Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2018 Apr;7(2):e00588.
doi: 10.1002/mbo3.588. Epub 2018 Feb 27.

Quantifying intracellular Mycobacterium tuberculosis: An essential issue for in vitro assays

Deisy Carolina Rodriguez  1   2 , Marisol Ocampo  2   3 , Luz Mary Salazar  1 , Manuel Alfonso Patarroyo  2   3
Affiliations
Review

Quantifying intracellular Mycobacterium tuberculosis: An essential issue for in vitro assays

Deisy Carolina Rodriguez et al. Microbiologyopen. 2018 Apr.

Abstract

Many studies about intracellular microorganisms which are important regarding diseases affecting public health have been focused on the recognition of host-pathogen interactions, thereby ascertaining the mechanisms by which the pathogen invades a cell and makes it become its host. Such knowledge enables understanding the immunological response triggered by these interactions for obtaining useful information for developing vaccines and drugs. Quantitative cell infection assay protocols are indispensable regarding studies involving Mycobacterium tuberculosis, which takes the lives of more than 2 million people worldwide every year; however, sometimes these are limited by the pathogen's slow growth. Concerning such limitation, a detailed review is presented here regarding the different methods for quantifying and differentiating an intracellular pathogen, the importance of mycobacteria aggregate dissociation and multiplicity of infection (MOI) in infection assays. The methods' differences, advantages, and disadvantages are discussed regarding intra and extracellular bacteria (on cell surface) differentiation, current problems are outlined, as are the solutions provided using fluorophores and projections made concerning quantitative infection assays.

Keywords: bacterial quantification; cytometry; fluorescence; infection assay; tuberculosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Mycobacterium tuberculosis H37Rv dissociation using tuberculin syringe with cut needle and rhodamine B staining. The first two photographs show characteristic groups without dissociation. White bar size is 100 μm
Figure 2
Figure 2
Overview of quantitative cell infection assay protocols, four ways of disaggregating mycobacteria are indicated with numbers
See this image and copyright information in PMC

References

    1. Agerer, F. , Waeckerle, S. , & Hauck, C. R. (2004). Microscopic quantification of bacterial invasion by a novel antibody‐independent staining method. Journal of Microbiological Methods, 59(1), 23–32. https://doi.org/10.1016/j.mimet.2004.05.008 - DOI - PubMed
    1. de Assunção, T. M. , Batista, E. L. , Deves, C. , Villela, A. D. , Pagnussatti, V. E. , de Oliveira Dias, A. C. , … Santos, D. S. (2014). Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide. Tuberculosis (Edinb), 94(4), 421–427. https://doi.org/10.1016/j.tube.2014.04.008 - DOI - PubMed
    1. Awuh, J. A. , & Flo, T. H. (2017). Molecular basis of mycobacterial survival in macrophages. Cellular and Molecular Life Sciences, 74(9), 1625–1648. - PMC - PubMed
    1. Benoit, M. , Desnues, B. , & Mege, J.‐L. (2008). Macrophage polarization in bacterial infections. The Journal of Immunology, 181(6), 3733–3739. https://doi.org/10.4049/jimmunol.181.6.3733 - DOI - PubMed
    1. Bermudez, L. E. , & Goodman, J. (1996). Mycobacterium tuberculosis invades and replicates within type II alveolar cells. Infection and Immunity, 64(4), 1400–1406. - PMC - PubMed

MeSH terms

LinkOut - more resources