All living cells are fluorescent and exhibit "fingerprint" spectral patterns that allow for their detection and identification. The Mycobacterium paratuberculosis that causes Johne's disease has a specific fluorescent pattern that may be used for its identification. In addition to its biochemically-based fluorescent spectral pattern, this microbe has a distinctive size and shape pattern that can also be detected through spectral analysis using light scatter measurements. Chemometric analysis techniques such as neural network algorithms allow for the detection of specific bacterial species even in mixtures of multiple bacteria. Some recent experience in the application of fluorometric spectral instrumentation to the identification of bacteria and other microbes in complex matrices will be presented.