial but long and flat, thepseudopodia being fewer in number and in one plane (Text-fig. 1,a, b, c). The cytoplasm of the healthy amoeba spreads out overa large surface area and consequently has but httle depth orthickness. This makes the examination of the living nucleusand the cytoplasmic contents easy. The average length I foundto be 420/i (Schaeffer (1916) gives 400/* as the average length):when the amoeba is stretched out to 420/x its width is about140//.. The cytoplasm is very finely granular, exceedinglymobile and flows with great rapidity. The flow or movementis always in the centre both in the main bulk of the creature andalong the pseudopodia. No forward movement of the cytoplasmat the sides can be detected. When the amoebae are fullystretched out there are no folds in the cytoplasm. The ecto-plasm is exceedingly sparse, forming only a thin skin round theendoplasm. Even at the tips of the pseudopodia ectoplasm isonly rarely seen. It is most conspicuous at the base of the

464 CATHEEINB HAYES

pseudopodia, that is, in the angle between the main body anda pseudopodium or between two pseudopodia.

Embedded in the cytoplasm is a number of crystals andmany minute particles which may be nascent crystals. These

TEXT-FIG. 1 a, h, and c.

Three outline drawings (not to scale) to show the usual forms assumedby Amoeba discoides when it has been a little time on a slideunder a cover glass, c.v., contractile vacuole; N., nucleus.

crystals resemble the characteristic bipyramidal ones foundin Amoeba proteus but they are much less truncatedthan those of Amoeba pro teus , also the angles or edgesare less sharp and the whole crystal has a more oval appearance.I cannot agree with Schaeffer that this amoeba is more stuffedwith crystals than Amoeba pro teus . During the manyyears that I have worked in the Notre Dame Laboratory I haveexamined innumerable Amoeba pro teus , and it is myexperience that under certain conditions of culture and agethe crystals are far larger and more numerous in Amoebaproteus than in Amoeba discoides. On the other hand,Sister Monica Taylor has often shown me specimens of Amoebaproteus containing very few and sometimes no crystals atall. A few years ago I carried out some experiments on the

AMOEBA DISCOIDES 465

crystals of A m o e b a p r o t e u s , but I have arrived at nosatisfactory conclusion about their formation or function. Ithink it certain that they grow in size and number as theamoeba grows in age. Schaeffer (1916) states that he has'never with certainty been able to find any other form ofcrystal', i.e. the dipyramidal' in this amoeba'. I have seen smallcubic crystals in several specimens, but these cubic crystalsare never as large as those seen in Amoeba p r o t e u s andtheir occurrence is much rarer. I have never seen in Amoebad i s c o i d e s the large square plate-like crystals which occur inA m o e b a p r o t e u s . Also in the cytoplasm of the young adultis a number of nutritive spheres. These are small (2 to 3/*),and of a definite greenish appearance. At this stage the nutritivespheres are quite structureless, and are not stained by Ehrlich'shaematoxylin.

I have been able to confirm all Schaeffer's published observa-tions on the living nucleus. It is normally single, disk-shapedwith rounded edges, and with an average diameter of about40/x by 15/x to 18p thick. One nucleus was 56/x in diameter,though why it should be so much above the average size I amunable to say. The two larger surfaces are generally concave(Text-fig. 2 a). The surface is smooth, without folds. Thenucleus is carried along in the flowing endoplasm, but generallyholds a position about one-third the length of the amoeba fromthe posterior end. If and when it is carried nearer the anteriorend it remains stationary while the cytoplasm flows over andround it, and thus it regains its normal position. While beingcarried along the nucleus is all the time rolling over and over.It is my opinion, however, that the nucleus of Amoebad i s c o i d e s rolls over more quickly and consequently moreoften, and so one obtains an edge view or ' elevation' view muchmore frequently, than in A m o e b a p r o t e u s . In rolling overthe nucleus sometimes bends on itself and may remain so forsome time, having thus the shape of a kidney-bean (Text-fig.2 6), as observed by Schaeffer (1916). As the cytoplasm ofAmoeba d i s c o i d e s is much less voluminous than that ofAmoeba p r o t e u s , and as it spreads this smaller volumeover a large surface area, the living nucleus is always visible, and

NO. 319 H h

466 CATHBBINB HAYES

the chromatin masses under the nuclear membrane and in thekaryosome are easily seen. The nucleus has a coarse mottledappearance. There appears to be a great deal of fluid of a mobilenature within the nucleus, and in this the karyosome changesposition so that sometimes it is central and at other times liesto one side. Indeed one gets the impression that the karyosomeitself contains mobile liquid readily changing position from onepart of it to another as the nucleus itself is being rolled about

TEXT-FIG. 2.

