ON PELOMYXA VIRIDIS. 357

On Pelomyxa viridis, sp. n., and on the Vesi-cular Nature of Protoplasm.

By

AlfreJ Glblb* Bourne , D,Sc.(Lond,), C.M.Z.S., F. l i .S. ,fellow of University College, London; Fellow of Madras University ;

Professor of Biology in the Presidency College, Madras.

With Plate XXVIII.

MY assistant, M.R.Ry. A. Sambasivan, B.A. (Madras),during the course of an exhaustive examination of the fauna of asmall tank (pond) in the neighbourhood of the PresidencyCollege discovered and drew my attention to numerous green" egg"like " bodies, of about -fa inch in diameter, which wereto be seen on the surface of the finely divided mud.

These bodies proved upon examination to be Rhizopods be-longing to the remarkable genus Pe lomyxa , and forming anew species of that genus, but differing in some importantparticulars from all hitherto described Rhizopods. I proposeto call the species P. v i r id i s .

The species is peculiarly interesting not only on account ofits great size (it is larger than any known form of the Lobosa),but also on account of the presence of chlorophyll and symbioticbacteria,1 and the assistance which this renders in the study ofits protoplasm.

1 The rod-like bodies here regarded as bacteria were described as crystalsof unknown composition by Greeff in his account of Pelomyxa palustris.Leidy describes them as exhibiting transverse striations. The symbiosis ofbacteria and amoeboid Protozoa has a special importance at the presentmoment in connection with MetschnikolPs doctrine of Phagocytosis.

858 ALFRED GIBBS BOURNE.

Hab i t a t and General Descript ion.

I have only found P. viridis in one tank, and this a tankwhich has not been known to dry up for many years.

When the mud from this tank is placed in a dish with alittle water and allowed to stand, several individuals of P.vir idis may generally be found close to the surface of themud ; if these are removed and the mud stirred up and againallowed to stand, it is not usual to find many other specimens.So that I believe that this Bhizopod lives close to the surface ofthe mud. I have never seen it crawling on the sides of thedish or on any other object; nor have I ever seen it muchspread out while in the mud, but when mounted on a slide with acover-glass it readily spreads itself out and becomes very flat,and soon commences to exhibit active amoeboid movements.Pig. 1 represents a mounted specimen, partially spread outand magnified about four diameters. It is difficult to conveyany idea of the size to which such an organism may attain, asso much depends upon the extent to which it is spread out. Ihave seen specimens so much spread out that they wouldaverage as much as £ inch in diameter. The specimens are ofvery varying sizes, but I have never found very small speci-mens ; I have never, in fact, been able to find a specimen by theaid of the microscope which I had not previously picked outwith the naked eye, although I have searched many slides ofthe mud for this purpose. It may be that small individualsare to be found at some other time of year, or it may be thatI have not, in spite of my search, happened to alight upon asmall specimen.

"When viewed by reflected light individuals vary a gooddeal in colour, from the rich transparent green of a gelatinouslichen to that of a faded leaf. I presume that the chlorophyllundergoes some degeneration. I have never found anyapproach to the red colour found in Euglena or Haemato-coccus. There is often a whitish, more opaque-looking spot,such as has been shown in the middle of fig. 1. This is causedby an aggregation of sand particles, and it is often more clearlydefined than in my figure. When seen by transmitted light

ON PELOMTXA VIEIDIS. 359

with a low power snch regions naturally appear dark, butwith polarised light they appear as bright spots, owing to thedoubly refracting character of the sand particles. A numberof these particles is always present, but at times their numberis so enormous that there is almost as much sand as proto-plasm. I have watched an individual so filled collect thegreater number of these particles at one spot, and then simplypour them out from the protoplasm. An individual may thusget rid of hundreds of these particles in a minute or two, butthe process of collecting them together preparatory to theirextrusion is slow. I believe that there is a periodical whole-sale extrusion of these particles. These particles have all theappearance of sand ; they are of very varying size and of veryirregular shape, and are insoluble in all ordinary reagents;they are crystalline in nature, and, as stated above, are doublyrefracting.

