Modification of MTT Assay for Precision and Repeatability and Its Mechanistic Implication

Abstract

The discovery of antibiotics is one of the key achievements of modern medicine. Antibiotic use has contributed enormously to human health. However, microorganisms can evolve and adjust to changes in their environment and have subsequently developed several protective mechanisms to reduce their susceptibility to antibiotics 1. More and more potent antibiotics lose their efficacy over time, rendering them useless for conditions they could once successfully treat 2-4. Therefore, there is an urgent need for screening and development of new antibacterial agents effective against resistant strains 5-8. Screening of active substances plays a crucial role in drug research and development. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay has been widely adopted for low cost, rapidity and high throughput format 9. In this assay, the yellow and water-soluble MTT is converted to the purple and water insoluble MTT formazan in the presence of mitochondrial dehydrogenase as shown in Scheme-I 10. As a result, the amount of formazan dye produced is directly proportional to the number of metabolically active cells and indicates the reducing potential of the cells 11. The MTT assay uses frequently, while the reported conditions and parameters of the assay vary widely. Thus attempts to modification of MTT assay are never ceased 12-15. Incubating cells with MTT,

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives

“…Although Mosmann (1983) originally used acid isopropyl alcohol for extraction, Carmichael et al (1987) found that this solvent resulted in low optical absorption values and suggested the use of DMSO or mineral oil as alternatives. In strongly acidic media, the presence of the cationic MTT formazan results in a complete disappearance of the absorption at 575 nm (Wang et al, 2014). The acidic pH used in solubilization solvents has two effects: it modifies the absorption spectrum of the cationic formazan and also changes the absorption of any pH indicator if present.…”

“…In addition to its use on mammalian cell cultures, the colorimetric MTT assay is employed for testing the viability of protozoan (Dias et al, 1999) and sperm cells of several species (Aziz, 2006;Van der Berg, 2015). Microbiological viability assay applications of tetrazolium salts are also well known and widely used (Thom et al, 1993;Bhupathirajua et al, 1999;Wang et al, 2014;Villegas-Mendoza et al, 2015;Grela et al, 2015), mainly for biofilms (Pérez et al, 2010;Trafny et al, 2013). MTT, CTC, XTT, and INT are the most widely used tetrazolium salts for assessment of microbial viability.…”