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

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.

Intracellular Accumulation of Novel and Clinically Used TB Drugs Potentiates Intracellular Synergy

“…However, very few studies have been conducted using M. tuberculosis H37Rv as the infectious agent while examining compound accumulation and intracellular efficacy within the same experimental system. The infected macrophage assay was developed based on the uninfected assay used in this study and according to methods described in several publications ( 8 , 43 , 49 , 84 , 86 – 90 ). The approach utilized a standardized 20× MIC compound dose comparison to incorporate the assay into our screening cascade.…”

Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

“…Optical clearing procedures that use detergent such as TritonX-100 within the CUBIC-1 and CUBIC-L reagents may also impact on the ability of the Auramine O and Rhodamine B dyes to bind to the mycolic acids that form the cell wall of Mtb . Others though have used Auramine O and TritonX-100 successfully 36 , 37 albeit with much lower concentrations of TritonX-100. Fluorescence in situ hybridization (FISH) dyes or immunofluorescence could offer a solution for these limitations 35 .…”

Impact of type 2 diabetes on the capacity of human macrophages infected with Mycobacterium tuberculosis to modulate monocyte differentiation through a bystander effect

“…The single cell suspension was prepared according to the reported protocol 14 and bacterial concentration was determined by optical density at 600 nm. For binding assays, Mtb was stained with 25 µ m CFSE (C34554; Invitrogen Life technologies; Eugene, OR, USA) for 30 min, using conditions standardized by our laboratory based on a previous report, 50 and bacteria staining was checked by flow cytometry for each experiment.…”