Enumeration of marine microbial organisms by flow cytometry using near‐ UV excitation of Hoechst 34580‐stained DNA
Enumeration of marine microbial organisms by flow cytometry using near‐ UV excitation of Hoechst 34580‐stained DNA
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使用 Hoechst 34580 染色 DNA 的近紫外激发，通过流式细胞术对海洋微生物进行计数
DOI：
10.1002/lom3.10454
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Selph, Karen E.
中科院分区：
文献类型：
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作者：
Selph, Karen E.
描述了一种使用低功率固态近紫外激光器对活的或保存的样品中的海洋异养细菌和浮游植物进行计数的流式细胞术方法。该方法使用Hoechst 34580对海水样品中微生物细胞的DNA进行染色。这种染料最佳激发波长为375 nm，与Hoechst 33342不同，后者需要约350 nm的激发波长，仅在更昂贵的激光器上可用。Hoechst 34580方法的浮游植物丰度与未染色样品的丰度相当，并且当通过Hoechst 33342染色方法进行分析时。利用这种新方法，非色素海洋细菌和浮游植物的丰度，同时在一个单一的样品Hoechst发射波长（~ 450 nm）是很好地分离的叶绿素和藻红蛋白荧光的发射波长。这种新方法与采用已建立的Hoechst 33342和SybrGreen I方法获得的细菌丰度相似。丰度接近105 cells mL− 1的群体的精密度估计值（变异系数）为1- 3%，在103 cells mL−1的较低细胞浓度下增加至3-9%。Hoechst 34580方法简单，不需要加热或用RNA酶预处理，可用于未保存和甲醛保存的细胞，适合在海上使用便携式、紧凑型、低功耗的流式细胞仪。
A flow cytometry method for enumerating marine heterotrophic bacteria and phytoplankton in a living or preserved sample using a low power solid state near‐ultraviolet laser is described. The method uses Hoechst 34580 to stain DNA in microbial cells in seawater samples. This stain is optimally excited at 375 nm unlike the similar Hoechst 33342, which requires ~ 350 nm excitation only available on more expensive lasers. Phytoplankton abundances from the Hoechst 34580 method are comparable to those of unstained samples and when analyzed by the Hoechst 33342 staining method. With this new method, nonpigmented marine bacteria and phytoplankton abundances are obtained simultaneously in a single sample as the Hoechst emission wavelength (~ 450 nm) is well separated from the emission wavelengths of chlorophyll and phycoerythrin fluorescence. Bacteria abundances are similar between this new method and those obtained with established Hoechst 33342 and SybrGreen I methods. Precision estimates (coefficient of variation) on populations with abundances near ~ 105cells mL−1are 1–3%, increasing to 3–9% at lower cell concentrations of 103cells mL−1. The Hoechst 34580 method is simple, requiring no heating or pretreatment with RNAse, can be used on unpreserved and formaldehyde‐preserved cells, and is amenable to at‐sea use with portable, compact, low power‐requiring flow cytometers.