The presence of these particles has not, I fancy, any specialsignificance, they occur in great numbers in the mud andare taken in with the food ; the animal, indeed, seems to exerciseno discretion as to what it takes in. They become collectedtogether as a mechanical result of slight movements, and wheuthe animal makes some more extensive movements are thrownout in great numbers.

Structure of the Protoplasm.

Viewed with an ordinary high power of the microscope themain mass of the organism appears of a green colour, while atthe periphery, from time to time, perfectly colourless regionsmay be seen. One's first impression is that there is a greenendoplasm and a colourless exoplasm (figs. 2 and 3). Withthe same magnifying power, in addition to the various proto-plasmic contents described elsewhere, what appear to begranules may be recognised abundantly distributed throughthe greater mass of the protoplasm. The colourless peripheralregions above mentioned are usually devoid of these granules.The granules all prove upon further examination to be bacteria,and but for their presence the protoplasm itself would appear

VOL. XXXII, PAET II I . NEW SER. A A

360 ALFRED GIBBS BOTTKNE.

absolutely non-granular. Exceedingly careful and repeatedobservations with a Powell and Lealand's -fa inch oil immersionobjective, Abbe's sub-stage condenser, and Engelmann's darkchamber, which last is invaluable in the glare usually pre-valent here, have enabled me to ascertain with great accuracyand clearness the real structure of the body substance.

The protoplasm is throughout a perfectly colourless andapparently homogeneous substance, and this substance, exceptin the peripherally placed portions which flow out from time totime to be sooner or later reabsorbed into the central mass, isdensely packed with spherules of a semi-fluid stroma, im-pregnated with what I have ascertained to be chlorophyll, andto these chlorophyll-bearing spherules the green colour of theorganism is due.

In these spherules we have not to do with chlorophyll-corpuscles, as they exhibit no phenomena of division, and are,moreover, more fluid than chlorophyll-corpuscles.

Their fluid character often becomes very evident after thedeath of the organism, when some of them may be usuallyobserved to run together into larger masses. Where thesespherules are packed closely together they assume the form ofregular polyhedrons. They seem about as fluid as the stromaof human red blood-corpuscles. I have come to the con-clusion that they correspond to the vacuoles or, as they arebetter termed, to distinguish them from other vacuoles,ve s i c l e s , which occur in so many specimens of protoplasmand give to such specimens a vesicular character.

The fact that they contain in P. v i r i d i s a substance im-preguated with chlorophyll, a state of things hitherto un-observed, has enabled me to throw some new light upon theirnature and upon the organization of the body substance.

The word protoplasm is used above in the sense in whichBiitschli1 uses the word p l a sma . I t designates the substancewhich Leydig speaks of as the spongioplasma, as distinguishedfrom hyaloplasma (chylema, Strasburger). In other words,I consider that the substance within the vesicles is to be

1 Bronn, 'Protozoa,' p. 1392.

ON PELOMYXA VIRIDIS. 361

regarded as an entoplastic product of the protoplasmic activityrather than as any portion of the protoplasm itself. Thechlorophyllogenous stroma occupying the vesicles of P.v i r id is is no more entitled to be regarded as a portion of theprotoplasm than are chlorophyll bodies or oil-globules, or anyother structures ordinarily spoken of as protoplasmic contents.It is, of course, the presence of the chlorophyll which leads meto this conclusion. If I am correct in regarding these chloro-phyllogenous structures as the homologues of the vesiclesfound in other protoplasms, the condition of P. v i r id is lendsstrong support to Biitschli's theory that the plasma is the sub-stance which forms the envelopes of these vesicles only, or,as Butschli has termed it, the substance forming the scaffold-ing of a honeycomb (Substanz des Wabengerusts), and does notinclude their contents (Wabeninhalt),

The structure of P. v i r id is is, on the other hand, ratheropposed to Biitschli's most recent views with regard to proto-plasmic movements. If I understand rightly from the abstractavailable to me, Butschli believes that a movement starts by thebursting of some vesicle, a phenomenon which would naturallyescape observation in ordinary protoplasm, but of which, ifit is constantly occurring, I should surely have seen somethingin a protoplasm whose vesicular contents are brightly coloured.

