An Electron Microscope Study of a Small Free-livingAmoeba (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 inassociation with them. Double membranes are common in the cytoplasm, and mayshow granules along their outer borders. The nuclear membrane is a double-layeredstructure, with a honeycomb structure evident in tangential sections. The cell mem-brane 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, andGeiger, 1956; Manni, 1956; Pappas, 1956; Rudzinka, 1956; Sedar andRudzinka, 1956). Other amoebae, however, seem to have been neglected.Hartmanella astronyxis, a small species of soil amoeba, originally isolated byRay and Hayes (1954), has recently been maintained in this laboratory insterile culture. Since it is being used for a number of studies of cell growthand 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 detailedinvestigation 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) atpH 7-2. The fixed amoebae were then embedded in a methacrylate mixture(93% butyl methacrylate, 7% methyl methacrylate), or in a methacrylatemixture 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 hopedthat the addition of tetra-ethyl tin to the embedding medium would renderit dense enough to make the specimens appear light on a dark background(Deutsch, 1957); but in this we were disappointed, since it proved impossibleto dissolve a sufficient quantity of tin in the methacrylate mixture. However,some parts of the tissue were apparently 'stained' by the tin, and we haveincluded a few photographs of sections embedded in this medium. Thepossibilities of the method are being further investigated.[Quarterly Journal of Microscopical Science, Vol. 100, part 1, pp. 13-15, March 1959.]

14 Deutsch and Swarm—Electron Microscope

Sections were cut at 250 A, and examined in a Siemens Elmiskop I, atmagnifications 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, nucleo-lus, 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 morevesiculate appearance (fig. 1, c). The nuclear membrane consists of twoelectron-dense layers enclosing an electron-transparent one, the width of eachlayer being about 70 A (fig. 1, G). This double structure is most clearly seenin sections 'stained' with tin (fig. 1, F). Tangential sections of the nuclearmembrane (figs. 2, D; 2, F) show either a 'honeycomb' organization, thediameter of the pores being about 50-100 A, or a vesiculate appearance(compare Greider, Kostir, and Frajola, 1956; Pappas, 1956). The nucleolusis very electron-dense, and usually contains a reticulated inner region (fig. 1, B).

The cytoplasm contains many of the structures normally encountered inelectron micrographs. The mitochondria (fig. 2, B) resemble those found inother Protozoa (Manni, 1956; Sedlar and Rudzinka, 1956), containing numer-ous double membranes, somewhat contorted. There are food vacuoles and alarge number of small vesicles (fig. 1, A). There are also double membranesscattered throughout the cytoplasm, often with small dense granules arrangedalong their outer borders (figs. 2, c; 2, E; compare the endoplasmic reticulumof 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 themitochondria, but devoid of internal structure (see figs. 2, G; 2, H; compareRouiller and Bernhard, 1956). It is our impression that these bodies are onlyfound in cells near to mitosis, and that they may be in some way associatedwith the mitochondria (see arrows in figs. 2, G; 2, H).

The cell membrane consists either of two electron-dense layers enclosingan electron-transparent one (figs. 1, D; I, E) or, less often, of a number ofalternating dense and transparent layers (fig. 2, A). The layers are about70 A thick.

We are indebted to Mr. A. E. G. Dunn for his most skilful assistance, andto the Melville Trust for Cancer Research who provided the microscope.

FIG. I (plate). A, low-power view of H. astronyxis, showing nucleus, nucleolus, food vacuolessmall 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.)

FIG. I

K. DEUTSCHa^M. M. SWANN

FIG. I

K. DEUTSCHWM. M. SWANN

FIG. 2

K. DEUTSCHaniM. M. SWANN

*J L - ^ 1 I

FIG. 2

K. DEUTSCHaWM. M. SWANN

Study of Hartmanella 15

REFERENCESANDRESEN, N., 1956. Comp. rend. Trav. Lab. Carlsberg, Ser. chim., 29, 435.BAIRATI, A., and LEHMANN, F. E., 1951. Pubbl. Staz. Zool. Napoli, 23, Suppl. 192.

1954. Experientia, 10, 173.COHEN, A. I., 1957. J. biophys. biochem. Cytol., 3, 859.DEUTSCH, K., 1957. Proc. Roy. Phys. Soc. Edinburgh, 26, 7.GREIDER, M. H., KOSTIR, W. J., and FRAJOLA, W. J., 1956. J. biophys. biochem. Cytol., 2,

Suppl., 445.LEHMANN, F. E., MANNI, E., and GEIGER, W., 1956. Naturwiss., 43, 91.MANNI, E., 1956. Boll. Soc. ital. Biol. sper., 32, 113 and 115.PALADE, G. E., 1955. J. biophys. biochem. Cytol., I, 567.PAPPAS, G. D., 1956. Ibid., 2, Suppl., 431, and 2, 221.RAY, D. L., and HAYES, R. E., 1954. J. Morphol., 95. 159-ROUILLER, C , and BERNHARD, W., 1956. J. biophys. biochem. Cytol., 2, Suppl., 355.RUDZINKA, M. A., 1956. Ibid., 2, Suppl., 425.SEDAR, A. W., and RUDZINKA, M. A., 1956. 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.)