Enumeration of bacterial colonies in an agar plate is simple in concept, but automated colony counting is difficult due to variations in colony color, size, shape, contrast, and density, as well as colony overlap. Furthermore, in applications where high throughput is essential, it is critical to employ a fast and user-friendly automated technique that doesn t compromise counting accuracy. While commercial products exist that can count bacterial colonies, they can be cost-prohibitive for small laboratories. We present here a colony counting program, NIST s Integrated Colony Enumerator (NICE), designed to count dark colonies from multiple regions of interests on an agar plate. Images can be cost effectively acquired by a digital camera or a desktop scanner and imported into NICE. High throughput standardized assay formats such as the multiplexed opsonophagocytic killing assay can be readily counted by NICE.