appearance of the irregular spheres isolated from SW1 and SW4 resembled Archaea, but more information was essential to determine their taxonomic status. In some instances, particularly where heavy metals may be detrimental to DNA integrity, it may not be possible to obtain amplifiable DNA, or if DNA is obtained it may not sequence properly. If it is not possible to obtain DNA for identification from cultures, fluorescence in situ hybridization (FISH) is used to identify the taxonomic group. An example of this approach is given in Figure 1.7. The controls include DAPI stain, to identify Bacteria and Archaea, a nonsense sequence probe (NON338) and no probe added. Both samples and controls were processed identically and at the same time. Negative results were obtained with the controls and indicated that if the Archaea-specific Arch915 probe labeled cells, the labeling when positive indicated the cells were Archaea. In this sample from a culture of mixed rods and spheres only the spheres were labeled by the probe, indicating that they were members of the taxonomic group Archaea, although no further information on their taxonomy is available from this direct labeling technique.
Figure 1.6 Sulphur Works iron vent SW4 culture, pH 4.5, 80 °C. (a) Phase contrast. Silica, iron and 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
