Department of Biology, Ouachita Baptist University.
Department of Mathematical Sciences, University of Arkansas.
J Vis Exp. 2023 Jun 30(196). doi: 10.3791/65482.
Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.
噬菌体是自然环境的重要组成部分，它们具有强大的塑造细菌种群的能力。为了了解噬菌体如何与放线菌等生长缓慢的细菌宿主相互作用，需要一种简单可靠的方法来定量测量噬菌体存在时细菌的长期生长情况。分光光度微孔板读数仪可实现高通量重复测量，但长时间孵育小体积可能会带来技术挑战。本规程改编了标准的 96 孔微孔板，允许在不进行亚采样的情况下进行噬菌体和细菌的共培养 96 小时，每隔 8 小时使用分光光度吸光度值记录细菌生长情况。使用 R 对这些光密度值进行分析，得出感染指标，包括生长抑制百分比、相对毒力和 Stacy-Ceballos 指数。这里概述的方法提供了一种有效的方法来进行和分析延长时间的微孔板生长曲线实验，并包括了减少蒸发和盖子凝结的修改。这些方案促进了基于微孔板的缓慢生长细菌宿主与其噬菌体之间相互作用的测定。
