School of Civil and Environmental Engineering, Nanyang Technological University, Blk N1, 50 Nanyang Avenue, Singapore 639798, Singapore.
Microbiol Res. 2011 Feb 20;166(2):129-35. doi: 10.1016/j.micres.2010.01.007. Epub 2010 Feb 20.
Assessment of physiological states of individual bacterial cells can be useful in the monitoring of the biotechnological processes. Physiological heterogeneity of Escherichia coli population by respiration activity and membrane potential during growth and starvation in batch cultures was evaluated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and bis-(1,3-dibutylbarbituric acid) trimethine oxo (DiBAC₄)(3) fluorescent probes in combination with flow cytometry. The shares of CTC-reducing cells (CTC(+)-cells) and cells with positively charged outside cytoplasmic membrane which were not stained by DiBAC₄(3) (ΔΨ(+)-cells), were 90% and 95% in the exponential phase of batch culture, respectively. After short-term starvation for 10 h, the shares of CTC(+)-cells and ΔΨ(+)-cells in the samples taken from the exponential phase dropped to 78% and 72%, respectively. After long-term starvation for 40 days, the share of CTC(+)-cells dropped to 5%, whereas the share of ΔΨ(+)-cells was about 50%. The conclusions from this research are as follows: (a) the physiological heterogeneity of bacterial population increased after starvation; (b) the cell respiratory activity is more sensitive to starvation than the cell membrane potential; (c) a probe for the cell membrane potential DiBAC₄(3) is more suitable than a probe for the cell respiratory activity CTC in the detection of viable indicator bacteria in environment.
评估单个细菌细胞的生理状态在生物技术过程监测中可能是有用的。使用 5-氰基-2,3-二苯基四氮唑氯化物 (CTC) 和双-(1,3-二丁基巴比妥酸) 三甲烯氧 (DiBAC₄)(3) 荧光探针结合流式细胞术，评估了分批培养中大肠杆菌种群的呼吸活性和膜电位的生理异质性。在分批培养的指数生长期，CTC 还原细胞（CTC(+)-细胞）和未被 DiBAC₄(3) 染色的带正电荷的细胞质膜外细胞（ΔΨ(+)-细胞）的比例分别为 90%和 95%。经过 10 小时的短期饥饿后，取自指数生长期的样品中 CTC(+)-细胞和 ΔΨ(+)-细胞的比例分别降至 78%和 72%。经过 40 天的长期饥饿后，CTC(+)-细胞的比例降至 5%，而 ΔΨ(+)-细胞的比例约为 50%。从这项研究中得出以下结论：(a) 饥饿后细菌种群的生理异质性增加；(b) 细胞呼吸活性比细胞膜电位对饥饿更敏感；(c) 在检测环境中的存活指示菌时，细胞膜电位探针 DiBAC₄(3)比细胞呼吸活性探针 CTC 更合适。