Diagrammatic representation of the nucleus to show (a) 'elevation'view with rounded edge and concave faces. (6) the nucleus bent.

in the endoplasm. This hypothesis is confirmed by the varietyof appearances which one comes across when studying a largenumber of fixed specimens. In many of the young adults thenucleus lies in clear cytoplasm, yet I am not prepared to callthis clear space a vacuole. There does not seem to be anydefinite boundary line round it as there is, for instance, roundthe contractile vacuole. It seems to me to be a region of veryclear and very mobile cytoplasm free from cytoplasmic in-clusions, which gradually merges into the ordinary cytoplasmcontaining crystals, nutritive spheres, &c.

In most specimens there is a single contractile vacuole whichreaches a diameter of 30 /i. It is however quite a common thingto find two contractile vacuoles, a primary one at its maximumsize and a secondary one beginning to grow. As the primary onebursts the secondary takes its place, while another secondary isformed at once. The contractile vacuole is generally formed

AMOEBA DISCOIDBS 467

near the nucleus, and moves along with it until it is ready toburst when it lags behind the nucleus and takes up a positionnear the posterior end of the amoeba. In this position it burstsvery slowly and very gently without causing any apparentdisturbance 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 inyoung specimens. The irregularity may, of course, be due tothe imprisoned conditions in which it is necessary to examinethe amoebae.

A m o e b a d i s c o i d e s feeds on Flagellates of various sizes,on green unicellular plants, and on Eotifers. When the latterare plentiful in the culture they generally form the staple foodof the amoebae and it is quite a regular occurrence to find asmany as nine while occasionally twelve Rotifers may be seeningested by one individual amoeba. The variety and the amountof food which an individual amoeba can contain at the same timein its cytoplasm is truly amazing.

I have a culture at present (July 1937) in which, though itcontains a plentiful supply of the above-named organisms, theamoebae have taken to ingesting long pieces of filamentousalgae. Whether these latter are wholly digested or ejectedundigested I am as yet unable to say.

DESCRIPTION OF THE AMOEBAE WHEN THE CULTURE IS

BECOMING SENESCENT.

Viewed by reflected light the amoebae at this stage (i.e. oldadults) are white and opaque. In transmitted light the crystalsare seen to be large and very numerous. The nutritive sphereswhich at the beginning of the 'optimum' period were smalland inconspicuous, are now large (5/J. to 6/x) and exceedinglynumerous, filling up much of the endoplasm. Their definitegreen appearance tints the whole A m o e b a . The spheres tendto collect together into groups of from twenty to thirty, andsuch a group is often seen at the tip of a pseudopodium to theexclusion of crystals and other cytoplasmic inclusions (Text-fig. 3). Schaeffer (1916) was of the opinion that the number ofthese spheres which he designated 'so-called excretion spheres'

468 CATHERINE HAYES

depended on the amount of food digested by the amoeba. Iconfirmed that observation in 1924 on Amoeba p r o t e u s(see Taylor, 1924). Now, however, in the light of much moreexperience and intensive observation, I think that the amountof food eaten by the amoeba is not the only factor at work. Togive but one out of many instances, in the early part of 1936,owing to stress of other work, my culture of Amoeba dis-coides was neglected. Whilst not actually starved, it was

TEXT-PIG. 3.

Outline drawing of Amoeba discoides (not to scale) to showhow the nutritive spheres collect into a group at the end of apseudopod. n.s., nutritive spheres.

decidedly underfed, yet when I resumed work on it in July Ifound that all the amoebae were stuffed with very large nutritivespheres. These amoebae were then about six months old, andfrom that time onwards they began to disappear graduallyfrom the culture.

The nutritive spheres grow in number and in size as theamoebae grow in age. When a culture is healthy and under-going its normal cycles the amoebae are always found to containmany large nutritive spheres just before the so-called ' depres-sion' periods. It has been shown for Amoeba p r o t e u s(Taylor, 1924) that the nutritive spheres are intimately con-nected with the formation of encysted young amoebae. As willbe shown later, I have had evidence of this being true also forAmoeba d i sco ides .

