Cites Plate IV, Journal of Physiology 20 (4-5) (1896). Figs. 1-14 of M. Greenwood, 'On Structural Change in the Resting Nuclei of Protozoa. Part I. The Macronucleus of Carchesium Polypinum'.
Description:

Explanation of Plate IV (figs. 1-14):

'The outlines of the nuclei, of the macrosomes and of the contained vacuoles are, as a rule, traced by means of the camera lucida. The exceptions are indicated below.

In all the figures, n = macronucleus.

mac = macrosome.

mic = microsome.

membr = nuclear membrane.

seg = segment of macronucleus.

v = vacuole contained in macrosome.

No attempt has been made to represent the structure of the cell substance, and in some figures, sketched simply for change in macrosomatic substance, the microsomes are not indicated.

Fig. 1. Diagrammatic sketch of Carchesium polypinum, to show position of macronucleus, coexistence of microsomes and macrosomes and varying shape and size of the latter.

Fig. 2. Macronucleus of Carchesium. a. Fresh specimen faintly stained with osmic-methylene blue. The microsomes are abundant. b. Specimen more deeply stained; the underlying macrosomes appear.

Fig. 3. To show extreme contrast in nuclear structure. a. Freshly mounted specimen, intermittent bacterial nourishment; the microsomes hide other structures. b. After long treatment with white of egg, the macrosomes are large and highly vacuolate, the microsomes indistinguishable.

Fig. 4. Reaction of unmasked iron with potassium ferrocyanide in Carchesium. a. Section stained with eosin, macrosomes demonstrated by oxyphile reaction. b. Another section of the same series unstained, the macrosomes are rarely distinguishable; the vacuoles which show (v), indicate that the Prussian blue stain has been localized. c. Carchesium. I episty. stained by long exposure to pot. ferrocyanide. This has decomposed and combined like a stain with the macrosomatic matter which is actually poor in iron.

Fig. 5. Macronucleus of Spirostomum. Localization of large macrosomes in small segments of nucleus, and distinct vacuolation of these.

Fig. 6. Demonstration of nuclear membrane in early stages segmentation of macronucleus. Carchesium.

Fig. 7. Macronucleus of Carchesium. a and b showing nuclear membrane, a showing massive macrosomes under rich diet. b, c and d various stages of vacuolation; in c the nucleus is watery and the microsomes distinct.

Fig. 8. Macronucleus of Carchesium. Macrosomes in axial row stained with Canon's fluid.

Fig. 9. Carchesium polypinum. Diagrammatic; to show maximum extension of macronucleus under rich diet (a), and contracted form (b) with scanty nourishment.

Fig. 10. Macronucleus of Carchesium. Heidenhain's iron-haematoxylin stain. a. Transverse section of nucleus; macrosomes unequal in size and arrangement, microsomes obscure. b. union of macrosomes to imperfect network. c. large size of free macrosomes and rare vacuolation.

Fig. 11. a. Irreguilarly filamentous macrosomes of large size fuising to network, to contrast with, b. fine filaments which characterize nucleus of fission.

Fig. 12. Extreme form of dotted or granular macrosomes (basophile granulation): microsomes standing out clearly from nucleochyme.

Fig. 13. Segments of macronucleus freed,-to show varying condition of macrosomes. a solid, b slightly, c extremely vacuolate.

Fig. 14. Macronucletis of Carchesium. To show substance of macrosomes gathered to vacuolate, axial strand.

Figures 3, 9 b and 13 are drawn with the free hand. The magnification of the majority of the figures is Leitz obj. 2 mm. comp. oc. 6, but Figs. 7 d, 12, are drawn with comp. oc. 4, and Figs. 4, 5, 7a and 7b with Leitz oil imm. 1/12. Zeiss. oc. 3.' (453-454)

 

Figs. 1-3, 7 and 12 in text:

'It is... convenient and I thinik not unfair to choose for introductory description a polype taken from a healthy colony which has been but recently caught and mounted. In such a polype (which has had abundance of water and air, and sufficient bacterial food) the long curving nucleus lies peripherally in the cell, forming an imperfect spiral through its substance or more rarely an encircling zone (Fig. 1).

