Electron Microscopy of Small Cells: Mycoplasma hominis

Transcription

1 JOURNAL of BAcTRiowOY, Dc. 1969, p Copyright American Society for Microbiology Vol. 100, No. 3 Printed In U.S.A. NOTES Electron Microscopy of Small Cells: Mycoplasma hominis JACK MANILOFF Departments of Microbiology and ofradiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York Received for publication 14 August 1969 The size, ultrastructure, and reproduction of Mycoplasma hominis species H39 were studied by electron microscopy. These are the smallest known cells. Mycoplasma hominis species H39 has one of less-steel grids were suspended on a stainless-steel the smallest cellular genomes (9) and, hence, screen in organ culture dishes (60 by 15 nm; would be expected to be among the smallest Falcon Plastics). A drop of an exponential-phase cellular systems. To study the size, cytology, and culture was put on the grids, and fresh medium mode of replication of such small cells, thinsection and negative-staining electron microscopy When the medium reached the level of the screen was slowly added to the well below the screen. have been used. containing the grids and culture, the drop of For thin-section studies, an exponentially culture was drawn down by surface forces, depositing the cells on the grids. The dishes were growing liquid culture of M. hominis species H39 (6) was added to an equal volume of cold then incubated at 37 C and the cells, in contact 12.5% glutaraldehyde (in 0.1 M cacodylate) and with liquid medium below the grid, grew into fixed in the cold for 2 hr. After harvesting by microcolonies. The grids were then fixed in 6.25% centrifugation (10 min at 14,000 X g) some samples were postfixed with osmium tetroxide (4). silicotungstic acid. glutaraldehyde and negatively stained with 2% The pellets were then dehydrated, embedded in A typical microcolony is shown in Fig. 5, Epon, sectioned, uranyl-stained (8), and examined on a Siemen's Elmiskop I (converted to tubule. where two cells can be seen to be connected by a IA) electron microscope. Since these micrographs of exponentially growing cells must contain cells distributed of all The cells appear as short filaments or rods (Fig. 1 and 2) about 0.25 jum wide. The only stages of growth, it is possible to construct a membraneous structure is the bounding unit probable life cycle from them. The daughter M. surface membrane, about 8.0 to 8.5 nm thick. hominis species H39 cells are small coccoids. Each The intracytoplasmic material appears as granules cell grows and elongates, and replication occurs of several sizes and densities, and as dispersed by a binary fission event. At this stage the two nuclear material. dividing daughter cells are about 0.25 by 0.33 Some cells appear to be two parts, connected,um; these are then the smallest known cells. As by a thin tubule (Fig. 2 and 3). The tubule, which the daughter cells move apart, they remain connected by a tubule of membrane-bounded cyto- is about 17 to 19 nm thick, is two back-to-back unit membranes. These tubules can also be seen plasm. The tubule becomes longer and thinner, in whole cells fixed with glutaraldehyde and negatively stained with 2% silicotungstic acid (Fig. eventually breaks. Cells in all of these stages can appearing as two back-to-back membranes, and 4). It should be noted that the tubule diameter be seen in Fig. 2. The growing cells probably is less than the 30-nm value which Robertson tend to stick together, giving rise to the clumps (11) states should be the smallest diameter for seen in phase-contrast microscopy of cell suspensions (10). membrane structures. To examine growing cells, M. hominis microcolonies were grown on electron microscope grids. M. hominis species H39 are similar to those found The ultrastructural features reported here for For these studies, Formvar-carbon coated stain- for AM. neurolyticum (12) and for M. hominis 1402

2 VOL. 100, 1969 NOTES 1403 A,.- FIG. 1. Exponentially growing cells ofm. hominis species H39, glutaraldehyde fixed. X 40,S00.

3 .. ;~~~~~~~~~~~~~~~~~~-A,-_...:...,3, 1404 NOTES J. BACTrERIOL. ''a 4 t., Fio. 2. Exponentially growing cells of M. hominis, glutaraldehyde fixed. The ultrastructural features and life cycle stages, described in text, can be clearly seen. X 162,000.

4 VOL. 100, 1969 NOTES 1405 $o t i ; L-, s.i b FIG. 3. Membrane tubules in dividing M. hominis cells. (a) Glutaraldehyde and osmium fixation. The tubule membrane can be seen to be continuous with the surface cell membrane. X 121,500. (b) Glutaraldehyde fixation. X 240,000. (c) Glutaraldehyde and osmium fixation. Section through the tubule, with the cells at either end out of the plane of section. X 162,000.

5 1406 NOTES J. BACrERIOL. t... if v 1- #%L.-5v 4p. T...V.1, rae. 4> S v, -. ka' :4jft a s s '.... ' '-,1 I'w 1%.., * la FIG. 4. Glutaraldehyde fixed cells, negatively stained showing attached membrane tubule. (a) X 32,400. (b) X 40,500.

6 VOL. 100, 1969 NOTES 1407 A ii.,-. VP 0 FiG. 5. M. hominis microcolony grown on grid, glutaraldehyde fixed and negatively stained. Two cells can be seen dividing still connected by a membrane tubule (arrow). X 32,400.

7 1408 NOTES strain HEp-2 (1). This cytological picture distinguishes these species from those Mycoplasma which grow as rods, filaments, and streptococcallike chains (J. Maniloff, Bacteriol Proc., p. 78, 1968). The binary fission mode of replication found for M. hominis has also been noted for M. orale (2), M. pneumoniae (3), M. gallisepticum (7), M. laidlawii (J. Maniloff, in preparation), and suggested for several other Mycoplasma (5). I wish to thank Madalyn Smith for her excellent technical assistance. This investigation was supported by American Cancer Society grant no. E-471 and by the University of Rochester Atomic Energy Project (report no. UR ). LITERATURE CITED 1. Anderson, D. R., and M. F. Barile Ultrastructure of Mycoplasma hominis. J. Bacteriol. 90: Furness, G Analysis of the growth cycle of Mycoplasma orale by synchronized division and by ultraviolet irradiation. J. Infect. Dis. 118: Furness, G., F. J. Pipes, and M. J. McMurtrey Analysis of the life cycle of Mycoplasma pneumoniae by synchronized division and by ultraviolet and X irradiation. J. Infect. Dis. 118: Kelienberger, E., A. Ryter, and J. Sechaud Electron J. BACTERIOL. microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4: Kelton, W. H Growth-curve studies of Pleuropneumonia-like organisms. Ann. N.Y. Acad. Sci. 79: Maniloff, J Electron microscopy of Mycoplasma. Bacteriol. Proc., p a. Maniloff, J Use of blood agar plates to study Mycoplasma physiology. Microbios. 1: Maniloff, J., and H. J. Morowitz Ultrastructure and life cycle of Mycoplasma gafllsepticum A5969. Ann. N.Y. Acad. Sci. 143: Maniloff, J., H. J. Morowitz, and R. J. Barrnett Studies of the ultrastructure and ribosomal arrangements of the Pleuropneumonia-like organism A5969. J. Cell Biol. 25: Morowitz, H. J., H. R. Bode, and R. G. Kirk The nucleic acids of Mycoplasma. Ann. N.Y. Acad. Sci. 143: Razin, S., and B. J. Cosenza Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope. J. Bacteriol. 91: Robertson, J. D Unit membranes, p In M. Locke (ed.), Cellular membranes in development. Academic Press Inc., New York. 12. Zucker-Franklin, D., M. Davidson, and L. Thomas The interaction of Mycoplasmas with mammalian cells. I. HeLa cells, neutrophils, and eosinophils. J. Exp. Med. 124: