University of Albertagrid.17089.37, Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, Canada.
Appl Environ Microbiol. 2022 Aug 9;88(15):e0082222. doi: 10.1128/aem.00822-22. Epub 2022 Jul 20.
Surface plating on agar and most probable number (MPN) are the standard methods for determining bacterial viability but both have limitations. Here we present a novel cell count method, high-throughput MPN (htMPN), that uses a chip-based digital PCR instrument to accelerate and to improve the quantification of viable or sublethally injured cells. This method tracks growth of up to 20,000 individual bacterial cells on a single chip. Single cells were grown in the individual wells of the chip at their optimal temperature until the cell density was high enough to detect the fluorescent signal with cell-permeant or cell-impermeant DNA-intercalating fluorescent dyes. This method based on microfluidic devices implemented in digital PCR equipment was equivalent to surface plating in determining cell counts of Escherichia coli, Salmonella enterica serovar Typhimurium, Fructilactobacillus sanfranciscensis, Pseudomonas putida, and vegetative cells but not spores of Bacillus subtilis. Viable E. coli could be enumerated within 7 h. Culture of strict aerobes was restricted to strains that are capable of nitrate respiration; organisms requiring complex media that also contain double-stranded DNA were detected after treatment of growth media with DNase before inoculation. Our approach not only monitors the frequency distribution of bacterial growth and determines cell counts with high reliability but also detected heat-injured cells of Typhimurium that escaped detection by the surface plating. Overall, the method accelerates detection of viable bacterial cells, facilitates automation, and offers new possibilities for the analysis of individual bacterial cells. htMPN uses chip-based fluorescence acquisition and is a simple and compact tool for automatic viable cell enumeration with applications in microbiological research. This method applies to a wide range of anaerobic or facultative anaerobic species and improves accuracy by reducing the number of pipetting steps. In addition, the method offers an additional tool for single-cell microbiology. The single cell time-to-detection times have been used as an important criterion for the physiological state of bacterial cells after sublethal stress, and htMPNs support the acquisition of such data with an unprecedented number of cells. In particular, htMPN provides an anaerobic environment and enables a long incubation time to increase the recovery rate of sublethally injured cells. Given its reproducibility and reliability, our approach can potentially be applied to quantify viable cells in samples from environmental, clinical, or food samples to reduce the risk of underestimation of the number of viable bacterial cells.
琼脂表面镀覆和最大可能数（MPN）是测定细菌活力的标准方法，但都有其局限性。在这里，我们提出了一种新的细胞计数方法，高通量 MPN（htMPN），它使用基于芯片的数字 PCR 仪器来加速和改善活细胞或亚致死损伤细胞的定量。该方法可在单个芯片上跟踪多达 20,000 个单个细菌细胞的生长。单细胞在芯片的各个孔中在其最佳温度下生长，直到细胞密度足够高，能够用细胞通透性或非细胞通透性 DNA 嵌入荧光染料检测到荧光信号。这种基于微流控装置的方法在数字 PCR 设备中实现，与表面镀覆法在测定大肠杆菌、肠炎沙门氏菌血清型 Typhimurium、果糖乳杆菌、恶臭假单胞菌和枯草芽孢杆菌营养细胞但不是孢子的细胞计数方面等效。可在 7 小时内对活大肠杆菌进行计数。严格需氧菌的培养仅限于能够进行硝酸盐呼吸的菌株；在接种前，用 DNA 酶处理生长培养基，然后检测复杂培养基中需要双链 DNA 的生物。我们的方法不仅可以监测细菌生长的频率分布，并且可以高度可靠地确定细胞计数，还可以检测到表面镀覆法漏检的 Typhimurium 热损伤细胞。总的来说，该方法加速了活细菌细胞的检测，促进了自动化，并为单个细菌细胞的分析提供了新的可能性。htMPN 使用基于芯片的荧光采集，是一种简单紧凑的自动活细胞计数工具，在微生物研究中有广泛的应用。该方法适用于多种厌氧或兼性厌氧物种，并通过减少移液步骤来提高准确性。此外，该方法为单细胞微生物学提供了一种额外的工具。单细胞检测时间已被用作亚致死应激后细菌细胞生理状态的重要标准，htMPN 支持以空前数量的细胞获取此类数据。特别是，htMPN 提供了一个厌氧环境，并允许长时间孵育以提高亚致死损伤细胞的回收率。鉴于其可重复性和可靠性，我们的方法有可能应用于从环境、临床或食品样本中定量活细胞，以降低低估活细菌细胞数量的风险。
