Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and (14)C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

通过流式细胞术（flow cytometry）分析的细菌前向光散射强度随其干重变化，且符合理论预期。本研究基于瑞利-甘斯（Rayleigh-Gans）理论构建标准曲线，以适配细胞形态与排列状态。该标准曲线以小型海洋生物寡养旋孢菌（Cycloclasticus oligotrophus）的终末稀释培养分离株进行校准，其干重通过碳氢氮（CHN）元素分析与14C-乙酸盐掺入法测定。我们将固定细胞因甲醛蓄积所导致的前向光散射强度增量纳入了该标准曲线。当考虑培养基折射率差异以及不同物种间固定效应的差异后，通过流式细胞术测得的多种细菌的细胞干重，与通过库尔特计数器（Coulter Counter）体积及浮力密度计算得到的细胞干重结果一致。该一致性验证了本标准曲线的有效性，并支持了“细胞在流道中呈排列状态”的假设。针对三种细菌的混合样本，依据前向光散射强度与DNA结合型4′,6-二脒基-2-苯基吲哚（DAPI）荧光强度进行分析，我们共解析出多个细胞亚群。该混合样本的总生物量为340 μg/L。含单条染色体的寡养旋孢菌亚群的平均干重最低，为0.027 ± 0.008 pg/细胞。通过干重、库尔特计数器体积及浮力密度的测量结果计算可知，该分离株的干重占湿重的14%~18%，而大肠杆菌（Escherichia coli）的该比值为30%。本方法适用于干重范围0.005~约1.2 pg、最低浓度可达10³个细胞/mL的细菌细胞，可用于测定混合细菌种群的生物量分布，具备独特的应用价值。