Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal


Bacteria have been found associated with a variety of ancient samples1, however few studies are generally accepted due to questions about sample quality and contamination. When Cano and Borucki2 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 109.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Halite crystals taken from the dissolution pipe at the 569 m (1850 ft) level of the Salado Formation (air intake shaft18 for the Waste Isolation Pilot Plant, Carlsbad, New Mexico) in October 1998.
Figure 2: Phylogenetic tree developed from the 16S rDNA sequence of unknown 2-9-3 compared with several other species of the family Bacillaceae.

Similar content being viewed by others


  1. Grant, W. D., Gemmell, R. T. & McGenity, T. J. Halobacteria: the evidence for longevity. Extremophiles 2, 279–287 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Cano, R. J. & Borucki, M. Revival and identification of bacterial spores in 25 to 40 million year old Dominican amber. Science 268, 1060–1064 (1995).

    ADS  CAS  PubMed  Google Scholar 

  3. Denner, E. B. M. et al. Halococcus salifodinae sp. nov., an archaeal isolate from an Austrian salt mine. Int. J. Syst. Bacteriol. 44, 774–780 (1994).

    Article  Google Scholar 

  4. Huval, J. H. & Vreeland, R. H. in General and Applied Aspects of Halophilic Bacteria. Vol. 201 (ed. Rodriguez-Valera, F.) 53–62 (Plenum, New York, 1991).

    Google Scholar 

  5. Norton, C. F., McGenity, T. J. & Grant, W. D. Archaeal halophiles (halobacteria) from two British salt mines. J. Gen. Microbiol. 139, 1077 –1081 (1993).

    Article  CAS  Google Scholar 

  6. Greenblatt, C. L. et al. Diversity of microorganisms isolated from amber. Microb. Ecol. 38, 58–68 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Lambert, L. H. et al. Staphylococcus succinus sp. nov., isolated from Dominican amber. Int. J. Syst. Bacteriol. 48, 511– 518 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Vreeland, R. H. & Powers, D. W. in Microbiolology and Biogeochemistry of Hypersaline Environments (ed. Oren, A.) 53–74 (CRC, Boca Raton, Florida, 1999 ).

    Google Scholar 

  9. Vreeland, R. H. & Rosenzweig, W. D. in Enigmatic and Extreme Microorganisms. (ed. Seckbach, J.) 387– 398 (Kluwer, Delft, 1998).

    Google Scholar 

  10. Croft, J. S. Upper Permian conodonts and other microfossils from the Pinery and Lamar Limestone Members of the Bell Canyon Formation and from the Rustler Formation, west Texas. Thesis, Ohio State Univ. (1978).

  11. Walter, J. C. Paleontology of the Rustler Formation, Culberson County, Texas. J. of Paleontol. 27, 679–702 (1953).

    Google Scholar 

  12. Renne, P. R., Steiner, M. B., Sharp, W. D., Ludwig, K. R. & Fanning, C. M. 40/39 Ar and U/Pb SHRIMP dating of latest Permian tephras in the Midland Basin Texas. EOS 77, 794 (1996).

    Google Scholar 

  13. Renne, P. R., Sharp, W. D. & Becker, T. A. 40Ar/39Ar dating of langbeinite [K2Mg2(SO4)3] in late Permian evaporites of the Salado Formation, Southeastern New Mexico, USA. Mineral. Mag. 62A, 1253– 1254 (1998).

    Article  ADS  CAS  Google Scholar 

  14. Hardie, L. A., Lowenstein, T. K. & Spencer, R. J. The problem of distinguishing between primary and secondary features in evaporites. Sixth Int. Symp. On Salt 1, 11–39 (1983).

    Google Scholar 

  15. Roedder, E. The fluids in salt. Amer. Mineral. 69, 413 –439 (1984).

    CAS  MATH  Google Scholar 

  16. Lowenstein, T. K. & Hardie, L. A. Criteria for recognition of salt-pan evaporites. Sedimentol. 32, 627–644 (1985).

    Article  ADS  CAS  Google Scholar 

  17. Lowenstein, T. K. Origin of depositional cycles in a Permian “saline giant”: the Salado (McNutt zone) evaporites of New Mexico and Texas. Geol. Soc. Am. Bull. 100, 592–608 (1988).