With regard to the homology which I have assumed to existbetween the vesicles of ordinary protoplasm and those ofP. v i r id i s with their chlorophyllogenous contents, it is quiteclear, in the first place, that these are the structures whichrender the protoplasm of P. vir idis vesicular. There exist,it is true, in addition to them a number of vacuoles of varyingsizes, but of these there are after all. comparatively few, and ifthey alone were present the protoplasm could not be calledvesicular. There is no evidence that these vacuoles containany substance other than water.

We can undoubtedly distinguish in P. v i r id i s between twovarieties of non-contractile fluid-containing spaces (nicht con-tractile Fliissig-Keitsraume), the vacuoles containing waterand the vesicles having chlorophyllogenous contents.

362 ALFRED GIBBS BOURNE.

The vesicles vary very little in size, being always aboutE 0*0 0 inch in diameter.

The vacuoles, on the other hand, vary greatly in size, somebeing almost as small as the vesicles, while others attain fiveor six times that diameter.

No distinction has, as far as I am aware, been hitherto drawnbetween vacuole and vesicle in any so-called " vesicular" or" vacuolised" protoplasm.

Biitschli,1 writing ten years ago, says of non-contractilevacuoles, " Seltener hingegen wird ihre Zahl so betrachtlich,dass das sie trennende Plasma nur noch ein Maschenwerk vonScheidewanden zwischen ihnen herstellt, das Plasma eineschaumige oder alveolare Beschaffenheit annimmt. Einderartiges Verhalten begegnet uns z. b. gewohnlich beiPelomyxa, auch bei gewissen Amb'ben tritt ahnliches mehroder weniger deutlich hervor."

Of Biitschli's most recent views l e a n only judge from M.Yves Delage's2 note on the models of protoplasm with whichButschli has been recently experimenting, and if I understandrightly in, for instance, the soap and xylol model, the plasma(Biitschli) is represented by the soap and the chylema (Stras-burger) by the xylol. So that the contents of both vacuoleand vesicle would be termed chylema, but there is no wordas to the possibility of the chylema being other than a singlesubstance.

Now, in P. v i r id is , two distinct substances take the placeof the xylol of Biitschli's model, the contents of the vacuolesand those of the vesicles, and were it not for the presence ofthe chlorophyll in one of them these would not be opticallydistinguishable one from the other.

Two species of Pe lomyxa have been hitherto described,P. p a l u s t r i s , Greef,3 and P. vi l losa (Amoeba vil losa),Leidy.* The protoplasm of both these species has been de-

1 Bronn, ' Protozoa,' p. 101.3 ' Arch. Zool. Experimentale,' 1889.3 'Arch. f. mikr. Anatomie,' Bd. x, 1874.4 'Fresh-water Rhizopods of North America,' Washington, 1879.

ON PELOMtfXA VIRTDIS. 363

scribed as highly vesicular, and Gruber,1 in speakiug ofP. villosa, which he found in Germany, says, " Zunachstfallen die zahlreichen Fliissigkeitsvacuolen ins Auge, welcherden grossten Bestandtheil des Korpers ausmachen und dem-selben ein Schaumiges Aussehen verleihen. Diese Vacuolen,die von wechselnden Umfange Sind, liegen eingebettet indem homogenen Plasma, welches tnehr oder weniger feineScheidewander zwischen ihnen bildet, ahnlich eine Intercellu-larsubstanz zwischen den einzelnen Zellen eines Gewebes."

I think it probable that in these forms we have both vacuolesand vesicles, but as the vesicles are filled with colourlesssubstance no distinction has been drawn between them. Itwill be interesting to re-examine these forms to ascertain iffrom the relative size and frequency any distinction can bedrawn. The importance of the question lies in this, that thevesicles have probably an intimate relation to the structure ofthe protoplasm, and possibly to the production of amoeboidmovements; while the vacuoles vary from time to time in sizeand number, and have nothing to do with the ultimate structureof the protoplasm.

