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

A hallmark of bacterial populations cultured in vitro 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 Escherichia coli 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，其典型特征为生长均一性：绝大多数菌体的生长速率、细胞尺寸与胞内物质均保持一致。然而近期研究表明，即便处于指数生长期（exponential growth phase）的菌体，在常用于衡量细胞生长的各项指标上也可能存在异质性。细菌异质性对于细菌应对抗生素等环境胁迫的响应机制具有重要研究价值。例如，抗菌持留（antimicrobial persistence）现象便与一小部分进入休眠状态的菌体亚群相关，此时抗生素无法对其发挥杀伤作用。尽管目前已开发出多种可识别细菌培养中单个非增殖菌体的方法，但针对此类菌体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，可同时对多个营养琼脂（nutrient agar）平板上的细菌菌落生长过程进行自动化图像采集与分析。本研究同时阐述了该平台的硬件与软件设计细节，涵盖温控培养箱（temperature-controlled growth chamber）、高分辨率扫描仪（high-resolution scanner）以及用于提取并绘制菌落延滞时间（lag time）与生长动力学（growth kinetics）曲线的图形用户界面（graphical interface）。我们采用大肠杆菌（Escherichia coli）K12野生型菌株开展实验，以验证该平台搭建方案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留性的影响提供全新研究视角。