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Methods for preparation of high-titer purified bacteriophage suspensions for use in environmental experiments

机译：用于环境实验的高滴度纯化噬菌体悬浮液的制备方法

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Riverbank filtration (RBF) can effectively remove pathogens while providing more consistentwater quality to subsequent treatment processes. EPA's LT2ESWTR provides regulatory creditfor pathogen removal by RBF. Many laboratory studies have attempted to physically model RBF,but experimental scale, media heterogeneity, and variable flow paths and fluxes have made itdifficult to relate laboratory outcomes to field performance. Accordingly, field studies have beenrelied upon to assess pathogen removal by RBF. In many such studies, organisms are introducedinto the susbsurface and their removal over specified travel distances is evaluated. PRD1 andPRD-like bacteriophages have been extensively used in many such RBF assessments because ofits high stability in aqueous and geologic media and structural and functional similarities tomammalian adenoviruses. Simple and reliable methods for bacteriophage isolation, propagationand enumeration are well established. However, methods for the production of large volumes ofhigh-titer purified phage suspensions that are often required for studying transport in porousmedia are scant. Recent methodological advances are dispersed in the literature. Based onliterature review and our own experimental results with the PR772/E. coli system, we propose aseries of reliable, cost effective and time saving procedures which can be used, with minoradjustments, to propagate, concentrate and purify any bacteriophage. The main focus of this workis to ensure maximum host sensitivity and high bacteriophage yields, effective particleconcentration, and maximum total organic carbon removal from the final lysate to produceoptimal bacteriophage suspensions for use in performance demonstrations.Plating trials achieved ～10x higher PR772 titers than liquid propagations. A few repetitions of theplating method will produce enough phage for use in many studies. When many liters of high titerphage suspension are required, liquid propagation is essential but it should be preceded bypropagation by plating to ensure consistent production of an initial phage suspension, which canthen be used to seed large volumes of liquid media for large-scale propagation. To ensuremaximum yields, a high phage concentration and short contact time (high multiplicity) methodfor liquid propagation of the PR772/E.coli system is recommended. A method using PEG8000and NaCl is a reliable choice for phage concentration since it is fast, gentle, efficient (highrecoveries), easy to use and removes undesirable impurities from phage lysates. Furtherpurification can be achieved by using Vertrel XF followed by centrifugation. The success of theproposed sequence of procedures greatly depends on key details specified in the materials andmethods. To avoid phage sensitivity losses, it is critical to ensure the host bacterium is properlycultured and stored.
机译：河岸过滤（RBF）可以有效去除病原体，同时为后续处理过程提供更一致的水质。 EPA的LT2ESWTR为RBF清除病原体提供了监管信誉。许多实验室研究尝试对RBF进行物理建模，但是实验规模，介质异质性以及可变的流动路径和通量使得将实验室结果与现场性能联系起来变得困难。因此，已经依靠现场研究来评估通过RBF去除病原体。在许多此类研究中，将有机体引入到表层，并评估了它们在指定行进距离内的去除情况。由于PRD1和PRD样噬菌体在水性和地质介质中的高度稳定性以及与哺乳动物腺病毒的结构和功能相似性，因此已广泛用于许多此类RBF评估中。建立了简单可靠的噬菌体分离，繁殖和计数方法。然而，用于研究在多孔介质中运输的经常需要的用于生产大量高滴度纯化的噬菌体悬浮液的方法很少。最近的方法学进展分散在文献中。基于PR772 / E的文献综述和我们自己的实验结果。在大肠杆菌系统中，我们提出了一系列可靠，具有成本效益和节省时间的程序，这些程序可以在不做任何调整的情况下用于繁殖，浓缩和纯化任何噬菌体。这项工作的主要重点是确保最大的宿主敏感性和高噬菌体产量，有效的颗粒浓度以及最大程度地从最终裂解液中去除总有机碳，以生产用于性能演示的最佳噬菌体悬浮液。镀敷试验的PR772滴度比液体繁殖高出约10倍。重复几次铺板方法将产生足够的噬菌体，以用于许多研究。当需要许多升高滴度的噬菌体悬浮液时，液体的传播是必不可少的，但在进行繁殖之前应先进行平板接种以确保始终产生初始噬菌体悬浮液，然后将其用于播种大量液体培养基以进行大规模繁殖。为了确保最大产量，建议使用高噬菌体浓度和短接触时间（高多重性）的方法来传播PR772 / E.coli系统的液体。使用PEG8000和NaCl的方法是噬菌体浓缩的可靠选择，因为它快速，温和，高效（高回收率），易于使用并从噬菌体裂解物中去除了不良杂质。通过使用Vertrel XF然后离心可以进一步纯化。提议的程序顺序的成功很大程度上取决于材料和方法中指定的关键细节。为了避免噬菌体敏感性损失，确保宿主细菌正确培养和储存至关重要。

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