Stable isotope ratios as a tool in microbial forensics--Part 1. Microbial isotopic composition as a function of growth medium

The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced. In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium. In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 +/- 2.0% per thousand (parts per thousand), and in nitrogen, 4.5 +/- 0.7% per thousand. We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water. A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.     

Stable isotope ratios as a tool in microbial forensics--Part 2. Isotopic variation among different growth media as a tool for sourcing origins of bacterial cells or spores

Since the anthrax attacks of 2001 the need for methods to trace the origins of microbial agents has become urgent. The stable isotope ratios of bacteria record information from both the nutrients and the water used to make their culture media and could potentially be used to provide information about their growth environment. We present a survey of carbon (C), nitrogen (N), and hydrogen (H) stable isotope ratios in 516 samples of bacteriological culture media. The observed variation was consistent with expected isotopic variation in the plant and animal products upon which the media are based. The variation is sufficient to translate into substantial isotope variation in cultures grown on different batches of media, and thus to allow investigators to determine whether seized media could have been used to produce seized bioweapons agents.     

Stable isotope ratios as a tool in microbial forensics--part 3. Effect of culturing on agar-containing growth media

Stable isotope ratios of hydrogen and oxygen in microbes have been shown to be functions of the corresponding isotope ratios of the water with which the culture medium was prepared, and thus to contain a potential geographic signal. Water can evaporate from agar (solid) media during culturing, changing its isotope ratios. Here we describe the effect of drying on the isotope ratios of water extracted from agar media and the H and O stable isotope ratios ratios of Bacillus subtilis spores cultured on agar. The delta2H vs delta18O relationship of water in Petri dish agar was surprisingly constant during evaporation regardless of the ambient relative humidity, making it possible to calculate the approximate isotope ratios of the original water, even in significantly evaporated agar. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched.