Environmental Microbiology (2003) 5(11), 1094–1102 doi:10.1046/j.1462-2920.2003.00509.x © 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2920Society for Applied Microbiology and Blackwell Publishing Ltd, 20035 1110941102Original Article97 k years old Halobacterium salinarum M. R. Mormile et al. Received 7 March, 2003; revised 24 June, 2003; accepted 24 June, 2003. *For correspondence. E-mail mmormile@umr.edu; Tel. (+1) 573 341 6346; Fax (+1) 573 341 4821. Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions Melanie R. Mormile, 1 * Michelle A. Biesen, 1 M. Carmen Gutierrez, 2 Antonio Ventosa, 2 Justin B. Pavlovich, 3 Tullis C. Onstott 3 and James K. Fredrickson 4 1 Department of Biological Sciences, University of Missouri-Rolla, Rolla, MO 65401,USA. 2 University of Seville, 41012 Seville, Spain. 3 Princeton University, Princeton, NJ 08544, USA. 4 Pacific Northwest National Laboratory, Richland, WA 99352, USA. Summary Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years. Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls). U-Th disequilibrium dating indicates that these halite layers were deposited at 9600 and 97 000 years before present (ybp) respec- tively. Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-spe- cific 16S rDNA primers or placed into haloarchaea culture medium. Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloar- chaea-containing prepared crystals (positive con- trols), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (nega- tive controls) resulted in no growth. Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobac- terium salinarum. DNA–DNA hybridization experi- ments confirmed that BBH 001 was closely related to H. salinarum (81–75% hybridization) and its ascription to this haloarchaea species. The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites. Introduction A variety of microorganisms have been isolated from salt deposits by enrichment of aseptically crushed halite crys- tals (Dombrowski, 1960; Norton et al., 1993). Rigorous surface sterilization and controls (Norton et al., 1993) pre- cluded the possibility of laboratory contamination and demonstrated that the isolates were endolithic. Bulk sam- pling methods cannot determine, however, whether cul- tured microorganisms were derived from primary or secondary fluid inclusions or from intercrystalline grain boundaries or intracrystalline fractures. Incontrovertible claims of antiquity can only be made for halophiles iso- lated from single phase, primary fluid inclusions in radio- metrically dated salt crystals. Consequently, assertions of viable ancient halophiles (Dombrowski, 1960; Norton et al., 1993) have been difficult or impossible to verify. Even with the rigorous surface sterilization procedures and use of sterile drill bits to breach brine inclusions (Vreeland et al., 2000), there have been a number of concerns expressed over the integrity of the crystal stud- ied (Hazen and Roedder, 2001) and the lack of genetic differences between modern and ancient microorganisms (Graur and Pupko, 2001; Nickle et al., 2002). As a means of achieving greater specificity in the isolation of endolithic haloarchaea from brine inclusions, this report presents the development, evaluation, and application of a method for extracting microorganisms from single phase, pri- mary fluid inclusions, while under direct microscopic surveillance. The impetus for the development of this in situ extrac- tion approach is twofold. First, it aims to reduce the opportunity for biological contamination by bacteria intro- duced into the crystal structure via microscopic fissures. For bulk crushed salt samples, crystals must be immersed in sterilization and wash solutions several times for surface sterilization before enrichment. This induces dissolution of the salt and potentially kills tar- geted endolithic microorganisms in addition to external microorganisms. Drilling into the interior of salt crystals with a sterile drill bit, and extracting bacteria directly from fluid inclusions, reduces the possibility of contamination by exogenous bacteria. Second, the in situ extraction approach offers enhanced specificity over the bulk sam- ple crushing technique. In situ extraction enables the isolation of microscopically detected cells or cell-like par- ticles from primary fluid inclusions as opposed to har-