An image acquisition and analysis system are disclosed. The system enables high throughput, objective analysis of microbial samples over days or weeks. The system may accommodate upwards of twelve 96- or 384-well plates simultaneously (liquid or solid media). The system may acquire and analyze a large number of samples in a short period of time. For example, over 384 samples per minute or 18,432 samples per hour. The system hardware may include a multi-spectral imager (fluorescence and bright field detection), electro-mechanical assemblies, and an optional high-resolution stage. The system may automate image acquisition, image data processing, simplify data storage, and enable automated analysis tools to significantly reduce the manual labor and time associated with such tasks. The system may allow for quick processing and analysis of data into clear phenotypic classes. The analysis capabilities may include colony growth, colorimetry, and structural morphology assays, and automated phenotype classification capabilities.