Effect of growth media on the MTT colorimetric assay in bacteria
Effect of growth media on the MTT colorimetric assay in bacteria
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DOI：
10.1371/journal.pone.0219713
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发表时间：
2019-08-27
期刊：
影响因子：
3.7
通讯作者：
Benov, Ludmil
中科院分区：
文献类型：
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作者：
Benov, Ludmil
通过代谢活性细胞将四唑盐还原成有色甲瓒产物广泛用于评估细胞活力。最常用的四唑化合物是MTT [3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑]。在哺乳动物细胞培养物中进行的关于MTT的细胞还原位点和机制的许多研究已经确定了影响甲瓒产生的各种参数，并可能导致高估/低估活细胞或治疗效果。尽管缺乏原核细胞的此类数据，但MTT测定通常用于微生物学研究，这通常导致矛盾的结果或对数据的误解。本研究的目的是调查生长培养基的成分和生长条件如何影响微生物细胞的甲瓒形成。结果表明，MTT还原依赖于培养基的氨基酸组成。几种氨基酸增强革兰氏阳性和革兰氏阴性细菌的甲瓒生产，组氨酸具有最强的效果。本研究的结果表明，MTT试验获得的数据应谨慎解释，特别是当使用不同的生长培养基或处理影响代谢途径时，并且应在特定条件下进行MTT试验可靠性的评价，以避免错误的结果。用悬浮在补充葡萄糖的缓冲液中的细胞进行测定将消除代谢物的影响，并将在与MTT孵育期间限制细胞分裂。另一个需要考虑的关键因素是选择合适的溶剂来溶解甲瓒晶体。
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.