I distinguish in it four elements:

1. The nuclear membrane, which, structureless and always marked off from its surroundings by contour rather than colour, is demnonstrable with difficulty in this stage.

2. The nucleochyrne. And besides these constituents which have not been made the subject of great controversy in the literature of the nucleus, formed elements of two kinds.

3. The nucleochyme is crowded with fine highly refractive granules (Figs. 1, 2, 3 a, 12).

4. Larger masses of substance lie among these, rounded in shape, or bluntly angular. Some may abut on the nuclear membrane but the majority lie more deeply and thus in optical section are hidden by the smaller granules except at a certain focus (Figs. 1, 2, 7 c and d).

I have been unable to demonstrate in the nucleus of Carchesium any structural constituent which cannot be included in this classification.' (428-429)

 

Figs. 1-3, 7 and 14 in text:

'Protomacrosomes. These are complex mobile bodies especially prominent in the majority of stained specimens. At times they are spherical or bluntly angular in shape and lie freely; at other times they form an irregular or imperfect network or fuse to an axial strand; when free they may be equal in size or equidistant but they are commonly uinequal and so grouped that small macrosomes lie towards the periphery of the nucleus, larger bodies being gathered to its core (Figs. 1, 2 b, 7 a, 14).

The complex character and action of these macrosomes are suggested;- (1) by their reaction with dyes for, staining selectively with eosin, they may also colour deeply with methylene blue; the eosin stain emphasizes their smooth aspect, while with methylene blue they are less homogeneous and may even show stained granulation;- (2) by their remarkable capacity for vacuolation; this activity finds expression in the deposition of small drops of fluid centrally; these may grow centrifugally, and encroach on the unchanged substance of the macrosome which survives in extreme cases as a mere shell-like investment. Vacuolation may be induced in any phase of arrangement of the macrosomes, but is most striking when they lie freely and are relatively massive (Figs. 3 b, 7 b, c, d, 14).' (450)

 

Figs. 1, 4, 7-8 and 10 in text:

'The second formed element is that in which structural variations are especially striking. In the well nourished polype which I am considering now it is arranged in small masses or lumps, distinguishable from the granules of the nucleochyme by configuration, disposition, and comnmonly by size and affinity for dyes. These bodies are spherical, elongated, or bluntly angular, the largest forming an irregular axial row along the nucleus (Figs. 1, 7), smaller fragments lying more peripherally (Fig. 10 a). I have implied that their outlines and smooth aspect become clear in unstained nuclei, only at a certain focus; all the nuclear stains which I have used however differentiate them more or less clearly from the granules of the nucleochyme.

Picrocarmine, hamatoxylin, saffranin colouir them deeply, they reduce gold from its soluitions and retain very persistently M. Heidenhain's iron-hamatoxylin stain (Fig. 10). They are deeply blue after treatment with Canon's fluid (Fig. 8), and stain slowly but selectively with dilute "osmic methylene blue  [note: 'By this term I refer to the modification of Loeffler' s methylene blue mentioned above, p. 429.']." On the other hand the mixed reagent known as Ehrlich-Biondi's fluid gives a sharply contrasting picture, the blue or violet granules of the nucleochyme forming a basis in which are imbedded bright red spheres or lumps. A comparatively deep stain is also taken up from weak alcoholic solutions of eosin (Fig. 4 a). ' (431-432)

 

Figs. 1, 4, 7-8 and 10-12 and 14 in text:

