minerals form a heterogeneous base for cell growth in culture. Arrowheads (a,b) indicate same cell location. (b) DAPI stain. Cells appear as blue fluorescent spheres mainly surrounding edges of steam deposits. (c) Phase contrast. Faint red tinged siliceous particle with few cells seen, other than at lower right edge. (d) DAPI stain. Same small cell group evident at lower right edge in phase contrast and DAPI images; blue DAPI stained cells, not seen by phase contrast, appear dispersed throughout the deposit particle. Bar A–D, 5 µm. (Image credit: the authors).
Figure 1.7 FISH labeled culture pH 4.5, 80 °C, isolated from iron vent, Sulphur Works SW4. (a) Phase contrast. (b) DAPI stain. (c) Fluorescent image of cells labeled with Archaea-specific probe Arch915-CY3 probe. Labeled cells were all bright orange archaeal spheres. None of the rod-shaped bacterial cells (A,B, arrowhead) were labeled by the probe (c). Only spherical Archaea were fluorescent. Arrowheads (a-c) mark location of a typical bacterial rod nonlabeled in C. Bar A–C, 5 µm. (Image credit: the authors).
A salt cave liquid enrichment, Hawai’i H5, was obtained at pH 4.5, 55 °C. The culture was a mixture of thin filaments, wide rods about 0.5 µm in diameter, and pleomorphic cells. Further work with more selective conditions (ionic species, pH, temperature) and solid surface isolations may assist in obtaining pure cultures of the organisms present.
1.5 Cell Structure of Isolates
For scanning electron microscopy, cells fixed in glutaraldehyde and osmium were examined in an FEI Quanta 450 SEM. Images were recorded digitally. When individual cells were observed in the light microscope and cells tumbled, they appeared as irregular spheres. Similarly, in the scanning electron microscope cell isolates showed a generally irregular spherical SEM appearance (Figure 1.8). Most cells were less than 1µm in size and the nonsulfur cave isolate SW1 appeared as large (Figure 1.8a) and small groups (Figure 1.8a, insets). Paired and single cells more typically were seen in light microscope images (Figure 1.4a, b). The SW4 iron vent cells grown at pH 3 were highly concentrated and readily seen in SEM images as paired or single cells (Figure 1.8b). The cells appeared to be irregular spheres. At higher magnification, irregular ridges and depressions were prominent (Figure 1.8b, inset). This can be typical of later stages of growth when competition for food, and increased surface to volume ratios exist. The origin of ridges and depressions remains uncertain and requires further study to eliminate the possibility of artifact resulting from sample preparation. In the field of view shown here, the iron vent SW4 isolate had a few cell pairs that appeared to be held together by thin cell-to-cell bridges, presumably resulting from cell division. Though somewhat difficult to discern, one example marked by an arrowhead, is more readily visible in the inset. The thin cell bridge measured ~0.07 × 0.5 µm and tapered at both ends where it connected to cells. We did not determine that cells were motile as no flagella were seen, though some Archaea with flagella are known to be motile [1.9].
Figure 1.8 Scanning electron microscope images of steam vent isolates. (a) Cell cluster from nonsulfur steam cave SW1 with irregular spheres grown at pH 4.5, 80 °C. Left inset, SW1 phase contrast; right inset, SW1 DAPI stain. (b) Archaea grown from iron vent SW4 appear in the SEM mainly as pairs and single cells; arrowhead marks inset cell pair. Inset, ridges, depressions, and thin cell-to-cell connections appear. Cells grown at pH 3, 80 °C. Bar A, 3 µm. Bar B, 10 µm. Bar insets A (phase-DAPI stain), 5 µm. Bar inset B, 1 µm. (Image credit: the authors).
It seemed unusual that the two new Lassen isolates grew only at 80 °C, and not above. Yet, the habitat temperatures for nonsulfur cave (SW1) and iron vent (SW4) were 87 and 85.5 °C, respectively. Furthermore, in prior years the habitat temperatures were both above 90 °C. Temperature drops off quickly even at short distances away from the cave or vent opening. Thus, locations providing gases, nutrients or energy sources may not lie directly in the path of venting steam, possibly explaining the discrepancy between the higher environmental temperature and the lower isolation temperature. It may also be the case, as reported for hot springs [1.5], that different temperature strains exist within natural populations bridging a broader range of temperatures than that of the isolates obtained in our study.
1.6 Environmental Models
Models of the Lassen SW4 iron-oxidizing environment and the Hawai’i H5 salt cave environment are presented below (Figure 1.9 and 1.10) to provide an overview of these habitats.
Figure 1.9 Iron-oxidizing environment. Heated rainwater from magmatic heat convection produces steam that contacts lava and extracts soluble Fe(II). (1) Fe(II) is solubilized in the steam water and (2) rises with steam through porous lava fissures and cracks and reaches the iron vent openings of SW4, as the Fe(II) contacts air (3) Archaea utilize the Fe(II) in their growth by oxidizing the iron to Fe (III). Air oxidation also takes place and adds Fe(III) to the iron-rich layer formed on the solid rocks within the iron vent. (Image credit: the authors).
Figure 1.10 Salt cave environment. (1) Rainwater recharge of the Hawaiian ground water passes slowly through the porous lava rich ground. Fractured lava is heated by the underlying magma and as (2) rainwater descends through crevices and fissures