Department of Biochemistry, Faculty of Medicine, Kuwait University, Kuwait City, Kuwait.
PLoS One. 2019 Aug 27;14(8):e0219713. doi: 10.1371/journal.pone.0219713. eCollection 2019.
Reduction of tetrazolium salts to colored formazan products by metabolically active cells is widely used for assessment of cell viability. Among the tetrazolium compounds most commonly used is MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Numerous studies about sites and mechanisms of cellular reduction of MTT, performed in mammalian cell cultures, have identified various parameters that affect formazan production and can lead to overestimation/underestimation of viable cells or effects of treatment. Irrespective of lack of such data for prokaryotic cells, the MTT assay is commonly used for microbiological studies, which often leads to contradictory results or misinterpretation of data. The aim of this study was to investigate how components of growth media and conditions of growth, affect formazan formation by microbial cells. Results showed that MTT reduction depended on the amino acid composition of the medium. Several amino acids potentiated formazan production by Gram-positive and Gram-negative bacteria, with histidine having the strongest effect. Results of this study demonstrate that data obtained with the MTT test should be interpreted with caution, particularly when different growth media are used or treatments affect metabolic pathways, and that evaluation of the reliability of the MTT assay under specific conditions should be performed, to avoid erroneous results. Performing the assay with cells suspend in glucose-supplemented buffer would eliminate the effects of metabolites and will limit cell division during incubation with MTT. Another critical element to be considered is the choice of a proper solvent for dissolution of formazan crystals.
四唑盐被代谢活跃的细胞还原为有色的formazan 产物，被广泛用于评估细胞活力。最常用的四唑化合物之一是 MTT[3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐]。在哺乳动物细胞培养物中进行的关于 MTT 细胞内还原的位点和机制的大量研究，已经确定了各种影响 formazan 产生的参数，这些参数可能导致对活细胞的高估/低估或处理效果。尽管没有针对原核细胞的此类数据，但 MTT 测定法常用于微生物学研究，这常常导致矛盾的结果或对数据的误解。本研究旨在研究生长培养基的成分和生长条件如何影响微生物细胞的 formazan 形成。结果表明，MTT 的还原取决于培养基的氨基酸组成。几种氨基酸增强了革兰氏阳性和革兰氏阴性细菌的 formazan 产生，其中组氨酸的作用最强。本研究的结果表明，应该谨慎解释 MTT 试验获得的数据，特别是当使用不同的生长培养基或处理方法影响代谢途径时，并且应该在特定条件下评估 MTT 试验的可靠性，以避免错误的结果。用含有葡萄糖的缓冲液悬浮细胞进行测定可以消除代谢物的影响，并限制在与 MTT 孵育期间的细胞分裂。另一个需要考虑的关键因素是选择适当的溶剂来溶解 formazan 晶体。