Fig. 6 represents a fragment of the protoplasm in a specimenwhich was deeply stained with osmic acid. At x is seen pro-jecting at the surface a small portion of the hyaline protoplasm,devoid of all contents, stained brown and rendered granular bythe osmic acid. The rest of the drawing shows the vesiclesonce occupied by the chlorophyllogenous substance boundedby the protoplasm. The chlorophyll has been dissolved, butwhether any substance now remains in the vesicles I cannotdetermine; there is nothing which is stained by osmic acid.With all other fixing reagents which I have used the vesicularstructure disappears.

The protoplasm of P. v i r id i s appears then to be perfectlyhomogeneous, and small portions of it may at times be observedat the periphery of the organism free from all contents, but thegreat mass of it forms a mere scaffolding for the numerousvesicles, and is, moreover, densely packed with bacteria

1 ' Zeit. f. w, Zoo!.,' Bd. xli, p. 189.

364 ALFRED GIBBS BOURN Jl.

('' crystals " of Greef), to say nothing of its various other con-tents. The vesicles contain a fluid substance impregnatedwith chlorophyll. The vesicles and the bacteria are to beregarded as bodies contained in the protoplasm, and the lattermay flow out leaving all its contents behind. When the proto-plasm does flow out in this way some of the bacteria soon follow,and may then be seen to start an active movement; and if the out-flow continues, the superficial vesicles leave the central massand may be seen isolated in the hyaline protoplasm (fig. 5).

Pro toplasmic Movements, Pseudopodia.Specimens of P. viridis will often remain for a long time

absolutely quiescent; in such a condition the animal is, if notflattened by a cover-glass, fairly spherical in shape, and the greenvesicles extend close up to the periphery. They always remainembedded in the protoplasm, so that they do not actually cometo the surface j and, indeed, there is at the surface somethingmore than the mere envelopes of the most superficially placedvesiculae, or else the margin of an optical section would pre-sent a sinuous curve, the sinuosities of which corresponded toa row of vesicles, which is not the case.

In specimens where movements are taking place these move-ments are usually very active, and most so at the periphery,although they are by no means confined to that region.In this matter P. viridis agrees with other species ofPelomyxa.1

Long-continued movements often take place without theprotrusion of any pseudopodia. Owing to its great size, P.viridis, and, indeed, other species of Pelomyxa, move aboutfrom place to place, at any rate while under observation, lessfrequently than smaller amoeboid organisms.

When the movements, which result neither in the protrusionof a well-marked pseudopodium nor iu a translation of theorganism, are taking place, the margin of an optical sectionconsists of a hyaline layer, the movements in which have awave-like, undulating appearance. It looks as though the

1 Bronn, 'Protozoa,' p. 97, foot-note.

ON PELOMYXA VIBID1S. 365

hyaline protoplasm protruded at some one spot, leaving all itscontents behind, and then spread out laterally, while a sharplydefined line seems to mark the original boundary; this lineafterwards disappears, giving one at first the impression thatthe protoplasm, which has, so to speak, overflowed the originalboundary but remained in contact with it, is separated from therest of the protoplasm, except at the spot whence it actuallyprotruded, by a sort of pellicle, which is subsequently dissolved.Gruber1 has figured such an appearance for P . vi l losa, andsays, " Wenn an einem ausgetretenen Protoplasmafortsatzdie Vacuolen und die Kornchen einige Zeit durch eine scharfeLinie von der Sarkode getrennb bleiben, so beruht dies daraufdass das Pseudopodium durch Zusammenfliessen des Plasmasueber diese Stelle hinentstand, und dass die feine peripherischeSchicht von Protoplasma, welche vorher die Grenze gegen dasumgebende Medium bildet, und welche jetzt von der Massedes Pseudopodiums umflossen wird noch eine Zeit lang er-halten bleibt;" and regards this as further evidence of theexistence of a relatively hard cuticular layer or pellicle formedby the action of the water, a view which the same author putforward on a previous occasion.3

I cannot accept this interpretation of the facts. I do notbelieve that the hyaline protoplasm is protruded at one spotonly, and that there is lateral displacement of its particles;the undulating appearance is chiefly caused, like other wavemovements, by extrusions and retractions in a radial direc-tion. The appearance of the boundary line may be explainedby careful focussing, the line being at some slightly differentlevel from the protruding protoplasm, and its disappearancebeing due to a subsequent protrusion at that particular level.I am not at all sure how far the existence of any such pellicleas Gruber describes is in accordance with the views as to therelation of protoplasm to imbibition water.3