In April 1987 a culture of Amoeba d iscoides reared ina Petri dish was in an especially flourishing condition, containinga very large number of adults. The food organisms however inthis culture were sparse, and it was evident that in a short

AMOEBA DISCOIDBS 469

time starvation conditions would prevail. Consequently a sub-culture was made by transferring a portion of the material (aboutone-tenth of the whole) to a Petri dish containing an excellentculture of food organisms, Rotifers, Flagellates, &c. Some foodorganisms were also added to the parent culture. On examiningboth cultures three months later (July 1937) their conditionswere found to differ greatly. The parent culture contained nofood organisms. The amoebae, though still plentiful, hadbecome senescent—being black by transmitted light—full ofcrystals and large nutritive spheres—sluggish—refusing to gripthe substratum and flow like healthy individuals. In the sub-culture which still contained a good supply of food organisms,the amoebae had multiplied to an extraordinary degree. Theywere in beautiful condition, and as soon as placed under thecover-slip they gripped the substratum and flowed actively.The crystals and nutritive spheres were small. The lack offood in the one case had brought on senescence. The plentifulsupply of it in the other had warded off this condition, theamoebae being kept in good condition by repeated fission.However, this fission cycle will not continue indefinitely nomatter how plentiful the food supply, and there is evidentlystill much work to be done on the nutrition of the amoebae.

METHODS OP FIXING AND STAINING.

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

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

2. The amoebae were put on to the slides and given time tosettle, but instead of aeeto-carmine Bouin's fluid was used tofix them. After fixation in Bouin the amoebae were washed,

470 CATHERINE HAYES

stained in Ehrlich's haematoxylin, dehydrated, cleared in xyloland made permanent with Canada balsam, all by the irrigationmethod. Many beautiful preparations have been obtained bythis method.

3. At times when the amoebae were very plentiful in theculture large numbers of them were put into a Petri dish. Thenworking under the low power of the binocular with the help ofneedles and a fine pipette the amoebae were freed as much aspossible from the debris of the culture. This latter was as faras 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. Thematerial thus fixed was pipetted into centrifuge tubes wherethe washing, staining, dehydration, and clearing was carriedon. Finally, the amoebae were permanently mounted on slidesin Canada balsam. Bouin's fluid proved an excellent fixative,and Ehrlich's haematoxylin as a stain leaves nothing to bedesired.

DESCRIPTION OF THE FIXED AND STAINED NUCLEUS.

The fixed and stained nucleus varies in diameter from 21/xto 45fx, with an average diameter of about 36ju.. It is surroundedby two membranes. Of these, the outer is very definite andsharply differentiated, looking like a distinct blue skin in thestained preparations. The inner membrane is much less definite.There is a clear space between the two membranes (figs. 3, 4,7, 8, 11, PI. 32). Immediately within the inner membrane, infact close against it, lie the large chromatin blocks (figs. 2-12,PL 32). In optical sections of the nuclei these blocks appearvery regularly arranged and equally spaced. There is generallya second layer of chromatin blocks within the outer one, occa-sionally there is a third such layer, but the blocks of these innerlayers are not so regularly arranged as those of the outer. Thechromatin blocks stain easily in Ehrlich's haematoxylin—eachblock standing out clear cut and richly coloured like fullydifferentiated chromatin, reminding the observer of a chromo-some of a metazoan nucleus. The karyosome which carriesmasses of chromatin lies within the chromatin blocks region.

AMOEBA DISCOIDBS 471

As a rule its diameter almost equals that of the nucleus. Some-times, however, when the latter is seen in 'plan', the karyosomecannot be distinguished from the rest of the nucleus, whichmeans that it extends right out to the inner nuclear membrane(fig. 6, PL 32). As has already been said in describing the livingnucleus, the karyosome takes up various positions in thenucleus, and the stained preparations show that it often ismuch bent on itself. That the nucleus, and even the karyosome,contain much fluid also becomes evident from an examinationof the stained preparations (figs. 3, 5, 6, 7, 11, PL 32). I thinkthere can be little doubt that there is also a layer of fluidbetween the inner and outer nuclear membrane.

DIVISION OF THE NUCLEUS.

I have fixed, stained, mounted, and examined a very greatnumber of Amoeba d i s c o i d e s at different times in thelife of the culture. The beautifully expanded condition of theamoebae, and the details of the structure which can be seenin the organisms present in the food vacuoles are evidence thatthe fixation is absolutely satisfactory. Many of the nucleiexamined are dividing—some just beginning to divide (figs. 5and 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 whateverof mitotic division. The division of the nucleus as a whole isamitotic, and so far as I can see a very simple, straightforwardcase of amitosis. The cleavage divides the nucleus as a wholeas well as the karyosome into two parts. Each of these partsforms a new daughter nucleus. In one nucleus which had sodivided, the two daughter parts had not separated from eachother yet there was evidence of a second division taking placeat right angles to the first. This observation was made on atemporary preparation in aceto-carmine, so I can only representdiagrammatically what I saw (Text-fig. 4). In Amoebap r o t e u s it is possible to find four or even eight nuclei inindividual amoebae. In the present investigation I found onlyone specimen in which one of the newly formed daughter nucleihad already divided into two before the cytoplasm showed anysigns of division (fig. 11, PL 32).

