Department of Environmental Microbiology, Helmholtz-Centre for Environmental Research, Permoserstraße 15, D-04318 Leipzig, Germany.
onCyt Microbiology AG, Marchwartstrasse 61, 8038 Zürich, Switzerland.
Cells. 2023 Jun 6;12(12):1559. doi: 10.3390/cells12121559.
Cell density is an important factor in all microbiome research, where interactions are of interest. It is also the most important parameter for the operation and control of most biotechnological processes. In the past, cell density determination was often performed offline and manually, resulting in a delay between sampling and immediate data processing, preventing quick action. While there are now some online methods for rapid and automated cell density determination, they are unable to distinguish between the different cell types in bacterial communities. To address this gap, an online automated flow cytometry procedure is proposed for real-time high-resolution analysis of bacterial communities. On the one hand, it allows for the online automated calculation of cell concentrations and, on the other, for the differentiation between different cell subsets of a bacterial community. To achieve this, the OC-300 automation device (onCyt Microbiology, Zürich, Switzerland) was coupled with the flow cytometer CytoFLEX (Beckman Coulter, Brea, USA). The OC-300 performs the automatic sampling, dilution, fixation and 4',6-diamidino-2-phenylindole (DAPI) staining of a bacterial sample before sending it to the CytoFLEX for measurement. It is demonstrated that this method can reproducibly measure both cell density and fingerprint-like patterns of bacterial communities, generating suitable data for powerful automated data analysis and interpretation pipelines. In particular, the automated, high-resolution partitioning of clustered data into cell subsets opens up the possibility of correlation analysis to identify the operational or abiotic/biotic causes of community disturbances or state changes, which can influence the interaction potential of organisms in microbiomes or even affect the performance of individual organisms.
细胞密度是所有微生物组研究中一个重要的因素,因为相互作用是人们关注的重点。它也是大多数生物技术过程运行和控制的最重要参数。过去,细胞密度的测定通常是离线和手动进行的,这导致了采样和即时数据处理之间的延迟,从而无法及时采取行动。虽然现在有一些在线方法可以快速自动地测定细胞密度,但它们无法区分细菌群落中的不同细胞类型。为了解决这一差距,提出了一种在线自动化流式细胞术方法,用于实时高分辨率分析细菌群落。一方面,它允许在线自动计算细胞浓度,另一方面,可以区分细菌群落中的不同细胞亚群。为了实现这一点,将 OC-300 自动化设备(位于瑞士苏黎世的 onCyt Microbiology)与流式细胞仪 CytoFLEX(美国 Brea 的 Beckman Coulter)相耦合。OC-300 自动完成细菌样本的自动采样、稀释、固定和 4',6-二脒基-2-苯基吲哚(DAPI)染色,然后将其发送到 CytoFLEX 进行测量。结果表明,该方法能够可重复地测量细胞密度和细菌群落的指纹样模式,生成适用于强大的自动化数据分析和解释管道的数据。特别是,自动、高分辨率地将聚类数据划分为细胞亚群,为相关性分析提供了可能性,以确定群落扰动或状态变化的操作或非生物/生物原因,这可能会影响微生物组中生物体的相互作用潜力,甚至影响单个生物体的性能。