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Updated: Oct 19, 2025
A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Published on: January 17, 2014
Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
David A Barr1,2,3, Charles Omollo4, Mandy Mason4
1Wellcome Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town, 7925, South Africa. david.barr@liverpool.ac.uk.
Accurate counting of viable Mycobacterium tuberculosis is crucial. A new flow cytometry method using fluorescent dyes offers a precise alternative to traditional methods, improving tuberculosis research and drug development.
Area of Science:
- Microbiology
- Immunology
- Infectious Diseases
Background:
- Accurate quantitation of viable Mycobacterium tuberculosis (Mtb) is essential for understanding tuberculosis (TB) pathogenesis, transmission, and treatment efficacy.
- Current methods for viable Mtb enumeration, such as colony-forming units (CFU), are limited by clumping, differential culturability, and technical challenges with infectious agents.
Purpose of the Study:
- To develop and validate an absolute counting method for viable mycobacteria in liquid cultures using flow cytometry.
- To compare this new method with traditional CFU counting and assess its utility in standardizing experimental inocula and analyzing drug effects.
Main Methods:
- Development of an absolute counting method utilizing a bench-top flow cytometer with low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG).
- Enumeration of mycobacteria, including detection of cell aggregates, and comparison of flow cytometry counts with CFU counts under various growth conditions.
- Application of the flow cytometry method to assess drug pharmacodynamics, including time-kill curves for Mycobacterium bovis bacillus Calmette-Guérin (BCG) against different antibiotics.
Main Results:
- The CA+ cell counts from flow cytometry strongly correlated with CFU counts during exponential growth, serving as a real-time alternative for inoculum standardization.
- The flow cytometry method can detect and quantify cell aggregates, which are a source of variance in traditional methods.
- CFUs represent a sub-population of intact, metabolically active cells, with the proportion varying based on growth conditions.
- Flow cytometry revealed distinct time-kill kinetics for BCG treated with different drug combinations and highlighted the dynamics of morphologically distinct subpopulations.
Conclusions:
- The developed flow cytometry method provides a more accurate and precise way to enumerate viable mycobacteria compared to CFU counting.
- This technique improves the standardization of experimental inocula and offers deeper insights into mycobacterial population dynamics and drug responses.
- The method has significant implications for tuberculosis research, drug development, and understanding treatment outcomes.