'A comparison of Figs. 1, 7 and 10 will make it clear that the macrosomes vary in size. Not only is this inequality demonstrable in different nuclei so that the sparsely scattered fragments of Fig. 4 represent the relatively massive lumps sketched in Fig. 7 a, but in one nucleus an axial row of macrosomes flanked by others of much smaller size forms an arrangement as characteristic as is uniform distribution of macrosomatic substance (Figs. 10 a, 8, 12). A comparable but slighter disparity in the size of the nuclear macrosomes is demonstrated by Korschelt in the cells which he examined, and he points out firther that variations in shape are clear. In Carchesium too the shape is changeful; approximately spherical in many nuclei, in others the macrosomes may be bluntly angular, irregularly curved or elongate (Figs. 1, 11 a). Acute angles or filiform processes are not characteristic; there is rather mimicry in miniature of the rounded curves of lobate pseudopodia. And the mobility suggested by this likeness to recognized amoeboid structures is further indicated by variations in the union of the macrosomes. Infrequently, instead of scattered bodies an irregular network marked by like reactions is found (Figs. 4 c, 10 b) or an axial rod with or without blunt projections runs through the nucleus (Fig. 14). The network recalls Jickeli's description of a chromatic framework, massed here and there into knots in the nucleus of Infusoria; the axial strand was figured by Greef in Epistylis from the more imperfect histological preparations of twenty years ago, and it is very suggestive of the ribbon (cordon nucléaire) which curves or coils through the nuclear segments of Loxophyllum [note: 'It is interesting to note that this nuclear ribbon breaks into segments under treatment with some reagents. It may be that this ready cleavage, figured by Balbiani, indicates preexisting segmental structure.'] (Balbiani).' (438-439)

 

Figs. 2-3, 7 and 9 in text:

'It is less easy to apply discriminative stimulus or to produce specific lesion in Protozoa than in organisms of more differentiated build, but, endeavouring to exalt, depress, or inhibit respiratory or digestive activity I have come to certain preliminary conclusions: 

1. I find that bacteria, the natural food of Carchesium, produce and maintain what I regard as the most successfully balanced nutritive state. The cells are vigorous and transparent, have rounded, full outlines, the macrosomes are scattered and non-vacuolate or very slightly vacuolate, the microsomes are obvious, the nucleochyme is faintly oxyphile (Fig. 2 b).

2. With a complex fatty diet (in which non-coagulated proteid is present) - milk or yolk of egg suspended in water, there is an enormous increase in the granules of the cell-substance of Carchesium so that polypes thus fed become relatively opaque. The macronucleus takes up its position of maximum extension (Fig. 9 a), its macrosomes are arranged in larger masses and their substance is absolutely increased (Fig. 7 a). The basis of the nucleus is of dense aspect,-the microsomes and fluid do not stand off sharply, probably because of change in the latter, for in some cases, replacing the faint oxyphile reaction, is a violet or rose reaction with methylene blue. The macrosomes are slightly vacuolate, or may show no central deposit of fluid.

3. Under treatment with the coagulated white of egg, of which I have spoken, the phenomena which I first associated with its ingestion repeat themselves, but not always with equal rapidity or clearness. Vacuolation of the macrosomes and, often, fluidity of the substance of the nucleus generally, are characteristic of the condition set up. The microsomes may show clearly in their fluid surroundings, but are small; in rare cases I have not been able to see them (Figs. 7 d, 3 b).' (446)

 

Figs. 2-3, 12 in text:

