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Representatives of the genus Pyrodictium have been isolated from marine hydrothermal systems at Vulcano, Italy, and the Kolbeinsey ridge north of Iceland and from deep-sea "black smoker" samples at the Guaymas basin (Mexico) and the Mid-Atlantic ridge (TAG site) (9, 12). Pyrodictium strains grow at temperatures between 80 and 110°C at neutral pH under strictly anaerobic conditions. By their mode of metabolism, members of Pyrodictium are sulfidogenic facultative chemolithoautotrophs (9, 14). Pyrodictium is unique due to the formation of a network of hollow cannulae, about 25 nm in diameter, in which the cells are embedded (10, 11). Due to their small diameter, the cannulae are invisible under the regular phase-contrast microscope. Only groups of cells with constant distances between each other can be observed (12). By using a special dark-field microscope equipped with a 500-W xenon lamp (6), it was possible to visualize the network of Pyrodictium occultum (13). However, its mode of formation during cell growth was still unknown.

Other exceptional morphological characteristics in hyperthermophilic members of the domain Archaea are the "golf clubs" of members of the order Thermoproteales (15). These are protrusions which occur usually at one cell pole. They are observed during exponential growth phase at ambient (nongrowth) temperature under a regular microscopic slide. The type strain Thermoproteus tenax is a strictly anaerobic facultatively chemolithotrophic sulfidogen, which grows at temperatures between 80 and 96°C (15).

Growth and multiplication of microorganisms can be studied by time-lapse films (5, 8). Due to the extreme growth conditions needed for Pyrodictium or Thermoproteus, several specific modifications of a dark-field microscope and adaptations of the incubation unit were necessary. Here we present for the first time results of in vivo observations of growth and cell division of Pyrodictium abyssi TAG 11 and T. tenax.

Cultivation technique. P. abyssi TAG 11 was routinely cultivated as described previously (9, 12). Formate (0.1% [wt/vol]) was used as the electron donor. All in vivo growth studies were carried out in glass capillaries (1 mm [width] by 0.1 mm [height] by 100 mm [length]; Vitro Dynamics, Rockaway, N.J.), avoiding a gas phase. Thiosulfate (0.1% [wt/vol]) served as the electron acceptor (instead of elemental sulfur) to minimize light scattering in dark-field microscopy. T. tenax was grown according to reference 15 with thiosulfate (0.1% [wt/vol]) as the electron acceptor. The glass capillaries were coated with poly-L-lysine (7), to ensure cell adhesion. In an anaerobic chamber, culture media were inoculated with the corresponding organisms and used to fill the coated capillaries. Both ends of the capillaries were sealed by melting.

Microscope. For the investigations, an Olympus BX 50 microscope was placed inside a heatable polyacrylate chamber (Fig. 1). For phase-contrast microscopy, heatable 40× and 100× phase-contrast objectives (UPLFL40× PH/0.75 and UPLFL100× PH/1.25, respectively) were used, and a halogen lamp (100 W) served as the light source. To ensure high light intensity during dark-field observation, a 100-W mercury lamp was mounted in combination with UV absorption filters (GG series; Schott, Mainz, Germany). In addition, an electromagnetic shutter was used to minimize damage to the organisms caused by high light intensities (data not shown). Both the objective (UPLFL100×; 0.60 to 1.30) and the oil-immersion condenser were heated. Furthermore, a heatable stage was designed to ensure a constant temperature in the capillaries up to 98°C (±0.2°C). A charge-coupled device video camera with image integration (PCO, Kelheim, Germany) and a video capturing board (DC20; Miro Computer Products, Braunschweig, Germany) were used for video recording and frame grabbing. The data were processed with the Adobe Premiere 4.2 software package and finally transferred onto an S-VHS videotape. For a two-dimensional reconstruction of cell division and cannula development, images were processed with Corel Photopaint, Corel Draw, and Macromedia Extreme 3D.

View larger version (58K): [in this window] [in a new window]   FIG. 1.   Scheme of the microscope including heatable polyacrylate chamber and video camera. CCD, charge-coupled device.

Cell division and growth of cannulae of P. abyssi TAG 11. For our observations, a single cell in a capillary was selected. For about 110 to 115 min (temperature = 90°C), the cell diameter increased. During this time, one or more cannulae developed, usually forming loops with both ends attached to the cell surface, although the direct observation of insertion points was not possible. Within 2.5 min, the cell divided into two daughter cells (data not shown). After fission, both cells were connected by the cannulae (Fig. 2f and 2g, black arrow). About 2 h later, the next cell division, done nearly simultaneously by both daughter cells, took place. By elongation of the cannulae, the daughter cells increased their distance up to 30 to 150 µm. In addition, cannulae with only one insertion point were found (Fig. 2d and 2e, white arrow). Although the "free" end of such a cannula was often attached to the capillary surface, it is not clear if this attachment is artificial (e.g., as a result of the poly-L-lysine coating) or is a real function of the cannulae (3). From the experimental observations, growth at the proximal end of the cannulae can be inferred (data not shown). The elongation of the cannulae was determined to be between 1.0 and 1.5 µm/min, which was significantly faster than those of bacterial flagella (e.g., Salmonella: 0.16 µm/min, in vitro measurement [1]). In contrast, for eucaryotic microtubules (e.g., Xenopus eggs) rates of up to 20 µm/min were determined in in vitro experiments (2).

View larger version (139K): [in this window] [in a new window]   FIG. 2.   Cell division of P. abyssi TAG 11 and growth of cannulae. Frames were extracted from interval recording; for details, see the text. White arrows indicate cannulae with one insertion point and one free end; black arrows indicate cannula loops. Scale bar, 10 µm.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Two-dimensional reconstruction of cell division of P. abyssi TAG 11 and development of the network. Frames a to h were calculated from the data of Fig. 2a to 2h. White arrows indicate cannulae with one insertion point and one free end; black arrows indicate cannula loops.

Cell division of T. tenax. The cell division of T. tenax was observed by phase-contrast microscopy at a magnification of ×400 at a temperature of 85°C. In comparison to cultivation in serum bottles, the doubling time increased about twofold (up to 3.5 h) during growth in capillaries. After a cell of T. tenax had elongated to a final length of up to 10 µm (Fig. 4a), usually cell division initiated by an intensive vibration of the cell for about 2 min. Within a few seconds, the cell snapped off in the center (angle, 90 to 135°; Fig. 4b). As a result, the two daughter cells are arranged in a V shape, very similar to the form of cell groups commonly observed in cultures of coryneform bacteria (4). For this group of high-GC gram-positive bacteria, the "snapping postfission movement" or "snapping division" is very characteristic and is even used as a taxonomic feature (4). So far, it has not been described for members of the Archaea, and further investigations are necessary to check its distribution within this domain. In T. tenax, 2 to 5 min after snapping, the two daughter cells separated visually (Fig. 4c). For the next 3 h, they elongated again (Fig. 4d and 4e), followed by the next cell fission, lasting again only a few minutes (Fig. 4f, 4g, and 4h).

View larger version (88K): [in this window] [in a new window]   FIG. 4.   Cell division of T. tenax. Frames were obtained from time-lapse film. Scale bar, 20 µm.

Conclusions. In general, the combination of the modified microscope and culture technique turned out to be a powerful tool to study growth and cell division of anaerobic microorganisms up to temperatures of 98°C. Further applications can include motility of mesophilic to (hyper)thermophilic organisms or investigations with unicellular eucaryotes like flagellates or amoebae.