Diaper J P, Edwards C
Department of Genetics and Microbiology, The University, Liverpool, UK.
Microbiology (Reading). 1994 Jan;140 ( Pt 1):35-42. doi: 10.1099/13500872-140-1-35.
The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-IgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.
以金黄色葡萄球菌作为模式生物，评估了流式细胞术在湖水微观世界中检测和计数存活细菌的能力。营养琼脂上的菌落形成单位（c.f.u.）计数与通过流式细胞术检测罗丹明123染色细菌获得的计数没有显著差异，并且使用这些方法没有证据表明存在活的但不可培养的状态。然而，与营养琼脂相比，使用甘露醇盐琼脂估计时c.f.u.显著更低。用异硫氰酸荧光素标记的IgG染色后，金黄色葡萄球菌也通过免疫荧光法进行计数。免疫荧光法估计的菌数与吖啶橙直接计数法之间没有显著差异，并且与活力估计不同，仅观察到总细胞数略有减少。还通过流式细胞术测量了金黄色葡萄球菌存活期间蛋白质和核酸含量的变化，以研究饥饿群体中可能出现的任何潜在异质性。发现流式细胞术测定结果与各自的化学测定结果显著相关。这些结果证明了流式细胞术在检测饥饿期间的活细菌以及研究大分子含量变化方面的能力。它们还说明了使用适当方法检测环境样品中活细菌的重要性。
