The Assessment of Viability of M. Tuberculosis after Exposure to CPC Using Different Methods

Abstract

Settings. National Institute for Research in Tuberculosis, Chennai. Objective. To assess the proportion of metabolically active cells of Mycobacterium tuberculosis after exposed to CPC using FDA-EB vital staining and viable counts on LJ medium. Mycolic acid content in M. tuberculosis after exposure to CPC was estimated using HPLC. Methods. Clinical isolates of M. tuberculosis and standard reference strain M. tuberculosis H37Rv were used for FDA-EB, viable count, and HPLC. Results. FDA/EB consistently stained 70-90% of log phase cells as green and the remaining cells as red-orange. After CPC treatment, 65-70% of the cells stained red-orange. The viability counts were comparable to 0-day controls. Synthesis of mycolic acids in mycobacteria was reduced when exposed to CPC using HPLC due to the decreased metabolic activity of the organisms. Conclusion. The cells are metabolically inactive during storage with CPC but these cells grew well on LJ medium after removal of CPC. The viability of M. tuberculosis was maintained in CPC with minimal reduction. Mycolic acid content was reduced if the cells of M. tuberculosis were treated with CPC for 7 days. All the above findings provide yet another evidence for the damage of cell wall of M. tuberculosis.

Figure 1

FDA-EB staining using Fluorescence microscopy.

Figure 2

Determination of mycolic acid content of M. tuberculosis after exposure to P-CPC (H37RV).

Figure 3

Determination of mycolic acid content of M. tuberculosis after exposure to P-CPC (clinical isolate).