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If gutta-percha cartridges are contaminated with bacteria prior to obturation then contamination of the root canal system may result. Successful treatment not only depends on bacterial elimination but also depends on prevention of recontamination. This study was motivated by the observation that endodontists frequently use single use gutta-percha cartridges on multiple patients. The goal of this study was to determine if cross contamination occurred when a single gutta-percha cartridge in the Calamus™ Flow System was used on multiple patients. An effective recovery method was established during a pilot study using Enterococcus faecalis as a sample bacterium. Microbial recovery was obtained using standard media. The Calamus™ heated gutta-percha delivery system was evaluated to determine the percentage of cartridges with recoverable microorganism under four conditions. Results showed that for cartridges tested immediately after removal from manufacturer’s packaging, the observed negative outcome was 0/10 (0%). For cartridges used on a single patient with an alcohol wipe after use, the negative outcome was 0/20 (0%). For cartridges used on a single patient with no alcohol wipe after use, the negative outcome was 1/20 (5%). For cartridges used on three patients with an alcohol wipe after each use, the number of observed negative outcomes was 0/20 (0%). Using 95% score confidence intervals, the results indicate that the percentage of cartridges with recoverable microorganism under the four conditions is below 27.8% for cartridges sampled from manufacturer’s packaging, 16.1% for cartridges used on a single patient with a alcohol wipe after use, 23.6% for cartridges used on a single patient with no alcohol wipe after use, and 16.1% for cartridges used on three patients with an alcohol wipe after each use. Evidence from this study indicates using Calamus™ flow cartridges on multiple patients vs. single patient does not lead to an increased contamination risk.