Rychlik I, Cardova L, Sevcik M, Barrow P A
Veterinary Research Institute, Hudcova 70, 621 32, Brno, Czech Republic.
J Microbiol Methods. 2000 Nov;42(3):255-63. doi: 10.1016/s0167-7012(00)00199-8.
We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.
我们已经表明，鼠伤寒沙门氏菌及其同基因的nuoG和cydA突变体的生长、饥饿和群体异质性可以通过流式细胞术进行监测。对细菌细胞进行了未染色分析，以及用罗丹明123、碘化丙啶和吖啶橙染色后的分析。在未染色的培养物中，可以区分有鞭毛和无鞭毛的细胞。nuoG和cydA突变体用罗丹明染色较少，证实了它们在产生膜电位方面的缺陷。在第4天到第14天之间，所有菌株中碘化丙啶染色增加，这与膜完整性降低有关。吖啶橙染色显示，nuoG和cydA突变体在稳定期的发育受到抑制。此外，直到第28天，一小部分细胞显示出高RNA和DNA水平。为了确定这些细胞是否代表更适合长期存活的亚群，我们通过OD值和活菌计数来测量群体的生长。由于在整个研究过程中，野生型和突变株的OD值都增加了，而活菌计数逐渐减少，我们提出即使在非常陈旧的培养物中，也一定有一群细胞在进行复制。
