Improved enumeration and kinetic study methods for Mycobacterium avium complex

Studies involving Mycobacterium have historically relied upon plate count data for enumeration, and optical density analysis for determination of kinetic growth parameters. Plate count enumeration is a labor, material and space intensive process, requiring long durations for results, with experiments subject to peril if plates become desiccated or contaminated during extended incubation. Optical density measurement is also a labor intensive process, requiring extensive manual effort to obtain a steady data stream, which is also subject to inherent error caused by changes in cell morphology or decay. In addition, both methods are subject to error caused by clumping of Mycobacterium, which can cause a colony forming unit (CFU) to represent 1 to 25 individual cells, or more, and which influences optical density measurement by the micro-scale heterogeneity it causes.;This dissertation provides two methods that improve our ability to enumerate and kinetically study the behavior of Mycobacterium avium Complex (MAC), and possibly Mycobacterium in general. The first involves a method to accurately and precisely enumerate MAC using BACTEC BD MGIT 960 over a broad range of inoculates ranging from 1 to 108 CFU. The method minimizes the potential for error caused by excessive handling and optimizes the cell preparation technique to ensure consistency of results and minimization of cell aggregation. Compared to the standard plate count method, this method minimizes the potential for loss of results due to contamination or desiccation of sample, and reduces assay time by 50 to 90 percent.;The second method quantifies the growth kinetics of MAC, which includes estimation of maximum substrate utilization rates from an electron acceptor perspective and maximum specific growth rate. The method includes estimation of the fraction of electron donor used for energy to fuel cell growth and maintenance needs, which allows conversion of data to the typical nomenclature of electron donor substrate utilization rates.