472 CATHERINE HAYES

OBSERVATIONS ON THE LIFE-HISTORY OF AMOEBA

DISCOIDES.

So as to make quite clear what has to follow in this sectionI think it advisable to give at this point some readings from therecord of the culture A.14/11/84. The culture was started in the manner explained at

the beginning of this paper (p. 461).12/12/84. No adult amoebae could be found when the culture

was examined with the low power of the Greenough

TEXT-FIG. 4.

The 'mother' nucleus had divided into two 'daughter' nuclei(a) and (b). (a) is shown by a broken line lying beneath (6). Thesetwo daughter nuclei had not separated yet (6) had begun todivide at right angles to the plane of the original division.

binocular. An examination by the ordinary microscope wasnot made.

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

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

11/10/35. Many large adults were present. These when examined

AMOEBA DISCOIDES 473

with the microscope were found to be in an active healthy-condition.

30/11/35. Amoebae were not nearly so plentiful as when lastexamined. It was evident that the culture was approachinga ' depression' period.

4/1/36. Very few adults could be found. Many young amoebaepresent, 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 back-ground. When examined with the microscope the amoebaewere found to be stuffed with crystals and very largenutritive spheres. As these amoebae were about six monthsold, the culture was carefully watched during the nextfew weeks. The number of adult amoebae grew less andless, but before they had all finally disappeared the culturecontained numbers of micro-amoebae. These micro-amoebae will be described later.

(Note.—The term micro-amoeba is used for the small amoebaerecently emerged from the cyst and undergoing develop-ment.)

From a study of this record it is evident that, as has alreadybeen said, there are times when the culture contains no adultamoebae but such times are only so-called ' depression' periodsfor during them numbers of micro-amoebae can be found.These young amoebae grow up and in their turn increase innumber by fission.

The question now to be considered is the formation of theseyoung amoebae, and though I have not yet seen all the stagesdescribed for Amoeba p r o t e u s (see Taylor, 1924), thoseI have seen are, I think, sufficient to prove that agamontogonyin Amoeba d i sco ides is similar to that in Amoebap r o t e u s .

The 'old' adult with its large nutritive spheres is the aga-mont. When an agamont has been fixed and stained in Ehrlich'shaematoxylin it becomes evident that there are two types ofspheres in its cytoplasm—the ordinary large deeply stainednutritive spheres and other still larger spheres which are

474 CATHERINE HAYES

definitely differentiated into palely stained and deeply stainedregions. These larger spheres are the developing agametes. Inthe process of differentiation they use up the nutrient materialof the nutritive spheres which latter in consequence lose theirstaining capacity. In a fully formed agamete it is possible todistinguish the new cytoplasm which is pale-staining from thechromatin which stains more deeply (fig. 13, PI. 32). Dr.Monica Taylor (1924) has shown how in Amoeba p r o t e u sthe chromatin blocks escape from the nucleus and becomeassociated with the nutritive spheres to form the rudiment ofthe agametes.

I have not seen the chromatin blocks actually escaping fromthe nucleus of Amoeba d i s c o i d e s . I have, however, foundspecimens, containing fully differentiated agametes, in whichthe nucleus had no visible membrane (fig. 12, PL 32). Nowordinarily the nuclear membrane is a conspicuous structurewhich, stains very readily. It seems reasonable then to concludethat in these specimens the nuclear membrane has dissolved inorder to set the chromatin blocks free into the surroundingcytoplasm. Here these masses of chromatin uniting with thenutritive spheres differentiate round themselves a certainamount of cytoplasm and become fully formed agametes (fig. 13,PL 32). When the agamont disintegrates the agametes are setfree 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 firstthese micro-amoebae are circular in general outline, from 20/Jto 25/x in diameter, with a great number of very short bluntpseudopodia radiating in all directions (Text-fig. 5, a and b).The cytoplasm is clear and streams very slowly, so that thereis little change in the creature's position on the slide. Thenucleus is very conspicuous, the pale green-looking karyosomestanding out clearly, the rest of the nucleus forming a clearzone round it. At this stage the young nucleus of the micro-amoeba does not roll over in the cytoplasm, it is always seenin ' plan'. The contractile vacuole which lies beside the nucleusis also a conspicuous feature. It grows rapidly, bursts regularlyand very gently. Many of those micro-amoebae contain no food

AMOEBA DISCOIDES 475

vacuoles while others are stuffed with them, the former, I con-clude, being those most recently emerged from the cysts.

In the next stage the blunt pseudopodia have been withdrawnand replaced by a single large pseudopod so that the amoebae

TEXT-FIG. 5.

Free-hand drawings of very young Amoeba discoides to showthe gradual change in shape as they grow older. The drawingsare not to scale, but the amoebae were measured and the greatestdiameter of a, 6, and c and greatest length of d, e, and/, given in ft.N., nucleus; c.v., contractile vacuole; f.v., food vacuole.

are no longer circular but oblong in outline, about 30/x to 40/*long (Text-fig. 5 d, e,fj. A great part of this pseudopodium—the anterior portion of it—consists entirely of clear cytoplasm(Text-fig. 5 d, e, / ) . The cytoplasm flows rapidly so that the

476 CATHERINE HAYES

little creature travels along at a considerable rate. The nucleuswhich is very conspicuous, and still seen always in plan, seemsto act as a dividing centre for the cytoplasm which flows roundit in two streams, one on either side (Text-fig. 5 / ) . The con-tractile vaeuole beside the nucleus behaves as in the earlier

TEXT-FIG. 6.

Outline drawings of two young Amoeba discoides when theyhave become adult in form. The figures are not drawn to scalebut the longest axis as measured in y. is indicated.

stage, while the food vacuoles are larger and more numerousthan they were in that stage.

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

SUMMARY.

1. A large free-living amoeba found by Mr. Harry Watkinsonin the tropical fish tanks of Mr. Albert Sutcliffe of Grimsby has

AMOEBA DISCOIDES 477

been identified as Amoeba d i s c o i d e s (Schaeffer, 1916) =M e t a c h a o s d i s c o i d e s (Schaeffer, 1926).

2. Prom the inoculation material obtained from these tanksAmoeba d i s c o i d e s has been successfully cultivated in theNotre Dame Training College Laboratory by a techniquesimilar to that used for the cultivation of Amoeba p r o t e u s :wheat being the pabulum employed. In contrast to what obtainsin the cultivation of Amoeba p r o t e u s , however, Amoebad i s c o i d e s flourishes more luxuriantly in shallow Petri dishes,than in deeper troughs.

3. The nucleus in the resting and dividing stages is described;division is amitotic.

4. The more important cytoplasmic contents, includingnutritive spheres, and crystals are likewise described.

5. The life-history has been worked out. The adult amoebabecomes an agamont giving rise to agametes which eventuallygrow into adult amoebae, the life-cycle occupying roughlyabout four months.

6. Descriptions of the nucleus of the newly hatched anddeveloping amoebae are deferred.

I wish to offer my sincerest thanks to Professor Graham Kerrunder whom this work was begun, and who has continued fromafar to watch over it with ever kindly interest and encourage-ment and who has read the paper in typescript.

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

In conclusion I would like to express my appreciation of herskill and of the care and trouble bestowed by Miss Brown Kellyin the execution of the original drawing of fig. 1, PL 81.

EEFEKENCES.

Carter, Lucy A., 1919.—"Some Observations on Amoeba proteus",'Proc. Roy. Phys. Soc. Edin.', 20.

Schaeffer, A. A., 1916.—"Specific and Other Characters of Amoebaproteus Pallas (Leidy), Amoeba discoides spec, nov., and Amoeba dubiaspec, nov.", 'Arch. Protistenk.', 37.

1926.—"Taxonomy of the Amoeba", 'Publ. Carnegie Inst.', no. 345.

478 CATHERINE HAYES

Taylor, Monica, 1923.—"Nuclear Divisions in Amoeba proteus", 'Quart.Journ. Micr. Sci.\ 67.

1924.—"Amoeba proteus: some new Observations on its Nucleus,Life-History, and Culture", ibid., 69.

1927.—"Development of the Nucleus of Amoeba proteus, Pallas(Leidy). (= Chaos diffluens (Schaeffer) )", ibid., 71.

EXPLANATION OP PLATES 31 AND 32.LETTERING.

N., nucleus; c.b., chromatin blocks of the nucleus; C.V., contractilevacuole; F. V., food vacuole (the organism being a large encysted Flagel-late); F.r., food vacuole (small Flagellates); s.c, cubic crystal; c, di-pyramidal 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 adultAmoeba d isco ides .

PLATE II.

All the figures were drawn from specimens fixed in Bouin's fluid andstained 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. 11.—Three nuclei from one amoeba. Cytoplasmic division has

not kept pace with nuclear division for one of the daughter nuclei of thefirst 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 d i sco ides .