Single-cell analysis of microbial population heterogeneity is a fast growing research area in microbiology due to its potential to identify and quantify the impact of subpopulations on microbial performance in, for example, industrial biotechnology, environmental biology, and pathogenesis. Although several tools have been developed, determination of population heterogenity in anaerobic bacteria, especially spore-forming clostridia species has been amply studied. In this study we applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer Pseudoclostridium thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we differentiated and enriched all sporulation-related morphologies of P. thermosuccinogenes. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined culture heterogeneity of P. thermosuccinogenes by discriminating between mature spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study sporulation dynamics and its impact on fermentation performance and product formation by P. thermosuccinogenes.

微生物种群异质性的单细胞分析是微生物学中一个快速发展的研究领域，这是因为它有潜力识别和量化亚群对例如工业生物技术，环境生物学和发病机理中微生物性能的影响。尽管已经开发了几种工具，但是已经充分研究了确定厌氧细菌，尤其是形成孢子的梭状芽胞杆菌物种中种群异质性的方法。在这项研究中，我们将单细胞分析技术（例如流式细胞仪（FCM）和荧光辅助细胞分选（FACS））用于形成孢子的琥珀酸盐生产菌假单胞菌热琥珀酸基因。通过将FCM和FACS与荧光染色相结合，我们分化并丰富了所有与孢子形成有关的形态体育产琥珀酸。为了评估代谢活跃的营养细胞的存在，染料碘化丙锭（PI）和羧基荧光素二乙酸酯（cFDA）的混合物测试得最好。侧向散射（SSC-H）与代谢指示剂cFDA染料的结合提供了最佳的孢子形成群体分离。基于此协议，我们通过区分成熟孢子，前孢子，暗相和亮相内生孢子以及营养细胞群体，成功确定了热琥珀酸单胞菌的培养异质性。今后，该方法可用于进一步研究孢子形成动力学及其对热琥珀酸杆菌的发酵性能和产物形成的影响。