    Article  ADS  CAS  Google Scholar 

  18. Holt, R. M. & Powers, D. W. Geological mapping of the air intake shaft at the Waste Isolation Pilot Plant. Report no. DOE/WIPP 90-051, 1–90 (U. S. Department of Energy, Carlsbad NM, 1990).

  19. Lowenstein, T. K. & Spencer, R. J. Syndepositional origin of potash evaporites: petrographic and fluid inclusion evidence. Am. J. Science 290, 1–42 (1990).

    Article  ADS  CAS  Google Scholar 

  20. Holt, R. M. & Powers, D. W. in Geological and Hydrological Studies of Evaporites in the Northern Delaware Basin for the Waste Isolation Pilot Plant (WIPP), New Mexico (eds Powers, D. W., Holt, R. M., Beauheim, R. L. & Rempe, N.). Geol. Soc. Am. Guidebook 14, 45–78 (1990).

    Google Scholar 

  21. Powers, D. W. & Hassinger, B. W. Synsedimentary dissolution pits of halite of the Permian Salado Formation, southeastern New Mexico. J. Sed. Petrol. 55, 769–773 (1985).

    Google Scholar 

  22. Onstott, T. C., Mueller, C., Mikulki, K., Vicenzi, E. & Powers, D. W. 40Ar/39Ar laser microprobe dating of polyhalite from bedded, late Permian evaporites. EOS 76, S285 (1995).

    Google Scholar 

  23. Vreeland, R. H., Piselli, A. F., McDonnough, S. & Myer, S. S. Distribution and diversity of halophilic bacteria in a subsurface salt formation. Extremophiles 2, 321–331 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Rosenzweig, W. D., Peterson, J., Woish, J. & Vreeland, R. H. Development of a protocol to retrieve microorganisms from ancient salt crystals. Geomicrobiol. (in the press).

  25. Vreeland, R. H., Anderson, R. & Murray, R. G. E. Cell wall and phospholipid composition and their contribution to the salt tolerance of Halomonas elongata. J. Bacteriol. 160, 879–883 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Arahal, D. R, Marquez, M. C., Volcani, B. E., Schleifer, K. H. & Ventosa, A. Bacillus marismortui sp. nov., a new moderately halophilic species from the Dead Sea. Int. J. Syst. Bacteriol. 49, 521–530 ( 1999).

    Article  PubMed  Google Scholar 

  27. Heyndrickx, M. et al. Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int. J. Syst. Bacteriol. 48, 99–106 (1998).

    Article  Google Scholar 

  28. Garabito, M. J., Arahal, D. R., Mellado, E., Marquez, M. C. & Ventosa, A. Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species. Int. J. Syst. Bacteriol. 47, 735–741 ( 1997).

    Article  CAS  PubMed  Google Scholar 

  29. Waino, M., Tindall, B. J., Schumann, P. & Ingvorsen, K. Gracilibacillus gen nov., with description of Gracilibacillus halotolerans gen. nov. sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Int. J. Syst. Bacteriol. 49, 821–831 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Cournoyer, B. & Lavire, C. Analysis of Frankia evolutionary radiation using glnII sequences. FEMS Microbiol. Lett. 177, 29–34 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references


The authors acknowledge the following people who helped obtain the crystal samples for this research: D. Belski, N. Rempe, R. Carrasco, T. Garcia, D. Acevedo, S. Britain, E. Keyser, B. Kinsall, A. Morin and T. Padilla. This research was supported by the US National Science Foundation: Life in Extreme Environments Program (EAR Lexen).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Russell H. Vreeland.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vreeland, R., Rosenzweig, W. & Powers, D. Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature 407, 897–900 (2000).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing