ferruginea. At the present time four species of

Transcription

1 ELECTRON MICROSCOPY OF GALLIONELLA FERRUGINEA' A. E. VATTER AND R. S. WOLFE Electron Microscope Laboratory and the Department of Bacteriology, University of Illinois, For over 80 years prior to the work of Cholodny (1924, 1926) the twisted ribbons of ferric hydroxide found in iron-bearing waters were described as living organisms of the genera Gallionella and Spirophyllum. These early observations have been conveniently summarized and evaluated by Pringsheim (1949), so that at the present time Spirophyllum may be considered synonymous with Gallionella. It remained for Cholodny (1924) to reveal that these twisted ribbons of ferric hydroxide were actually fragments of inert stalks which had been secreted by bean-shaped apical cells. By allowing the organism to grow on cover-slips which had been immersed in appropriate natural waters it was possible to observe for the first time the intact cell and stalk. His excellent photographs have established the general morphology of Gallionella ferruginea. At the present time four species of Gallionella described from natural sources are recognized, but as suggested by Pringsheim (1949) perhaps these descriptions represent but a single species. Van Beneden (1951) has questioned Cholodny's interpretation of stalk secretion and has suggested that the twisted stalk represents a collapsed sheath from which a chain of cells has escaped. More recently, Berger and Bringmann (1953) have presented electron micrographs of stalk fragments from natural deposits of iron bacteria which suggest a finer netlike or fiber structure within the stalk, but much of the detail is obscured possibly because of inorganic chemical precipitation in the natural habitat. The present report is an extension of the morphological observations on Gallionella ferruginea and represents, in contrast to previous observations, the results of a study of seriallycultivated laboratory strains of the organism. The details of the cultivation method will be presented in a future communication. A brief report of this method and some of the morpho- 1 This work was supported in part by the National Science Foundation, Grant G1818. Received for publication January 27, 1956 Urbana logical observations have appeared (Vatter and Wolfe, 1955). MATERIAL AND METHODS Sessile colonies of Gallionela ferrupnea obtained by cultivation of the organism in a mineral salts medium were washed to remove salts of the medium prior to preparing grids for electron microscopy. Colonies to be washed were picked using a capillary pipette and placed in distilled water. Each colony was then picked up again in a like manner and deposited on a previously prepared carbon-coated, collodion membrane supported by a conventional wire grid. The excess water surrounding the colony was removed by careful blotting so that the preparation was air-dried quickly. Fixation was not employed. An alternative procedure for washing the colonies was also used. Here the colonies were allowed to remain in their original position in the culture tubes as the medium was removed and replaced by distilled water. Appropriate colonies were then picked and deposited upon grids as described above. Preparations to be metal shadowed were placed in a conventional shadowing apparatus and shadowed with chromium-gold alloy. Electron micrographs were made at magnifications from 1,000 to 20,000 X using an RCA- EMU-2E electron microscope. R;EULTS Structure of the 8talks. When the electron beam was first projected on a preparation of Gallionella ferrgnea in the electron microscope, it was immediately obvious that the stalks were composed of definite, linear, electron-opaque strands as illustrated in figures 1, 2, 6, and 7. As many as 80 of these strands have been observed in a single stalk. At high magnifications the individual strands which compose the stalk exhibited a segmented appearance (figures 3, 4, and 5). Since this pattern has been noticed in all preparations and was not observed to change in strands which were exposed to varying beam intensities, 248

2 rnr - ~ ~~~~~~~~- IN ~ :l..x.i -M. 3,_M.. _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. Figures 1-7 Figure 1. A stalk of Gallionella showing its filamentous and twisted nature. The mass of material near the center of the stalk is probably ferrous sulfide precipitate; X Figure 2. A negative print of a metal shadowed section of stalk showing the individual strands; X 25,000. Figures 3, 4, and 5. High magnification of the individual strands showing the segmented, secretion pattern; X 62,000. Figure 6. A large stalk secreted by several cells; X 6,000. Figure 7. Divided stalk showing terminal "ghost-like" cells; X 5,000. The line in each figure represents 1 micron unless otherwise indicated. 249

3 250 VATTER AND WOLFE [VOL I '13 W4 Figures 8-14 Figure 8. Transparent "ghost-like" cell of Gallionella exhibiting sparse granulation; X 25,000. Figures 9, 10, and 11. Dense, granular cells; X 27,000. Figure 12. End of a stalk showing terminal enlargements of the strands; X 50,000. Figure 13. Membrane-like cells illustrating multiple stalk secretion; X 28,000. Figure 14. An elongated cell apparently prior to division; X 12,000. The line in each figure represents 1 micron unless otherwise indicated.

