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together, the data support the hypothesis that the halophilic isolates from subterranean salt deposits may be the remnants of populations which inhabited ancient hypersaline seas; in addition, they provide strong evidence against the notion that the recovered strains could be the result of laboratory contamination, since the isolates were obtained independently from different locations.

Besides isolation of viable haloarchaea, PCR procedures were applied to rock salt from Bad Ischl, Austria. The salt was dissolved under sterile conditions, filtered through membranes (pore size 0.22 |im) and the residue on the filter was subjected to DNA extraction (Radax et al., 2001). Nucleotide primers, which were specific for archaeal 16S rRNA genes, yielded a single PCR product of the expected size, following separation of molecules by agarose gel electrophoresis. The purified haloarchaeal PCR products of several independent extractions of DNA from rock salt were used for the construction of clone libraries (Radax et al., 2001). Phylogenetic analysis revealed several clusters; about 37% of approximately 130 analysed clones had sequence similarity values of less than 95% when compared with established haloarchaeal species and the remaining clone sequences were 98-99% similar to isolates from rock salts of various ages, including cultured haloarchaea from British, Polish and Thai salt mines and with uncultured phylotypes (Stan-Lotter et al., 2004; Radax et al., 2001; McGenity et al., 2000). In a similar

Figure 3. Localisation of pre-stained haloarchaea in fluid inclusions of artificial halite. Cells were stained with the LIVE/DEAD BacLight kit prior to embedding. Low (left panel) and high (right panel) magnification of Halobacterium salinarum NRC-1 cells. Cells were observed with a Zeiss Axioskope fluorescence microscope.

Figure 3. Localisation of pre-stained haloarchaea in fluid inclusions of artificial halite. Cells were stained with the LIVE/DEAD BacLight kit prior to embedding. Low (left panel) and high (right panel) magnification of Halobacterium salinarum NRC-1 cells. Cells were observed with a Zeiss Axioskope fluorescence microscope.

experimental approach, using halite samples ranging in age from 11 to 425 millions of years, Fish et al. (2002) found haloarchaeal sequences and, in the older samples, also evidence for bacterial 16S rRNA genes which were related to several bacterial genera. These data suggested the presence of a probably very diverse microbial community in ancient rock salt.

During simulation of halite formation in the laboratory by drying salty solutions which contained microorganisms, the cells accumulated within small fluid inclusions (Fig. 3). The haloarchaea can be prestained with the fluorescent dyes of the LIVE/DEAD kit (Fendrihan et al., 2006), which indicated the viability status of a cell; this procedure also greatly improved the visualization of cells within crystals. The fluid inclusions were square or rectangular, as is common in the rectangular mineral halite, and the cells were rather densely packed within the fluid-filled spaces. From such experiments it appeared that the cells always accumulated in the fluid inclusions; there were no stained cells within the mineralic halite (Fig. 3).

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