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Applied and Environmental Microbiology, November 2005, p. 6524-6530, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6524-6530.2005

Differentiation of Spores of Bacillus subtilis Grown in Different Media by Elemental Characterization Using Time-of-Flight Secondary Ion Mass Spectrometry

John B. Cliff,^1* Kristin H. Jarman,¹ Nancy B. Valentine,¹ Steven L. Golledge,² Daniel J. Gaspar,¹ David S. Wunschel,¹ and Karen L. Wahl¹

Pacific Northwest National Laboratory, P.O. Box 999, Battelle Blvd., Richland, Washington 99352,¹ University of Oregon, 1252 University of Oregon, Eugene, Oregon 97403²

Received 17 March 2005/ Accepted 27 June 2005

We demonstrate the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in a forensics application to distinguish Bacillus subtilis spores grown in various media based on the elemental signatures of the spores. Triplicate cultures grown in each of four different media were analyzed to obtain TOF-SIMS signatures comprised of 16 elemental intensities. Analysis of variance was unable to distinguish growth medium types based on ⁴⁰Ca-normalized signatures of any single normalized element. Principal component analysis proved successful in separating the spores into groups consistent with the media in which they were prepared. Confusion matrices constructed using nearest-neighbor classification of the PCA scores confirmed the predictive utility of TOF-SIMS elemental signatures in identifying sporulation medium. Theoretical calculations based on the number and density of spores in an analysis area indicate an analytical sample size of about 1 ng, making this technique an attractive method for bioforensics applications.

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* Corresponding author. Mailing address: Advanced Radioanalytical Chemistry, National Security Directorate, Pacific Northwest National Laboratory, MS P7-07, P.O. Box 999, Battelle Blvd., Richland, WA 99352. Phone: (509) 373-9003. E-mail: john.cliff{at}pnl.gov.

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Applied and Environmental Microbiology, November 2005, p. 6524-6530, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6524-6530.2005

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