Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici C. Campus de Bellaterra, Cerdanyola del Vallès, Barcelona, Spain.
Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC), Cerdanyola del Vallès, Barcelona, Spain.
PLoS One. 2019 May 9;14(5):e0216292. doi: 10.1371/journal.pone.0216292. eCollection 2019.
Since 1959 with the proposal of Double Agar Layer (DAL) method for phage detection and quantification, many sophisticated methods have emerged meanwhile. However, many of them are either too complex/expensive or insensitive to replace routine utilization of DAL method in clinical, environmental and industrial environments. For that purpose, we have explored an alternative method for the detection and quantification of bacteriophages that fulfills the criteria of being rapid, simple and inexpensive. In this paper we have developed a method based on the analysis of optical density kinetics in bacterial cultures exposed to phage-containing samples. Although the decrease in optical density caused by cell lysis was one of the first observable consequences of the effect of viral infection in bacterial cultures, the potential of the method for the assessment of phage abundance has never been fully exploited. In this work we carry out a detailed study of optical density kinetics in phage-infected bacterial cultures, as a function of both, phage abundance and initial concentration of the host organisms. In total, 90 different combinations of bacteria/phage concentrations have been used. The data obtained provide valuable information about sensitivity ranges, duration of the assay, percentages of inhibition and type of lysing behavior for each phage concentration. The method described can detect, as few as 10 phage particles per assay volume after a phage incubation period of 3.5h. The duration of the assay can be shortened to 45min at the expense of losing sensitivity and increasing the limit of detection to 108 pfu/ml. Despite using non-sophisticated technology, the method described has shown sensitivity and response time comparable to other high-end methods. The simplicity of the technology and of the analytical steps involved, make the system susceptible of miniaturization and automation for high-throughput applications which can be implemented in routine analysis in many environments.
自 1959 年提出双层琼脂（DAL）方法检测和定量噬菌体以来，同时也出现了许多复杂的方法。然而，其中许多方法要么过于复杂/昂贵，要么不够灵敏，无法替代 DAL 方法在临床、环境和工业环境中的常规应用。为此，我们探索了一种替代方法来检测和定量噬菌体，该方法满足快速、简单和经济的要求。在本文中，我们开发了一种基于分析暴露于含噬菌体样品的细菌培养物的光密度动力学的方法。尽管细胞裂解引起的光密度降低是病毒感染对细菌培养物产生影响的最早可观察结果之一，但该方法评估噬菌体丰度的潜力从未得到充分利用。在这项工作中，我们详细研究了噬菌体感染的细菌培养物中的光密度动力学，作为噬菌体丰度和初始宿主生物浓度的函数。总共使用了 90 种不同的细菌/噬菌体浓度组合。获得的数据提供了有关每种噬菌体浓度的灵敏度范围、测定持续时间、抑制百分比和裂解行为类型的有价值信息。所描述的方法可以在噬菌体孵育 3.5 小时后检测到每个测定体积少至 10 个噬菌体颗粒。通过牺牲灵敏度并将检测限提高到 10^8 pfu/ml，可以将测定时间缩短至 45 分钟。尽管使用了不复杂的技术，但所描述的方法在灵敏度和响应时间方面表现出与其他高端方法相当的性能。该技术和涉及的分析步骤的简单性，使得该系统易于微型化和自动化，适用于许多环境中的高通量应用，可用于常规分析。