'The structural changes of the microsomes are obscure. At their largest these bodies are so small that I do not find it possible to trace their outlines through the camera lucida; they are smaller than many of the highly refractive granules which are scattered through the extra-nuclear protoplasm. Yet the effect which they produce in combination varies much. At times they crowd the nuclear substance, giving to it a coarsely granular aspect and obscuring the underlying macrosomes at any focus. At times again they are distinct but still more minute, standing out as bright spherical points (in formaline specimens) upon a background of some homogeneous medium; and there are conditions in which discrete granules cannot be distinguished, the basis of the nucleus has that dense but minutely irregular aspect which is described as ground glass (cp. Figs. 2, 3, 12). In short, it is clear that the microsomes vary in size but also that they and the medium in which they lie vary in refractive power, and we may ask, are they absent from the nucleus of ground glass aspect, or have they with the surrounding nucleochyme undergone such change that neither by formaline vapour, nor by the action of dyes can they be distinguished clearly? Bearing in mind how obscure at times may be the "rods" in the external border of intestinal cells, and noting how sharply the microsomes stand out from abundance of (apparently) watery nucleochyme [note: 'It is noteworthy too that in observing the microsomes the defining power of a lens is, within certain limits, more important than its magnifying power. I have sometimes used Leitz Apoch 2 mm. Compens. Oc. 4 with more effect than combinations which give higher magnification.'], I am inclined to think they are present possibly in some modified form or hidden by the increased density of their environment. A nucleus with macrosomes scattered through its perfectly limpid substance (Fig. 3 b) may probably be regarded as denuded of microsomes, but the condition is extremely rare and, I think, pathological. ' (442-443)

 

Fig. 3 in text:

'It was in the course of an experiment on the aggregation [note: 'cp. Phil. Trans. 1894, B. p. 359.'] of food particles in Carchesium that I was first struck by the phenomenon of vacuolation. A group of polypes (mounted in a hanging drop of water which held coagulated white of egg in suspension) had been watched by artificial light for some hours, and had shown marked secretory activity in the ingestion and partial solution of the proteid, with repeated ejection of insoluble residues. Killing the animal finally with "osmic-methylene blue" I found the macrosomes as highly vacuolate as those of Fig. 3 b, while massive non-vacuolate macrosomes characterized other polypes mounted freshly (as control) from a common stock. It is clear that the conditions of the experiment forbid immediate inference that vacuolation and the digestion of albumen are related as effect and proximate cause, for the illuminating and heating action of the transmitted light, and the limited fluid environment of the animal cannot be disregarded. I do find, however, that from a mass of experimental results, sometimes incomplete, not always concordant, certain deductions can fairly be made which show how intimate is the relation in Carchesium between nuclear structure and the nutritive condition of the mature cell.' (446)

 

Figs. 3-4, 8 and 10 in text:

'The nomenclature of Korschelt, insisting upon the salient if unexplained feature of contrasting size in the nuclear constituents does not prejudge the question of their composition or activities, and I have pointed out the notable likeness borne by the larval nucleus he describes to the macronucleus of Carchesium. Yet differences exist: I find no indication in Carchesium of permanent linin threads joining the larger masses of chromatophilous substance, and though it be bard to prove the absence of a delicate non-staining network it would seem capricious to assume that the bodies sketched in Figs. 3, 4, 8, 10, do not lie freely. Further, I have said (confirming Zoja) that in Carchesium the fine granules of the nucleus are basophile [cyanophile]; but the microsomes of the nuclei which Korschelt describes may combine with gentian violet and again with acid fuchsin, that is, with dyes of unlike constitution [note: 'Cp. Korschelt, loc. cit. Figs. 31, 32, 34.']. Because these differences exist and because I am speaking of protozoan cells in which specialization is yet new, I have chosen a modified terminology and speak of protornacrosomes and protomicrosomes. In the pages which follow, however, I propose to drop the distinctive but clumsy prefix where such abbreviation introduces no confusion.' (437)

 

Figs. 2-3, 5, 7 and 14 in text:

'When many small vacuoles co-exist, optical section of the nucleus shows that they lie deeply; further increase of fluid is centrifugal and may be so marked that only a thin bounding shell of unaltered matter remains (cp. Figs. 2 b, 3 b and 7). In cases of less extreme change and especially where the deposition of fluid has started at more than one centre, delicate partitions may stretch through the investing shell, and they have curiously rectangular attachments so that the contained vacuoles look polyhedral rather than spherical.