4 1956] ELECTRON MICROSCOPY OF G. FERRUGINEA 251 it is not believed to be an artifact of electron microscopy. The fragile, brittle nature of these strands, evidenced in figure 7, frequently made observations difficult. As first noted by Cholodny (1924) single stalks were secreted by several cells (figure 6), or as illustrated in figure 7, bifurcated stalks were formed by separation of the cells. Attempts to demonstrate material other than the strands in the stalk were unsuccessful. Cell structure. As previously observed by Cholodny (1924) the cells of Gallionellaferruginea were found to be very fragile, being easily lost. The majority of stalks observed in the electron microscope possessed no cells; washing of the colonies to remove salts of the medium resulted in the separation of most cells from the stalks. The detachment of cells was most pronounced in old colonies. Two general types of cells were observed in our preparations. One type shown in figures 11 and 14 was opaque to the electron beam, whereas the other type shown in figures 8 and 13 was less dense, resembling a cell membrane or ghost. Metal shadowed preparations indicated that the latter type of cell was essentially flat, whereas the denser cells were not collapsed. Figures 9 and 10 exhibit cells intermediate between these two extremes. Occasionally, as illustrated in figure 10, the granules were observed to possess flat adjacent sides. An opaque cell prior to division is illustrated in figure 14. Stalks from which the cells had been separated usually ended abruptly with no trace of cell attachment, but in some instances the strands exhibited terminal enlargements suggesting either a transition between strand and granule or a means of stalk attachment to a solid substratum. DISCUSSION Many of the early descriptions of Gallionella ferruginea (summarized by Pringsheim, 1949), such as fraying and thickness at the edges of the stalks, may be easily explained on the basis of the stalk structure illustrated here. Likewise, the occurrence of very large stalks in natural deposits may be explained as a separating of the individual strands of the stalk with an accompanying chemical precipitation of ferric iron common in iron-bearing waters. With these factors operating in the natural environment it is possible to visualize the formation of the bizarre stalk-fragments found in natural deposits containing Gallionella ferruginea. Since the width of an individual strand in a stalk remains approximately constant and has not been found on edge in micrographs, it may be inferred that the individual strands tend to be round. The mechanism causing twisting of the stalk requires additional study, for the micrographs of cells and stalks have not revealed a mechanism which explains this characteristic. On the other hand, we have made no observations which could support the unconventional hypothesis of van Beneden (1951) that the "stalks" are formed by a spiral chain of bacilli which after secreting a mucilaginous covering escape and leave behind the twisted "stalk." It is possible to interpret the gradation in density among the observed cells as representing cells in various stages of decomposition. The extreme flatness of the nongranulated cells presented in the electron micrographs stands in sharp contrast to nonfixed, air-dried cells examined by light microscopy, where the refractility of the cells gives a definite sense of depth to the cell. For this reason we have considered that the dense cells represent the true morphology of Gallionella ferruginea. Possibly the granules are secreted within the cell and are related to the segmented strands of the stalk. However, they may be entirely external to the cytoplasm, and may even represent a nonbiological chemical precipitation which in an unexplained way is selective for the cell surface. ACKNOWLEDGMENTS The authors wish to express appreciation to Dr. C. B. van Niel for his guidance in the initial experiments, and to Miss Sonia Kucera for assistance in culturing the organism. SUMMARY The stalks of Gallionella ferruginea were found to consist of definite linear strands which varied in number per stalk and which exhibited a segmented pattern. Dense cells possessing electron opaque granules as well as membrane-like or "ghost" cells were observed. Possible interpretations of these morphological features are discussed. REFERENCES BERGER, H. AND BRINGMANN, G Bisherige Anschauung uber die Morphologie von Gallionella und neuere elektronen-

5 252 VATTER AND WOLFE [VOL. 72 mikroskopische Befunde. Zentr. Bakteriol. Parasitenk., Abt. II 107, CHOLODNY, N Zur Morphologie der Eisenbakterien, Gallionella und Spirophyllum. Ber. deut. botan. Ges., 42, CHOLODNY, N Die Eisenbakterien, Beitrage zu einer Monographie. Pflanzenforschung Heft 4. Kolwitz, Verlag Gustav Fischer, Jena, Germany. PRINGSHEIM, E. G Iron bacteria. Biol. Revs., Cambridge Phil. Soc., 24, VAN BENEDEN, G Contribution nouvelle A l'etude des ferrobact6riac6es. Hydrobiol., 3, VATTER, A. E. AND WOLFE, R. S Observations on the growth and morphology of Gallionella ferruginea. Bacteriol. Proc. 1955, 35.