Bacteria have been found associated with a variety of ancient samples, however few studies are generally accepted due to questions about sample quality and contamination. When Cano and Borucki isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25-30 million-year-old amber, careful sample selection and stringent sterilization techniques were the keys to acceptance. Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2-9-3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation. Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus. Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation. Samples were rejected if brine inclusions showed physical signs of possible contamination. Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions. Sterilization procedures reduce the probability of contamination to less than 1 in 10(9).

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A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%-93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2-9-1(T) (= ATCC BAA-75 and JCM 11222) as written in the formal description.

Microorganisms have the ability to adapt to a wide range of NaCl concentrations. In general the NaCl tolerance shown by microbes far exceeds the salt tolerance of any other organism, procryote or eukaryote. There are at least three mechanisms available for adaptation to different salt concentrations. The first would be a passive one in which the cytoplasmic ion content would always equal that in the medium. A second mechanism which is used by many organisms involves concentrating compatible solutes to create an osmotic balance between the cytoplasm and the external environment. The third mechanism involves changing the cell physiology to control the movement of water allowing the cell to exist with an ionically dilute cytoplasm. This article will review the major developments and discuss the implications of increasing knowledge about salt tolerance in microorganisms.

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Following Recommendation 30b of the Bacteriological Code (1990 Revision), a proposal of minimal standards for describing new taxa within the family Halomonadaceae is presented. An effort has been made to evaluate as many different approaches as possible, not only the most conventional ones, to ensure that a rich polyphasic characterization is given. Comments are given on the advantages of each particular technique. The minimal standards are considered as guidelines for authors to prepare descriptions of novel taxa. The proposals presented here have been endorsed by the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Halomonadaceae.

Recent reports have described the isolation and analysis of living microbes and/or DNA fragments from halite crystals of significant geological age. This manuscript describes the isolation of six living strains of halophilic Archaea from Cretaceous (121-112 MYA) halite crystals. These 6 live strains represent the oldest Archaea isolated to date. This manuscript also presents the first isolation of representatives from two different archaeal genera in a single event. The data presented show that the organisms that inhabited these hypersaline environments today are similar to those present during the Cretaceous age. Considering the number of ancient samples that have now yielded living microbes or DNA fragments the evidence for long-term survival of microbes (at least within halite) is becoming increasingly definitive. While there are obviously still other trapped microbes to find, it may now be time to begin investigating the implications of these ancient microbes and the mechanisms that foster long-term survival.

An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism, designated strain PR5 T , is rod-shaped, non-motile and requires at least 12 % (w/v) NaCl to grow. The strain is highly thermotolerant : its temperature optimum is 50 SC and growth is possible up to 60 SC. Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S 2 -DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. Both C 20 ,C 20 and C 20 ,C 25 core lipids are present. The GMC content of the DNA is 63 3 mol %. According to 16S rDNA sequence data, strain PR5 T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5 T cannot be assigned to any of the recognized species within these genera. On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena. The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate. The type strain is strain PR5 T (l DSM 11552 T l ATCC 700275 T ).