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 40 Ca-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.The 2001 anthrax attacks in the United States have increased interest in developing analytical methods for determining the source of biological materials. In this regard, there is clear evidence that the chemistry of bacterial spores reflects their growth history. Whiteaker et al. (34) were able to distinguish spores grown on blood agar by detecting heme groups on the spore surfaces by matrix-assisted laser desorption ionization mass spectrometry. Horita and Vass (13) and KreuzerMartin et al. (16,17) have shown that the stable isotope signatures of bacteria reflect those of the medium in which they grew. Here we explore a method capable of distinguishing the growth media in which spores of the Bacillus anthracis surrogate Bacillus subtilis were prepared based on the elemental signature of the dried spores.Metals associate with bacteria in several fundamental ways. Major constituents of the culture medium can be incorporated into cells to maintain osmotic and ionic balance, and trace metals can be assimilated as part of enzymatic cofactors. Metals may also be adsorbed to surfaces of cells during any part of an organism's growth history (3, 6) or be precipitated as a consequence of bacterial metabolism (4). In addition, B. anthracis and other spore-forming bacteria accumulate metals in the spore core, complexed to dipicolinic acid, during the sporulation process (21,29,30). This process is apparently correlated to heat resistance (10,15,26), and this phenomenon has led to interest in the metal content of bacterial spores related to food safety, general survivability in the environment, and astrobiology (1,15,18,(20)(21)(22)(23).The first report directly indicating that Bacillus spore metal content could be altered by the metal content of the growth medium dates back to 1959 (27). Under normal conditions, Ca is by far the most abundant metal found associated with the spore, potentially making up more than 3% of the spore weight (21). It is localized primarily in the spore core and to a lesser ext...