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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The Delaware Basin is a broad asymmetric sedimentary trough in southeastern New Mexico and west Texas. Basin subsidence occurred from the Pennsylvanian into the Triassic. The basin also underwent tilting since the early Cenozoic. Layered evaporite units of Ochoan age in the basin are 1000 m thick. These evaporites are divided into three stratigraphic units (listed in order of increasing age): the Rustler Formation, the Salado Formation, the Castile Formation. These units, especially the Castile, are deformed along portions of the margin of the Delaware Basin and in some areas internal to the basin. In the northern Delaware Basin adjacent to the WIPP site, the term "Disturbed Zone" (DZ) has been applied to an area in which deformation structures are found in boreholes and from which chaotic seismig reflection data were obtained. The origin and timing of this deformation is considered important for the determination of This docunlent is PUBLI&'L%7 RELEASABLE Authorizing Official Date: 81/31 / W O d

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