M.Sc. / The global tuberculosis (TB) epidemic has resulted in the development of numerous methods for the enumeration and viability assessment of Mycobacterium tuberculosis (M.tb), as these methods play a key role in TB management and research. In this study the methods of quantitative culture (CFU), the microplate alamar blue assay (MABA), flow cytometry, the green fluorescent protein microplate assay (GFPMA) and quantitative PCR were investigated and compared for the enumeration and viability assessment of mycobacteria in culture. The MABA and the GFPMA were applied to the enumeration and viability assessment of mycobacteria post-infection. Quantitative culture was found to be simple and low in cost but was lengthy. The MABA, an economic and quick assay, was more sensitive for high mycobacterial concentrations. The flow cytometric enumeration of fluorescent mycobacteria was rapid and sensitive, but was dependent on access to a flow cytometer and therefore was costly. Flow cytometry facilitated enumeration but was limited concerning viability assessment. The GFPMA was a simple, rapid and cost effective assay. However, decreased sensitivity was observed for low mycobacterial concentrations. Quantitative PCR, although high in cost, was sensitive and rapid. The MABA and the GFPMA were useful for the enumeration of mycobacteria post-infection, with the former being the more sensitive method. This study serves as a reference of the methods available for the enumeration and viability assessment of M.tb. The advantages and disadvantages established for each of the methods investigated in this study enables an informed selection of the most appropriate method for a specific objective and research environment.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:1753 |
| Date | 30 November 2011 |
| Creators | Edmondson, Nicole |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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