ANUMBER of electron microscope studies have been made on Amoeba

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1 An Electron Microscope Study of a Small Free-living Amoeba (Hartmanella astronyxis) By K. DEUTSCH and M. M. SWANN (From the Department of Zoology, University of Edinburgh) With two plates (figs. I and 2) SUMMARY The fine structure of a species of small free-living amoeba, Hartmanella astronyxis, has been investigated. The mitochondria resemble those of other species of amoeba. Structureless bodies of about the same size as mitochondria are sometimes found in association with them. Double membranes are common in the cytoplasm, and may show granules along their outer borders. The nuclear membrane is a double-layered structure, with a honeycomb structure evident in tangential sections. The cell membrane is also double-layered, or occasionally multi-layered. INTRODUCTION ANUMBER of electron microscope studies have been made on Amoeba. proteus and Chaos chaos (Andresen, 1956; Bairati and Lehmann, 1951, 1954; Cohen, 1957; Greider, Kostir, and Frajola, 1956; Lehmann, Manni, and Geiger, 1956; Manni, 1956; Pappas, 1956; Rudzinka, 1956; Sedar and Rudzinka, 1956). Other amoebae, however, seem to have been neglected. Hartmanella astronyxis, a small species of soil amoeba, originally isolated by Ray and Hayes (1954), has recently been maintained in this laboratory in sterile culture. Since it is being used for a number of studies of cell growth and cell division, we felt it desirable to make a general study of its structure. The results of this work are presented below. The results of a more detailed investigation of the changes during mitosis will be presented in due course. MATERIAL AND METHOD The amoebae were fixed in 1% osmium tetroxide (or 10% formalin) at ph 7-2. The fixed amoebae were then embedded in a methacrylate mixture (93% butyl methacrylate, 7% methyl methacrylate), or in a methacrylate mixture to which tetra-ethyl tin had been added (4 parts butyl methacrylate, 1 part methyl methacrylate, 0-5 parts tetra-ethyl tin). It was originally hoped that the addition of tetra-ethyl tin to the embedding medium would render it dense enough to make the specimens appear light on a dark background (Deutsch, 1957); but in this we were disappointed, since it proved impossible to dissolve a sufficient quantity of tin in the methacrylate mixture. However, some parts of the tissue were apparently 'stained' by the tin, and we have included a few photographs of sections embedded in this medium. The possibilities of the method are being further investigated. [Quarterly Journal of Microscopical Science, Vol. 100, part 1, pp , March 1959.]

2 14 Deutsch and Swarm Electron Microscope Sections were cut at 250 A, and examined in a Siemens Elmiskop I, at magnifications of 8,000 and 40,000. The plates were enlarged photographically. RESULTS AND DISCUSSION A low power view of the amoeba is shown in fig. 1, A. The nucleus, nucleolus, food vacuoles, and mitochondria are readily visible. The nucleoplasm is mainly granular in osmium-fixed sections (fig. 1, B), while the addition of tin to formalin-fixed specimens gives a rather more vesiculate appearance (fig. 1, c). The nuclear membrane consists of two electron-dense layers enclosing an electron-transparent one, the width of each layer being about 70 A (fig. 1, G). This double structure is most clearly seen in sections 'stained' with tin (fig. 1, F). Tangential sections of the nuclear membrane (figs. 2, D; 2, F) show either a 'honeycomb' organization, the diameter of the pores being about A, or a vesiculate appearance (compare Greider, Kostir, and Frajola, 1956; Pappas, 1956). The nucleolus is very electron-dense, and usually contains a reticulated inner region (fig. 1, B). The cytoplasm contains many of the structures normally encountered in electron micrographs. The mitochondria (fig. 2, B) resemble those found in other Protozoa (Manni, 1956; Sedlar and Rudzinka, 1956), containing numerous double membranes, somewhat contorted. There are food vacuoles and a large number of small vesicles (fig. 1, A). There are also double membranes scattered throughout the cytoplasm, often with small dense granules arranged along their outer borders (figs. 2, c; 2, E; compare the endoplasmic reticulum of Palade (1955))- We have not, however, found any trace of a Golgi apparatus. A few cells show a less usual form of inclusion, about the same size as the mitochondria, but devoid of internal structure (see figs. 2, G; 2, H; compare Rouiller and Bernhard, 1956). It is our impression that these bodies are only found in cells near to mitosis, and that they may be in some way associated with the mitochondria (see arrows in figs. 2, G; 2, H). The cell membrane consists either of two electron-dense layers enclosing an electron-transparent one (figs. 1, D; I, E) or, less often, of a number of alternating dense and transparent layers (fig. 2, A). The layers are about 70 A thick. We are indebted to Mr. A. E. G. Dunn for his most skilful assistance, and to the Melville Trust for Cancer Research who provided the microscope. FIG. I (plate). A, low-power view of H. astronyxis, showing nucleus, nucleolus, food vacuoles small vesicles, and mitochondria. (Osmium.) B, nucleus, showing nucleolus with reticulated centre. (Osmium.) C, nucleus, showing vesiculate appearance when fixed with formaldehyde and 'stained' with tin. D, cell membrane showing double structure. (Osmium.) E, cell membrane showing double structure. (Formaldehyde, 'stained' tin.) F, nuclear membrane, showing double layer. (Osmium.) G, nuclear membrane, showing double layer. (Osmium, 'stained' tin.)

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5 Study of Hartmanella 15 REFERENCES ANDRESEN, N., Comp. rend. Trav. Lab. Carlsberg, Ser. chim., 29, 435. BAIRATI, A., and LEHMANN, F. E., Pubbl. Staz. Zool. Napoli, 23, Suppl Experientia, 10, 173. COHEN, A. I., J. biophys. biochem. Cytol., 3, 859. DEUTSCH, K., Proc. Roy. Phys. Soc. Edinburgh, 26, 7. GREIDER, M. H., KOSTIR, W. J., and FRAJOLA, W. J., J. biophys. biochem. Cytol., 2, Suppl., 445. LEHMANN, F. E., MANNI, E., and GEIGER, W., Naturwiss., 43, 91. MANNI, E., Boll. Soc. ital. Biol. sper., 32, 113 and 115. PALADE, G. E., J. biophys. biochem. Cytol., I, 567. PAPPAS, G. D., Ibid., 2, Suppl., 431, and 2, 221. RAY, D. L., and HAYES, R. E., J. Morphol., ROUILLER, C, and BERNHARD, W., J. biophys. biochem. Cytol., 2, Suppl., 355. RUDZINKA, M. A., Ibid., 2, Suppl., 425. SEDAR, A. W., and RUDZINKA, M. A., Ibid., 2, Suppl., 331. FIG. 2 (plate), A, cell membrane, showing multi-layered structure. (Osmium.) B, mitochondria, showing double membranes. (Osmium.) C, cytoplasmic double membranes, with granules along their outer border. (Osmium.) D, nuclear membrane in tangential section, showing honeycomb structure. (Formaldehyde, 'stained' tin.) E, cytoplasmic double membranes, with granules along their outer border. (Osmium.) F, nuclear membrane in tangential section, showing vesiculate appearance. (Osmium.) G, structureless bodies and mitochondria. Note the apparent association between the two. (Osmium.) H, structureless bodies and mitochondria. Note the apparent association between the two. (Osmium.)