Non Spore-Forming Bacteria: Sterility and Ultrastructure Study

Live organisms from containment laboratories must be inactivated with a valid protocol, or subsequently sterility tested prior to removal from containment and worked with in a lower level of containment. For bioforensic analysis, many specimens that are imaged using electron microscopy (EM) come from BSL2, BSL-3 and BSL-4 containment laboratories. Current literature documents many procedures for fixation of pathogenic organisms, but very few actually document sterility after fixation. For safety and bioforensic analysis, it has been necessary to identify the minimum times required to inactivate vegetative bacteria while preserving ultrastructure for EM. Recent information from this lab demonstrates that most bacterial endospores are inactivated in less than 240 minutes in Universal fixative (4% paraformaldehyde/1% glutaraldehyde (Brantner, et al. in press). Because spores are the most resistant form of bacteria, the data was useful in developing timelines for the treatment of vegetative cells. It has been considered necessary to treat samples containing pathogenic bacteria for periods up to 21 days for inactivation. In addition to potentially causing damage to the ultrastructure of samples, this prolonged fixation period increases the time required to process bioforensic samples. There are many protocols in the literature for inactivating bacterial samples, but they are either of long duration, or utilize harsh chemicals that destroy ultrastructure for subsequent analysis. This study demonstrates that all vegetative bacterial species, resuspended in fixative and tested, were successfully inactivated in 5 minutes. To standardize the laboratory protocols, the same samples were tested for 5, 15, 30, 60, 120, and 240 minutes.