Chemical and Biological Engineering Department, University of British Columbia.
Process Engineering and Applied Science, Dalhousie University.
J Vis Exp. 2022 Jul 27(185). doi: 10.3791/63849.
This study aimed to develop a repeatable, reliable, high-throughput protocol to monitor bacterial growth in 96-well plates and analyze the maximum growth rate. The growth curves and maximum growth rates of two bacterial species were determined. Issues including (i) lid condensation, (ii) pathlength correction, (iii) inoculation size, (iv) sampling time interval, and (v) spatial bias were investigated. The repeatability of the protocol was assessed with three independent technical replications, with a standard deviation of 0.03 between the runs. The maximum growth rates of Bacillus mycoides and Paenibacillus tundrae were determined to be (mean ± SD) 0.99 h ± 0.03 h and 0.85 h ± 0.025 h, respectively. These bacteria are more challenging to monitor optically due to their affinity to clump together. This study demonstrates the critical importance of inoculation size, path length correction, lid warming, sampling time intervals, and well-plate spatial bias to obtain reliable, accurate, and reproducible data on microplate readers. The developed protocol and its verification steps can be expanded to other methods using microplate readers and high-throughput protocols, reducing the researchers' innate errors and material costs.
本研究旨在开发一种可重复、可靠、高通量的方案，以监测 96 孔板中的细菌生长并分析最大生长速率。确定了两种细菌的生长曲线和最大生长速率。研究了包括（i）盖子冷凝、（ii）光路长度校正、（iii）接种量、（iv）采样时间间隔和（v）空间偏倚在内的问题。该方案的重复性通过三个独立的技术重复进行评估，运行之间的标准偏差为 0.03。确定分枝杆菌和嗜冷杆菌的最大生长速率分别为（平均值±标准差）0.99 h±0.03 h 和 0.85 h±0.025 h。由于这些细菌具有团聚的倾向，因此它们在光学上更难监测。本研究表明，接种量、光路长度校正、盖子加热、采样时间间隔和微孔板空间偏倚对于在微孔板读数器上获得可靠、准确和可重复的数据至关重要。所开发的方案及其验证步骤可以扩展到其他使用微孔板读数器和高通量方案的方法，从而减少研究人员的固有误差和材料成本。
