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).

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.

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.

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DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium. The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.