Thom S M, Horobin R W, Seidler E, Barer M R
Department of Microbiology, University of Newcastle upon Tyne, UK.
J Appl Bacteriol. 1993 Apr;74(4):433-43. doi: 10.1111/j.1365-2672.1993.tb05151.x.
The reduction of four tetrazolium cations (TCs), nitroblue tetrazolium (NBT), neotetrazolium (NT), methylthiazolyldiphenyltetrazolium (MTT) and iodonitrophenyltetrazolium (INT), by viable micro-organisms, immobilized on glass cover slips, was examined by light microscopy with a view to determining a systematic basis for applying these reagents as cytochemical indicators of microbial viability and activity. The potential value of histochemical information about TC reactions for developing their microbiological applications was also assessed. INT and MTT detected viable cells more readily than NBT and NT. In order to obtain cell-localized formazan, MTT required cobalt ions in the reaction mixture and INT reactions had to be assessed soon after mounting. In general, formazan deposition could be accelerated by the addition of glucose and an intermediate electron carrier (IEC) to the reaction mixture, although inhibitory effects of IECs were also detected. Cultures in exponential phase, in stationary phase and inhibited by chloramphenicol could be differentiated with MTT but not with INT. For some organisms, notably Candida albicans, Pseudomonas aeruginosa and Enterococcus faecalis. TC reactions proved to be a relatively insensitive means of demonstrating viability. Two parameters used in selecting TCs for histochemical reactions, lipophilicity and reducibility appeared to be predictive for the relative sensitivity of these reagents as indicators of cell viability. The concepts of substantivity, a measure of non-specific interactions between reagents and staining substrates, and TC oxygen sensitivity, the effect of competition between oxygen and TCs for electrons, were found to be relevant to formazan deposition in live microbes. These findings support the use of TCs as cytochemical probes of microbial activity in defined settings and the use of histochemical knowledge to support further development of these techniques.
通过光学显微镜检查固定在玻璃盖玻片上的活微生物对四种四氮唑阳离子（TCs），即硝基蓝四氮唑（NBT）、新四氮唑（NT）、甲基噻唑基二苯基四氮唑（MTT）和碘硝基苯基四氮唑（INT）的还原情况，目的是确定将这些试剂用作微生物活力和活性的细胞化学指标的系统依据。还评估了关于TC反应的组织化学信息在开发其微生物学应用方面的潜在价值。INT和MTT比NBT和NT更容易检测到活细胞。为了获得细胞定位的甲臜，MTT在反应混合物中需要钴离子，并且INT反应必须在封片后不久进行评估。一般来说，向反应混合物中添加葡萄糖和中间电子载体（IEC）可以加速甲臜沉积，不过也检测到了IEC的抑制作用。处于指数生长期、稳定期以及受氯霉素抑制的培养物可以用MTT区分，但不能用INT区分。对于一些微生物，特别是白色念珠菌、铜绿假单胞菌和粪肠球菌，TC反应被证明是一种相对不敏感的显示活力的方法。用于选择组织化学反应中TCs的两个参数，亲脂性和还原性，似乎可以预测这些试剂作为细胞活力指标的相对敏感性。发现直接性（一种衡量试剂与染色底物之间非特异性相互作用的指标）和TC氧敏感性（氧与TCs之间对电子竞争的影响）的概念与活微生物中甲臜沉积有关。这些发现支持将TCs用作特定环境中微生物活性的细胞化学探针，并利用组织化学知识来支持这些技术的进一步发展。
