| Summary: | Water safety and management programs (WSMP) utilize field measurements to evaluate control limits and monitor water quality parameters including <i>Legionella</i> presence. This monitoring is important to verify that the plan is being implemented properly. However, once it has been determined when and how to sample for <i>Legionella</i>, it is important to choose appropriate collection and processing methods. We sought to compare processing immediate and flushed samples, filtration of different volumes collected, and sample hold times. Hot water samples were collected immediately and after a 2-min flush. These samples were plated directly and after filtration of either 100 mL, 200 mL, or 1 L. Additionally, unflushed samples were collected and processed immediately and after 1, 24, and 48 h of hold time. We found that flushed samples had significant reductions in <i>Legionella</i> counts compared to immediate samples. Processing 100 mL of that immediate sample both directly and after filter concentration yielded the highest concentration and percent sample positivity, respectively. We also show that there was no difference in culture values from time 0 compared to hold times of 1 h and 24 h. At 48 h, there were slightly fewer <i>Legionella</i> recovered than at time 0. However, <i>Legionella</i> counts were so variable based on sampling location and date that this hold time effect was minimal. The interpretation of <i>Legionella</i> culture results depends on the sample collection and processing methods used, as these can have a huge impact on the success of sampling and the validation of control measures.
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