The routine quarterly killifish colony health surveillance program incorporates both molecular methods using whole fish (sentinels) and environmental samples (sump) for molecular analysis and whole fish for histopathology (sentinels). Adult postfiltration sentinel N. furzeri are placed in sentinel tanks in each rack for a minimum exposure period of 3 mo. Sentinels are euthanized by hypothermal shock (rapid chilling) or overdose with buffered tricaine methanesulphonate (MS-222, 1g/L) and submitted to IDEXX BioAnalytics (Columbia, MO) for individual evaluation by histopathology, pooled into groups of 5 for evaluation by real-time PCR, or analyzed individually in-house by conventional PCR for microsporidia. Each health monitoring round samples 2.7% of the entire fish colony. In addition, biofilter samples are collected from the sump of each rack as environmental samples and are processed individually by real-time PCR.