Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.

Figure ​Figure11 shows staining of archaea at pH extremes. S. acidocaldarius cells (Fig. ​(Fig.1b1b and c) were grown and stained at pHs 5.9 and 2.0, respectively, and Natronomonas pharaonis cells (Fig. ​(Fig.1a)1a) were grown and stained at pH 9.3; similar staining (not shown) was observed at pH 9.3 with Natronococcus occultus and at pH 2.0 with A. brierleyi, which is related to Sulfolobus (11). Since the Natronomonas and Natronococcus species had been grown in the presence of 4 M NaCl, the results suggested also that high ionic strength did not interfere with staining. This observation was corroborated with several haloneutrophilic archaea (Halobacterium sp. strain NRC-1, Halorubrum saccharovorum, Haloferax volcanii, Halococcus morrhuae), which were grown in media containing 4.0 to 4.28 M NaCl. Most haloarchaea contain in their cytoplasm high concentrations of salt, which range from 3.7 to 5.3 M KCl and 0.8 to 1.63 M NaCl (16). The LIVE/DEAD kit has been used so far in circumneutral media and solutions of low or only moderate ionic strength; the data provided here suggest its usefulness over a wide range of pHs and at high ionic strength at neutral or alkaline pH. It may be concluded that SYTO 9 penetrated cells well at alkaline pH, although its maximum fluorescence is to be expected at acidic pH (2). In summary, the dyes of the LIVE/DEAD kit permeated archaeal bilayer membranes, which contain C₂₀C₂₀ or, in the case of haloalkaliphiles, additional C₂₀C₂₅ diethers (9), as well as monolayer membranes from thermoacidophiles, which contain C₄₀C₄₀ tetraethers (13) and are notoriously low in permeability (5, 14, 29).

Haloarchaeal species, except halococci, are lysed and killed by placement in distilled water (9). This property was used for an evaluation of the reliability of the LIVE/DEAD kit. Unambiguous results were obtained with Halobacterium sp. strain NRC-1, which exhibited red fluorescence following incubation in water for 5 min (Fig. ​(Fig.2);2); no CFU were detected on agar plates, which were incubated for up to 4 weeks. Longer exposure to water (>5 min) caused complete disintegration of the cells, with the appearance of string-like structures and debris (not shown). Halococci were lysed only to a small extent when they were placed in water for up to 60 min, since 85 to 88% (of 750 counted cells) of H. dombrowskii H4 cells retained their green fluorescence and were still culturable (not shown). The results suggested that the membranes of noncoccoid haloarchaea became permeable within minutes upon exposure to water and that the resulting nonviability was readily apparent by the changing fluorescence of cells stained with the LIVE/DEAD kit, in contrast to halococci, which were rather resistant to the action of water. Halococci (H. dombrowskii H4, H. morrhuae, and H. salifodinae BIp) were permeabilized by treatment with 60% ethanol and stained with DAPI (18) afterwards, which preserved well their morphological features; subsequent staining with the LIVE/DEAD kit produced only red fluorescence and no culturable cells, indicating loss of membrane intactness. The LIVE/DEAD kit thus allowed rapid evaluation of the integrity of haloarchaeal membranes and the viability of cells following exposure to lethal treatments.

Ambiguous colors like yellow or orange have sometimes been observed in bacterial cells stained with the LIVE/DEAD kit (2). In our experiments, only coccoid haloarchaea, which generally grow in clusters and aggregates, showed occasionally intermediate yellowish colors; singly growing cells of noncoccoid haloarchaea and thermoacidophiles showed unambiguous (either red or green) fluorescence.

The possibility of inhibition of haloarchaeal growth by the kit's components and DAPI was examined. Cells of Halobacterium sp. strain NRC-1 and H. dombrowskii H4, both at an optical density at 600 nm of 1.0, were incubated with the LIVE/DEAD kit at the same concentrations as used for staining (0.01 mM SYTO 9, 0.03 mM propidium iodide). The CFU counts of Halobacterium sp. strain NRC-1 on five to seven agar plates per experiment were 6.3 × 10⁸ CFU/ml ± 6.4% (coefficient of variation) after 15 min of incubation and 2.8 × 10⁸ CFU/ml ± 9.0% after 24 h of incubation with the LIVE/DEAD kit. Cells of H. dombrowskii H4 showed more than 98% viable cells following incubation with dyes for 24 h (total of 350 counted cells). These results indicate that incubation with the LIVE/DEAD kit for up to 24 h did not markedly reduce the viability of these two haloarchaeal species. In contrast, DAPI is known to inhibit the growth of Halobacterium sp. strain GRB at concentrations of a few micrograms per milliliter (7) and that of Sulfolobus (17). We found that an even lower concentration (0.014 μM) inhibited the growth of H. salifodinae BIp when cells had been exposed for 2 h to DAPI.

We have previously isolated several novel haloarchaeal species from rock salt, which is believed to have been deposited about 280 million years ago (4, 23, 25) and which contains numerous as yet uncultured strains (19). We attempted to visualize directly the presence of halophilic microorganisms in dissolved rock salt and in samples from natural hypersaline brines. Figure ​Figure33 shows several particles about 1 to 2 μm long with green fluorescence in a sample of Dead Sea water (Fig. ​(Fig.3a3a and b) that had been stored at 4°C for about 3 years and in dissolved rock salt (Fig. ​(Fig.3c3c and d) from the salt mine in Altaussee, Austria, following staining with the LIVE/DEAD kit. According to their sizes and shapes, they could represent viable halophilic prokaryotes. Autofluorescent particles were not observed. Occasionally, hazy-appearing material was detected in dissolved rock salt, which produced weak red or green fluorescence (Fig. ​(Fig.3,3, e). In the Dead Sea water, rod-shaped particles of green fluorescence, including an apparently dividing cell (Fig. ​(Fig.3b),3b), were present; some particles showed a transient yellow fluorescence (Fig. ​(Fig.3a).3a). CFU densities in this sample were between 180 and 240/ml on agar plates containing M2S medium, thus corroborating the presence of viable halophilic microorganisms. CFU densities from dissolved rock salt varied between 10 and 130 ± 20/ml (see also reference 24). A direct correlation between fluorescent particles and CFU was obtained with a confocal scanning laser microscope, which affords precise volumes of fields of view; manual counting of fluorescent cells of pure cultures of Halobacterium sp. strain NRC-1, which contained 3.6 × 10¹⁰ CFU/ml ± 11%, according to streak plates (n = 12), yielded 3.9 × 10¹⁰ cells/ml ± 9% (n = 4). Older cultures, which contained about 20% dead cells, as estimated from red fluorescence, produced correspondingly lower values (7.8 × 10⁹ CFU/ml ± 10%; n = 4). Although these data were from a rather limited number of experiments, it could be inferred that no serious underestimation of the CFU densities of viable haloarchaea had occurred.

The search for life in the solar system and beyond is a goal of several space agencies in the 21st century (6). Extraterrestrial materials might be return samples or well-characterized meteorites; some of these are known to contain halite (28, 30, 32). Fluorescent dyes, such as those in the LIVE/DEAD kit, may be suitable agents for life detection experiments because of their high sensitivity and robustness in extreme environments. In contrast to DAPI, the LIVE/DEAD kit reagents were tolerated well by haloarchaea, and poststaining cultivation of detected particles should in principle be possible. If microbial forms of life with membranes and nucleic acids similar to those found on earth exist in extraterrestrial materials, the LIVE/DEAD kit dyes are promising for use in their detection.