The direct extraction of viable microbes from soil samples is critical for the application of many single-cell related technologies. However, there are many aspects of extraction technologies that can impact the viability and diversity of extractable cells from fresh or stored soil samples. In this study, physical dispersion method, chemical dispersion method, and Nycodenz density gradient medium concentration were optimized with two sequential rounds of cell extraction from four soil samples having diverse physicochemical properties. Cell viability was quantified with fluorescence staining and flow cytometry. The viable microbial community compositions in soil extractable cells and soil samples were assessed after selective removal of DNA from dead cells. Among the four different extraction and purification methods, a protocol that included physical blending, Tween 20 treatment, and centrifugation with 80% Nycodenz, had the highest cell viability and yield. Repeated extraction increased the yield but reduced the cell viability. The over- or under-represented taxa in extractable cells might contribute to the bias of the extractable microbial communities. Using the optimized cell extraction procedure, the effect of soil storage conditions (4 °C, −80 °C, and air-drying) on yield, viability, and community composition of soil extractable cells were assessed. Cell viability decreased in all stored soil samples, but significant decreases in cell yield was only observed in air-dried soil samples. Microbial community compositions changed significantly in all stored soil samples, with the least changes were observed in soil stored at 4 °C, confirming that 4 °C short-term storage is suitable for highly efficient viable cell extraction. Taken together, the developed method offers great potential for advancing our analyses and understanding of soil microbial ecology and the role of individual microbes.

从土壤样品中直接提取活微生物对于许多与单细胞相关的技术的应用至关重要。但是，提取技术有许多方面会影响新鲜或储存的土壤样品中可提取细胞的活力和多样性。在这项研究中，通过从四个具有不同理化特性的土壤样品中连续两次抽提细胞，优化了物理分散方法，化学分散方法和Nycodenz密度梯度培养基的浓度。细胞活力用荧光染色和流式细胞仪定量。在从死细胞中选择性去除DNA后，评估土壤可提取细胞和土壤样品中的微生物群落组成。在四种不同的提取和纯化方法中，包括物理混合，吐温20处理和80％Nycodenz离心的实验方案具有最高的细胞活力和产量。重复提取可提高产量，但会降低细​​胞活力。可提取细胞中分类单元的过多或不足代表可能导致可提取微生物群落的偏见。使用优化的细胞提取程序，评估了土壤储存条件（4°C，-80°C和风干）对可提取土壤细胞的产量，生存能力和群落组成的影响。在所有储存的土壤样品中，细胞活力均降低，但仅在风干的土壤样品中观察到细胞产量的显着降低。在所有储存的土壤样品中，微生物群落组成均发生了显着变化，在4°C的土壤中观察到的变化最小。证实4°C的短期储存适合高效活细胞提取。综上所述，开发的方法为推进我们对土壤微生物生态学和单个微生物的作用的分析和理解提供了巨大的潜力。