Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
J Microbiol Methods. 2012 Feb;88(2):297-303. doi: 10.1016/j.mimet.2011.12.015. Epub 2011 Dec 29.
Surface associated microbes have historically been difficult to accurately and effectively enumerate. In the current study, we propose a rapid and simple method for estimating abundance of surface associated microbial cells by fluorescence of SYBRGreen stained bacteria and in vivo chlorophyll a fluorescence of benthic diatoms in 24 and 48-well microtiter plates. The effectiveness of this high-throughput technique is demonstrated by assessing sensitivity of a clinical strain of Vibrio cholerae, a benthic bacterial isolate and the benthic microalgae Cylindrotheca closterium to three antibiotics--tylosin, lincomycin and ciproflaxacin. We report on the significant linear relationships between spectral chl a fluorescence and cell abundance and between microalgal growth rates derived from cell counts and fluorescence. Additionally, we provide a simplified and improved method for preparation of a silica gel matrix (SGM), which is an ideal plating media for fluorescence applications. These findings indicate that spectrofluorometry is an inexpensive tool for rapidly estimating abundance of surface associated microbiota and can be employed for assessing antibiotic sensitivity.
表面相关微生物历来难以准确有效地计数。在本研究中，我们提出了一种快速简便的方法，通过 SYBRGreen 染色细菌的荧光和 24 孔和 48 孔微量滴定板中底栖硅藻的体内叶绿素 a 荧光来估计表面相关微生物细胞的丰度。通过评估临床霍乱弧菌菌株、底栖细菌分离株和底栖微藻 Cylindrotheca closterium 对三种抗生素——泰乐菌素、林可霉素和环丙沙星的敏感性，证明了这种高通量技术的有效性。我们报告了光谱 chl a 荧光与细胞丰度之间以及细胞计数和荧光得出的微藻生长速率之间的显著线性关系。此外，我们提供了一种简化和改进的硅胶基质 (SGM) 制备方法，它是荧光应用的理想平板培养基。这些发现表明，荧光分光光度法是一种快速估计表面相关微生物群落丰度的廉价工具，并可用于评估抗生素敏感性。