If the density of the surrounding medium is increased, as1 ' Zeit. f. w. Zool.,' fid. xli, 1884, pi. xiii, fig. 2, and p. 190.3 'Zeit. f. w. Zool./ Bd. xxxvi, 1882, pp. 457—469.3 See Engelmann on " Protoplasmic Movement," in this Journal, vol. xxiv,

1884, p. 390.

366 ALFBEJD GIBBS BOURNE.

by the addition of a 1 to 2 per cent, solution of common salt,the protoplasm shrinks, owing to withdrawal of imbibitionwater. This might, of course, take place through the pellicle,but this is not probable, as such a layer must be impermeable,or difficultly permeable, to water, if it have the function ofprotecting the central protoplasm from the action of water;and, in any case, when a sudden shrinking took place, onemight expect to see some crenation of the surface, such asis exhibited by a human red corpuscle when it is exposed to amedium denser than its own plasma. No such crenationtakes place; but, on the other hand, some portions of theprotoplasm usually stick to the cover-glass and become tornoff, while some of the protoplasmic contents escape into thewater and are carried away by the current.

I have at times observed a similar phenomenon when a longpseudopodium was quickly withdrawn. A specimen of P.v i r i d i s , which has been moving about under a cover-glass,habitually leaves some portions of itself sticking to the glass.Appearances, such as are shown in fig. 2, constantly occurwhere a pseudopodium is retreating, and are due to the stick-ing of the superficial protoplasm to the glass.

I have also constantly seen villi, such as G-ruber1 hasfigured for P. v i l losa , formed in the same manner.

Such phenomena seem to me to render the existence ofany pellicle unlikely. Moreover, as will be described below,when a specimen of P. v i r i d i s is torn into pieces withneedles the water seems to penetrate and cause almost instantdisintegration. Now, if the protoplasm of these forms is inthe habit of forming a pellicle directly any new surface isexposed to the water, why does it not do so in this case ? I tappears more probable that so long as the protoplasm is alivethe amount of imbibition water which can be taken in isregulated, and that in the instance above cited the protoplasmis killed by a shock caused by the teasing, so that excess ofwater can at once penetrate and cause disintegration.

The pseudopodia are usually very blunt and lobose, and1 ' Zeit. f. w. Zool.,' Bd. sli, pi. xiii, fig. 3.

ON PELOMYXA VIRIDIS. 367

resemble those figured for P. p a l u s t r i s . When one of theselarge pseudopodia is about to be extruded the movementsaffect very deep-lying portions of the protoplasm; a wave-likebulging occurs, which gradually pushes out in a radial direc-tion ; an active current passes outward along its centre; allthe protoplasmic contents in the neighbourhood are carriedoutwards and, having arrived at the extremity, pass back againin the peripheral portions of the pseudopodium, which thusnarrows while it is elongating. The whole process gives onestrongly the impression that the impulse for the extrusion iscentral and deep-lying in its origin. I t is not that a smallpseudopodium is gradually increased in size, but the mass ofprotoplasm extruded is actually greatest at first and thengradually diminishes. Even when fully extruded the back-ward peripheral currents still continue, especially in theproximal portion of the pseudopodium, which thus becomesflask-shaped, joined to the main portion of the organism by anarrow neck.

All this time no peripherally placed colourless layer ap-pears, the vesicles and other protoplasmic contents beingpushed on up to the surface; but when this pseudopodiumcommences to retreat the contents pass backward faster thanthe protoplasm itself, and a colourless superficial layer is left.Fig. 3 represents the distal extremity of a pseudopodiumwhich has just commenced to retreat. If this withdrawal israpidly continued some of the colourless protoplasm will beleft sticking to the glass.