I note that the substance of the macrosomes may display this vacuolation whatever its arrangement. In such relatively large bodies as are sketched in Fig. 3 it is usually seen in the most striking and characteristic fashion, but small outlying macrosomes (where such are present) may show it, and vacuolar deposits may be seen in the coarse macrosomatic network, or the axial bar (Fig. 14). The vacuolation of a network has this result, that protomicrosomes impinge upon the outside of any bar or strand while its interior is hollowed by the deposition of axial, droplets, which tend to fuse to a continuous, fluid core. Where large and small macrosomes co-exist vacuoles may appear in the larger, while the smaller are homogeneous. 

A quite comparable difference is also demonstrable in the longjointed nucleus of Spirostomum. In the smallest joints or segments of this nucleus or at the junction of successive segments the largest macrosomes lie, and at times when they are clearly vacuolate others which, neighbouring or remote are smaller, may show no loss of homogeneous aspect (Fig. 5).' (440-441)

 

Figs. 3 and 12 in text:

'Many forms of lesion, notably lack of oxygen or excess of carbonic acid, bring out the nucleus in Carchesiuin, but inference drawn from the study of dying tissues cannot have great independent value, and I trust rather to the rapid fixative action of formaline vapour (used alone or followed by staining) and to that modification of Loeffler's methylene blue which has been used with effect by Kanthack and Hardy [note: 'The names of the Authors to whom I refer (with their appropriate works) are arranged in alphabetical order at the end of this paper.'] in their study of the activities of leucocytes. This fluid which, acting on fresh tissue, stains as it kills may yield preparations which are temporary indeed, but are quite without distortion or shrinkage. And both reagents harmonize with such fixatives as alcohol and corrosive sublimate in demonstrating the presence of minute granules in whose definite outline there is indeed no likeness to the irregularity of precipitated particles (Figs. 3 a, 12). These granules stain with gold chloride [note: 'I subjoin a brief statement of the experimental methods followed throughout these observations. Picrocarmine and saffranine were used... [see art.]'], with picrocarmine, and faintly with Heidenhain's iron-hamatoxylin; with eosin they colour only when the whole tissue of the cell is loaded indiscriminately with the stain; in Ehrlich-Biondi's fluid they become blue or blue-violet. With methylene blue they react but not intensely or selectively even when it is used in weak alcoholic or aqueous solutions; it may be said briefly that they show affinities for basic dyes but are not strongly basophile.' (429-430)

 

Fig. 4 in text:

'It is usually difficult to trace outlines of discrete granules in the deep blue stain which the mass or "stroma" of the nucleus exhibits, but this can be done sometimues; it is clear that optical arrangements (brilliant light and a widely open diaphragm) which emplhasize colour under the microscope impair definition, and that such an effect as that indicated in Fig. 4 b might well be induced by the superposition of minute blue granules, uniformly stained.' (431)

'I have implied that inorganic iron was absent from all parts of the nucleus; in specimens stained after the "unmasking" action of acid there is at most very slight indication of the existence of larger lumps or spheres in the general blue basis which is the resultant effect of minute stained granules. But colourless vacuoles may stand out clearly in tissue giving a marked "Prussian blue" reaction, I conclude then that the bodies which if they show no local deepening of tint also show no local paling do hold iron. It is clear that the content must be slight, and the effect of a dye like eosin superposed on the blue stain is confirmatory of this. Fig. 4a and b (which however represents a nutritive condition with which we are not concerned here) may be taken to illustrate this point. In Fig. 4b the nucleus has been stained by momentary treatnment with acid potassium ferrocyanide; Fig. 4a is taken from a rnember of the same series of sections which has been further stained with eosin. It is seen that the eosin not only reveals spherical lumps of matter which were obscured before, but that their proper blue tint has been too faint to give the eosin red a distinct violet tone.' (432)

