Yarrington Kaitlin D, Sánchez Peña Andrea, Limoli Dominique H
Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa.
Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa;
J Vis Exp. 2020 Aug 5(162). doi: 10.3791/61376.
Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time. Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.
微生物群落普遍存在于自然界中，但在单细胞水平上研究它们的相互作用却很困难。因此，已开发出一种基于显微镜的方法来观察两种细菌病原体之间的种间相互作用。本文展示了使用该方法探究运动性革兰氏阴性病原体铜绿假单胞菌和非运动性革兰氏阳性病原体金黄色葡萄球菌之间的相互作用。该方案包括在盖玻片和琼脂糖垫之间共同接种每种细菌，这将细胞维持在单一平面上，并允许在空间和时间上可视化细菌行为。此外，本文展示的延时显微镜非常适合观察两种或更多种细菌之间发生的早期相互作用，包括单一培养物中以及与其他物种共培养时细菌物种运动性的变化。由于显微镜设置中样本空间有限的性质，一旦细胞群体数量过高，该方案不太适用于研究细菌物种之间的后期相互作用。然而，该方案有几种不同的应用，包括使用染色对活细菌和死细菌细胞进行成像、通过荧光报告基因对基因或蛋白质表达进行定量，以及在单物种和多物种实验中追踪细菌细胞运动。
