Intracellular Mycobacteria Quantification
Intracellular Mycobacteria Quantification - Related Videos
15:28
A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
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Cited by 30 •
2014
Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.
11:57
Electroporation of Mycobacteria
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Cited by 55 •
2008
Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for...
02:20
Evaluating the Effectiveness of a Test Compound against Mycobacteria in Broth
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2025
This video demonstrates an in-broth assay to determine antibiotic efficacy against Mycobacterium tuberculosis. The bacterial culture is incubated with different concentrations of a bactericidal antibiotic. The bactericidal efficacy is measured by assessing the presence of viable bacteria using a resazurin assay.
06:08
A Charcoal Agar Resazurin Assay to Evaluate Test Compound Activity Against Mycobacteria
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2025
This video showcases the Charcoal Agar Resazurin Assay (CARA) for evaluating the activity of antimicrobial compounds against Mycobacterium tuberculosis. CARA aids in assessing the efficacy of these compounds by gauging their impact on bacterial growth and distinguishing between bactericidal and bacteriostatic effects.
11:54
Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator
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Cited by 9 •
2017
Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.
FAQs
Removing extracellular bacteria helps ensure that the recovered signal reflects bacilli associated with or residing within host cells. If organisms outside the cells remain, they can increase the measured burden and obscure intracellular survival. This separation improves comparisons of host-cell antimicrobial activity, bacterial persistence, and treatment effects across experimental conditions.
These readouts quantify recovered mycobacteria through complementary forms of evidence. Viable colony counts measure bacilli that can produce colonies, microscopy assesses the cellular distribution or presence of bacteria, and molecular measurements detect bacterial material. Selecting among them depends on whether the study emphasizes viability, cell-associated localization, or a molecular estimate of burden.
The host-cell type, mycobacterial strain, and experimental condition can all influence the measured burden. Comparisons are most informative when these variables are deliberately selected and applied consistently, allowing investigators to distinguish differences in bacterial survival or replication from differences caused by the cellular model or assay conditions.
A typical workflow begins by infecting host cells under controlled conditions, followed by removal of extracellular bacteria. The host cells are then lysed to recover intracellular bacilli, and the recovered material is quantified using viable colony counts, microscopy, or molecular measurements. Consistency across these stages is necessary for meaningful comparisons.
The approach is useful when investigators need to compare how effectively macrophages or other host cells limit mycobacterial survival. Measuring intracellular burden under defined conditions provides an outcome for evaluating host responses, differences among cell types or strains, and changes associated with experimental treatments. It therefore connects cellular immune activity with bacterial persistence.
Researchers can compare intracellular bacterial burden between treated and untreated experimental conditions to assess treatment-associated changes in mycobacterial survival or replication. Using the same infection, extracellular-bacteria removal, cell-lysis, and quantification procedures across groups makes the resulting measurements more suitable for evaluating antimicrobial efficacy and comparing responses among strains or host-cell models.