Pandey Santosh, Park Yunsoo, Ankita Ankita, Phillips Gregory J
Electrical and Computer Engineering, Iowa State University, Ames, IA, USA.
Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA.
HardwareX. 2021 Nov 25;10:e00249. doi: 10.1016/j.ohx.2021.e00249. eCollection 2021 Oct.
A hallmark of bacterial populations cultured 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 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.
培养的细菌群体的一个标志是其生长的同质性，即大多数细胞表现出相同的生长速率、细胞大小和内容物。然而，最近的研究表明，即使处于指数生长期的细胞，在通常用于测量细胞生长的变量方面也可能存在异质性。细菌的异质性对于细菌如何应对环境压力（如抗生素）具有重要意义。例如，抗菌持续性现象与一小部分进入休眠状态的细胞有关，在这种状态下抗生素不再有效。虽然已经开发出了识别细菌培养物中单个不生长细胞的方法，但对于这些细胞如何影响固体表面菌落的生长却较少受到关注。作为回应，我们开发了一个低成本的开源平台，用于同时对多个营养琼脂平板上的细菌菌落生长进行自动图像捕获和图像分析。文中包括了硬件和软件的描述，以及关于温度控制生长室、高分辨率扫描仪和用于提取和绘制菌落延迟时间及生长动力学的图形界面的详细信息。使用K12野生型菌株进行了实验，以证明我们装置的可行性和操作。通过自动跟踪菌落中的细菌生长动力学，该系统有潜力揭示关于理解微生物异质性对抗生素抗性和持续性影响的新见解。
