Scan4CFU: Low-cost, open-source bacterial colony tracking over large areas and extended incubation times

A hallmark of bacterial populations cultured <em>in vitro</em> is their homogeneity of growth, where the majority of cells display identical growth rate, cell size and content. Recent insights, however, have revealed that even cells growing in exponential growth phase can be heterogeneous with respect to variables typically used to measure cell growth. Bacterial heterogeneity has important implications for how bacteria respond to environmental stresses, such as antibiotics. The phenomenon of antimicrobial persistence, for example, has been linked to a small subpopulation of cells that have entered into a state of dormancy where antibiotics are no longer effective. While methods have been developed for identifying individual non-growing cells in bacterial cultures, there has been less attention paid to how these cells may influence growth in colonies on a solid surface. In response, we have developed a low-cost, open-source platform to perform automated image capture and image analysis of bacterial colony growth on multiple nutrient agar plates simultaneously. The descriptions of the hardware and software are included, along with details about the temperature-controlled growth chamber, high-resolution scanner, and graphical interface to extract and plot the colony lag time and growth kinetics. Experiments were conducted using a wild type strain of <em>Escherichia coli </em>K12 to demonstrate the feasibility and operation of our setup. By automated tracking of bacterial growth kinetics in colonies, the system holds the potential to reveal new insights into understanding the impact of microbial heterogeneity on antibiotic resistance and persistence.体外（in vitro）培养的细菌种群的典型特征之一是其生长均一性：绝大多数菌体展现出一致的生长速率、细胞尺寸与胞内物质组成。然而，近期研究表明，即便处于指数生长阶段的细菌细胞，在常用于表征细胞生长的各类指标上也可存在异质性。细菌异质性对于细菌应对环境胁迫（如抗生素）的响应过程具有重要意义。例如抗菌持久性（antimicrobial persistence）现象，便与一小部分进入休眠状态的细菌亚群相关——此时抗生素无法对这些细胞产生杀伤效果。尽管目前已开发出多种方法用于鉴定细菌培养物中的单个非生长细胞，但针对这类细胞如何影响固体表面菌落生长的研究却相对匮乏。为此，我们开发了一套低成本开源平台，可同时对多张营养琼脂平板上的细菌菌落生长开展自动化图像采集与分析工作。本研究详述了该平台的硬件与软件细节，同时涵盖了温控培养箱、高分辨率扫描仪以及用于提取并绘制菌落延滞期与生长动力学参数的图形用户界面的相关信息。我们采用野生型大肠杆菌（Escherichia coli）K12菌株开展实验，以验证该装置的可行性与操作流程。通过自动化追踪菌落的细菌生长动力学，该系统有望为解析微生物异质性对抗生素耐药性与持久性的影响提供全新的研究视角。