Besides these large pseudopodia, small ones are often foundwhich seem to take their origin in the superficial layer. Inthese latter the hyaline protoplasm is first protruded, and intothis a number of the bacteria appear to burst out, followedalmost at once by some of the vesicles, which often sepa-rate a good deal from one another aud appear as so manyisolated spherical green droplets; these are sometimes fol-lowed by the other protoplasmic contents (fig. 5). Thesepseudopodia are never prolonged to any extent.

The rate at which the large pseudopodia are protruded is

368 ALFRED GIBBS BOURNE.

considerable. I have found it to be about 075 mm. aminute. Engelmann1 states that a velocity of 0*5 mm. aminute is sometimes attained by an Amoeba, and may be con-sidered as exceptionally rapid. I have made my observationsat the ordinary temperature of the room; but this is about30° C. at this time of year, and is thus not far below theoptimum temperature for protoplasmic movement.

Nuclei .The number of nuclei present is enormous. I have en-

deavoured to estimate the number on stained preparations.I have made these preparations in various ways; one of themost satisfactory is to fix and harden on the slide withchromic acid (2 per cent, solution), followed by water andalcohol of increasing strengths; stain with picro-carmine,wash, and again treat with alcohol of increasing strengths;dehydrate, clarify, and mount in Canada balsam.

I put down the number of nuclei present in a large indi-vidual at 10,000.

They vary a little in size, but average, when living, about0'03 mm. in diameter.

It has struck me that there may be some connectionbetween the bulk of nuclear matter and the bulk of proto-plasm connected with it.

I calculate that in P. viridis all the nuclei taken togetheroccupy -gij of the total bulk of the organism. I have notmany data at hand for comparison with this calculation, butin a mammalian ovarian ovum the nucleus occupies about^ of the bulk of the ovum. So that 10,000 nuclei in P.viridis give relatively about the same bulk of nuclear mat-ter as is found in a mammalian ovum. The nuclei closelyresemble the nuclei of other species of Pelomyxa.

In a fresh condition they are spherical, and consist of anuclear membrane containing a perfectly clear and trans-parent fluid nuclear substance, in which are seen from nine totwelve highly refracting spherical nucleoli (fig. 7").

1 L. c, p. 379.

ON PELOMTXA. VIRIDIS. 369

I have never seen these enlarged or containing anything likea vacuole, a condition described by Greef for P. p a l u s t r i s ;nor have I ever seen anything in the protoplasm of P. v i r id i sresembling the " glanz-korper " of that author, or which mightbe nucleoli escaped from a nucleus.

I have frequently seen nuclei extruded from the protoplasm,usually along with food debris or sand particles; when soextruded they remain for some time unacted upon by thewater, but in time the water seems to penetrate the nuclearmembrane, and the nucleoli exhibit Brownian movements,and after some time come out into the water (fig. 9) ; butalthough I have watched them for a long time I have not ob-served them to undergo any change.

I have been unable by the use of any preservative or stain-ing process to make these nucleoli behave as does the singlenucleolus of so many Amoebae. This latter when treated withosmic, chromic, or picric acids, and subsequently with picro-carmine or alum carmine, stains deeply, while the nuclearsubstance remains clear and transparent, almost unstained.But when any one of these acids or even acetic acid is allowedto act upon the nuclei of P. v i r id i s , the nuclear substancebecomes opaque and granular, and the nucleoli disappear fromview on account of the now opaque character of the nuclearsubstance. When stained, either by osmic acid or by picro-carmine or alum carmine (preceded by fixing and hardeningreagents), and mounted in Canada balsam, they appear asquite different bodies from the fresh nuclei. To such anextent is this the case, that until I had actually watched asingle nucleus throughout the entire process I was not satis-fied that the bodies which I had taken to be nuclei in thefresh state were actually such bodies. What really happensis, according to the views of Pfitzner and others, that thechromatin substance, which is something distinct altogetherfrom the nucleoli, is brought into view by the staining process,and obscures to a certain extent the nucleoli. The nucleusstains diffusely all over, while very numerous minute granulesappear to stain more deeply.

370 ALFRED GIBBS BOURNE.

I have even after most careful examination of specimens,mounted whole and of sections, been unable to find any casesof nuclear division.

