The oxygen and hydrogen stable isotope ratios of precipitation and surface water carry a strong geographic signature, thus making them potentially useful in crime investigations. Stable isotope ratios provide a possible forensic link to media or source water in the event that a microbiological agent was recovered in the course of a criminal investigation. The oxygen and hydrogen stable isotope ratios would be of particular interest in such a case because of their potential to provide insights into the location of the water sources used to culture the microbes. The evaporation of medium water during the course of growth of a bacterial culture, however, might affect the O and H stable isotope ratios of the organisms produced later in the culture period relative to those first produced in the culture period, which changes the overall relationship of their isotope ratio values to those of the original culture water. The current study found that the isotopic behavior of water evaporating from agar-containing bacteriological media in Petri dishes was different from that of water that evaporated from open pools. Explanations for this are offered. The H stable isotope ratios of spores cultured on agar remained relatively unchanged as the agar dried, but the O ratio became significantly enriched. The hydrogen isotope ratios of spores apparently is relatively insensitive to evaporation, thus making it a more stable signal in linking spores to growth water, regardless of the mode of culturing. 4 tables, 5 figures, and 33 references