Abstract
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.