Other Protoplasmic Contents.I have already spoken of the chlorophyllogenous substance,

the permanent vacuoles, and the sand particles.In addition to these, I have observed in many individuals

small globular refractive bodies, about i6*00 inch in diameter,which, from the fact that they are blackened by osmic acid, Isuspect to be of a fatty nature. They are much more nume-rous at some times than at others.

I have on two or three occasions found all the specimensthen examined crowded with large (about -%fa inch in dia-meter) disc or lens-shaped, fairly transparent, but not highlyrefracting bodies, which, from the fact that all these specimenshad been feeding on some special substance, I believe to benutritive bodies of some kind. I have very little doubt fromthe appearance of the debris that the food on these occasionshad been Spirogyra filaments, but I was never fortunateenough to see them in the protoplasm in an undigested state.The protoplasm in all these individuals was simply crammedwith the food debris. It is probable that these specimens hadgot hold of some Spirogyra, of which there was a little on thesurface of the mud, and that it had proved a very easily digest-ible food, and that a great deal of nutriment had becomestored up in the protoplasm. The bodies under discussionstain a rich deep colour with iodine, but do not lose thecolour on warming. They are probably some amyloid sub-stance.

P. viridis takes in plenty of solid food of all kinds, Daph-nise, Ostracods, Diatoms, Naids, &c. I have also foundspecimens which had taken in two or three or more plants ofwhat I believe to beWolffia arrhiza (one of the Lemnacese),and which swarms in most of the tanks in Madras, and is by-the-bye often eaten, curiously enough, in enormous quantitiesby the tank frogs.

ON PBLOMTXA VIEIDTS. 371

The food sometimes lies in food vacuoles, while at othertimes the surface of the food particles is actually in contactwith the protoplasm.

Occasionally I have observed, when a large piece has lain ina large vacuole, pseudopodia protruded from the vacuolar walland reflected over the surface of the food particle.

Physiology.

I have tried but without success to demonstrate the activityof the chlorophyll. The animal does not readily allow ofmuch experiment, as it almost always dies after any manipula-tion. Treatment with iodine has, moreover, failed to demon-strate the presence of starch. The enormous number of thesymbiotic bacteria is correlated doubtless with the activity ofthe chlorophyll.

I have tried by putting them together under a cover-glassto get two individuals to fuse together, but without success.On the other hand, I have often observed two pseudopodia ofthe same individual form and fuse together at their distalextremities, so as to leave for a time a hole right through theanimal. Further, I have several times cut a specimen in halfwith a sharp scalpel, and kept the two halves living for sometime, and by placing the two halves close together haveinduced them to fuse together again.

I have also teased specimens into fragments with a pair ofneedles, but in that case the fragments have at once com-menced to die.

No information which we at present possess seems to me toexplain in any way why the halves of a divided individual orthe peripheral extremities of long pseudopodia protruded byany one individual should freely reunite, while separate indi-viduals will do nothing of the kind.

Gulliver's Views.—Since writing the above my attentionhas been drawn to a note on the minute structure of Pelo-myxa palustr is by Dr. G. Gulliver.1 The author believes, inthe first place, that the exoplasm is permanently differentiated

1 'Journal of the Royal Microscopical Society,' 1888, p. 11.

372 ALFRED GIBBS BOURNE.

from the endoplasm; and in the second place, that the" endoplasm " is composed of a number of cells. Both theseare very startling views, and while I feel sure that the conclu-sions are erroneous, I have observed certain phenomena whichmay have given rise to them.

Gulliver writes, " In the process of hardening this layer(the exoplasm) readily separates from the subjacent softerendoplasm." I have noticed in hardening specimens in awatch-glass for section-cutting, and also under the cover-glassfor subsequent staining and mounting as whole preparations,that the peripheral portion which is first attacked by thehardening reagent often becomes separated from the stillliving and shrinking central mass, another layer is thenattacked, and so the whole structure may harden in irregularlayers. The hardened portions break away with extremereadiness from the unhardened ones.