'There is also varying distinctness in the outlines of microsomes stained to demonstrate the presence of masked iron, and this when constant precautions are taken to avoid colouration by post mortem diffusion. The absence of "Prussian blue " in macrosomatic vacuoles tested for iron is, I think, some indication that the precautions may be effectual (Fig. 4). I suggest then, but quite without insistence, that the nucleochyme is sometimes but not always iron-free.' (443-444)

 

Figs. 6 and 9 in text:

'The mobility displayed by the macronucleus during the phenomena of reproduction is familiar, - its shortening before equatorial constriction when Carchesium multiplies by fission, its spherical or ovoid form in an encysted polype, the segmentation which constantly attends conjugation. But apart from these changes, with which a study of the "resting" nucleus has but indirect concern, I notice that at times there is variation from its typical form and position, and that in two directions. There is on the one hand a maximuim of extension; the nucleus has at such a time its most superficial position; it is long, curving through the cell in a rudimentary spiral, its substance is full, not shrinking readily under the action of reagents, but obscuring the nuclear membrane (Fig. 9 a). But on the other hand there is a shrunken or contracted form, lying more deeply withiin the endoplasm, showing (it may be) local irregularities of contour, or sharply bent upon itself. In this form shrinkage of substance or folding tends to bring out the nuclear membrane (Fig. 9 b). And lastly I notice, independently of the process of conjugation incipient, incomplete, and (in one case) complete segmentation (Fig. 6). In non-conjugating specimens of Paramcecium caudatum Jickeli has described a comparable nuclear segmentation bound up, in the case of Paramoecium, with continued absence of light.' (438)

 

Figs. 6 and 13 in text:

'In segmentation the nuclear contents split into approximately equal portions each holding one or more spherical macrosomes (Figs. 6, 13). These bodies are commonly solid but may be vacuolate, indeed, rarely they show vacuolation in its extreme form (Fig. 13). Segmentation, when attendant upon conjugation, is followed by degenerative change in the constituents of the freed segments, but of this I need not speak now, for it will be gathered from these brief statements that the variations in form, which I would emphasize, are not occasioned by imminent reproductive phenomena.' (445-446)

 

Figs. 11 and 14 in text:

'A combination of two of the characters which I have thus described in the macrosomes, namely, great inequality in size and partial vacuolation has led some observers to suggest that large nucleoli may break into small ones; Rhumbler would reverse the interpretation, inferrinig fusion rather than separation of the endosomes. But it would seem that in Carchesium both processes occur; the axial rod sketched in Fig. 14 can only have been formed by fusion: on the other hand in the macronucleus of any polype which is undergoing fission, the macrosomes are shortly filamentous (Fig. 11 b); thus it is clear that some of the bodies which are their precursors are larger than they.' (441-442)

 

Fig. 12 in text:

'In addition to these changes of shape, of size and union, the macrosomes of Carchesium exhibit marked changes of texture. One such, occurring rarely, has interested me because it hints at a complexity of build which may explain that dual affinity for dyes of opposite nature of which mention has been made. It is of such a kind that the macrosomes which as a rule are homogeneous appear turbid or even clearly dotted; there is a granulation of their substance which stains deeply with methylene blue, while some supporting basis remains colourless (Fig. 12). Now macrosomes stained with acid dyes are of especially homogeneous aspect, - smooth and uniform as one might picture coloured drops of myelin; may it not be that they are built up of at least two substances and that, rarely, the basophile constituent is separated out in this distinct granulation?' (439)

 

Fig. 13 in text:

'although badly nourished specimens of Carchesium may show highly vacuolate nuclear macrosomes, the appearance is exceptional and to be classed with the vacuolation which may mark, but does not typically mark, the freed segments of a nucleus (Fig. 13). The attenuate, irregular or shrunken nucleus of the ill-nourished (? or senile) polype is poor in macrosomatic matter, and irregular granularity replaces the minute, spherical microsomes, - in the moribund state there is hardly distinction and certainly not perfect organisation of the two formed substances.' (447)