Very curious appearances are sometimes thus produced, butI am convinced that the phenomenon is a mere accident inthe hardening process. The examination of living specimenscan leave no doubt as to the absence of any permanent differ-entiation of the body substance into exoplasm and endoplasm.With regard to the structures which Gulliver takes to be cellsbuilding up the "endoplasm," and which Lankester has sug-gested may be swarm-spores, I have formed a less definiteopinion. In a living specimen one can see no such structure,but in hardened specimens one does undoubtedly find smallrounded portions of the protoplasm lying in and yet separatedfrom the rest of the protoplasm. These bodies have, however,no definite and constant characters, they differ considerablyin size, sometimes include one nucleus, sometimes more thanone, and frequently none at all. It is difficult to detail theprecise appearances which lead me to the conclusion; but Iam much inclined to think that we have here merely an acci-dental rounding off of portions of the protoplasm, which takesplace during the hardening process. I have seen similarphenomena in specimens which have died from having beenkept mounted for a long time under a cover-glass. The body

ON PELOMYXA VIEIDIS. 373

substance in these large Pelomyxse forms a considerable mass,and this breaks up very readily after death into smalldroplets.

I find nothing in the structure of P. v i r id i s which wouldlead one to suppose that these organisms have any affinities,other than those usually assigned to them. Pe lomyxabelongs, as Butschli says, to the family Amoebsealobosa.

In spite of repeated endeavours I am unable to throw anylight upon the reproductive processes which may obtain inPelomyxa.

EXPLANATION OF PLATE XXVIII,

Illustrating Professor Alfred Gibbs Bourne's paper on"Pelomyxa viridis."

FIG. 1.—An entire specimen, partially spread out under a cover-glass,magnified about four diameters. In the left-hand lower corner is a Dapliniashell, which the animal had just extruded. The opacity of the central regionis caused by a great accumulation of mud or sand particles.

FIG. 2.—Extremity of a blunt pseudopodium, which is retreating. Smalldroplets of the colourless non-granular protoplasm are being left sticking tothe slide ; some of these will become detached as the pseudopodium retreatsfurther. The magnification is not sufficient to allow of the nuclei and vesiclesbeing distinguished, but a few of the larger vacuoles, v. »., are seen.

FIG. 3.—A long thin pseudopodium in the act of retreating, drawn to aneven smaller scale than the preceding figure.

FIG. 4.—Extremity of a blunt pseudopodium, more highly magnified. Theexistence of the peripheral layer of colourless protoplasm indicates that thepseudopodium is not being actively protruded, while its small extent andcomparative regularity shows that the pseudopodium is not being rapidlywithdrawn, n. n. Nuclei. /. v. Food vacuole, containing food ddbris. s,Naid seta. m. m. Sand or mud particles, v. v. Vacuoles. The small circleswhich are shown all over the figure, except in the colourless peripheral portion,represent the vesicles with their chiorophyllogenous contents. In places

374 ALFBED GIBBS BOURNE.

where, as over the food vacuole, marked f. »., there is only a single layer ofthese vesicles, the green coloration is very faint.

FIG. 5.—Portion of the periphery of an individual where there has just beena slight outflow of colourless protoplasm, and which will be withdrawn withoutthe protrusion of any pseudopodium. The bacteria (? crystals) seen comingout into the protruded protoplasm exhibit active (? Brownian) movements. Thegreen bodies represent the vesicles; two of these are seen coming out intothe protruded protoplasm.

FIG. 6.—View of a region precisely similar to that drawn in the precedingfigure, in an individual which has been treated with osmic acid. The colour-less protoplasm has stained; while the vesicular contents, now no longergreen, are not to be seen. It is seldom that the vesicular structure is so wellseen; usually, upon the application of reagents, the vesicles seem to collapse,and so no such network as is here shown can be seen.

FIG. 7-—View of a small portion of the central region of an individual,showing the same structures as in Fig. 4, with the addition of the bacteria.Three nuclei are drawn with their contained nucleoli.

FIG. 8.—A nucleus with an unusually large number of nucleoli.FIG. 9.—A nucleus which has been extruded with food debris, and which

has swollen under the action of the water, while its nucleoli have burst out.

N.B.—Figs. 5—9 are drawn to about the same scale as the line A B at thebottom of the plate, which represents x$s§ of an inch.

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