Department of Physics, Brown University, 182 Hope Street, Providence, RI, 02912, USA.
BMC Microbiol. 2024 Oct 10;24(1):403. doi: 10.1186/s12866-024-03547-3.
Bacterial growth rate, commonly reported in terms of doubling time, is frequently determined by one of two techniques: either by measuring optical absorption of a growing culture or by taking samples at different times during their growth phase, diluting them, spreading them on agar plates, incubating them, and counting the colonies that form. Both techniques require measurements of multiple repeats, as well careful assessment of reproducibility and consistency. Existing literature using either technique gives a wide range of growth rate values for even the most extensively studied species of bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This work aims to apply several methods to reliably determine the growth rate of a recently identified species of Enterobacteriaceae, called Enterobacter sp. SM3, and to compare that rate with that of a well-known wildtype E. coli strain KP437.
We extend conventional optical density (OD) measurements to determine the growth rate of Enterobacter sp. SM3. To assess the reliability of this technique, we compare growth rates obtained by fitting the OD data to exponential growth, applying a relative density method, and measuring shifts in OD curves following set factors of dilution. The main source of error in applying the OD technique is due to the reliance on an exponential growth phase with a short span. With proper choice of parameter range, however, we show that these three methods yield consistent results. We also measured the SM3 division rate by counting colony-forming units (CFU) versus time, yielding results consistent with the OD measurements. In lysogeny broth at 37C, SM3 divides every 21 ± 3 min, notably faster than the RP437 strain of E. coli, which divides every 29 ± 2 min.
The main conclusion of this report is that conventional optical density (OD) measurements and the colony-forming units (CFU) method can yield consistent values of bacterial growth rate. However, to ensure the reproducibility and reliability of the measured growth rate of each bacterial strain, different methods ought to be applied in close comparison. The effort of checking for consistency among multiple techniques, as we have done in this study, is necessary to avoid reporting variable values of doubling time for particular species or strains of bacteria, as seen in the literature.
细菌的生长速度通常以倍增时间来表示，常用的两种测定方法分别为：一种是测量生长培养液的光吸收值，另一种是在生长阶段的不同时间取样，稀释后铺在琼脂平板上，培养后统计形成的菌落数。这两种方法都需要进行多次测量，并仔细评估其重现性和一致性。即使是对大肠杆菌、铜绿假单胞菌和金黄色葡萄球菌等研究最为广泛的细菌物种，现有文献中使用这两种技术的结果也给出了广泛的生长速度值范围。本工作旨在应用多种方法可靠地测定最近鉴定的肠杆菌科 Enterobacter 种 Enterobacter sp. SM3 的生长速度，并将该速度与已知的野生型大肠杆菌菌株 KP437 进行比较。
我们扩展了常规光密度（OD）测量方法来确定 Enterobacter sp. SM3 的生长速度。为了评估该技术的可靠性，我们将通过拟合 OD 数据到指数增长、应用相对密度法以及测量在设定稀释倍数后的 OD 曲线变化来获得的生长速度进行了比较。应用 OD 技术的主要误差源是由于对短时间指数增长阶段的依赖。然而，通过适当选择参数范围，我们表明这三种方法可以得到一致的结果。我们还通过计数时间-菌落形成单位（CFU）来测量 SM3 的分裂率，结果与 OD 测量结果一致。在 37°C 的溶菌肉汤中，SM3 每 21±3 分钟分裂一次，明显快于大肠杆菌的 RP437 菌株，后者每 29±2 分钟分裂一次。
本报告的主要结论是，常规光密度（OD）测量和菌落形成单位（CFU）方法可以得到一致的细菌生长速度值。然而，为了确保每种细菌菌株测量生长速度的重现性和可靠性，应该在紧密比较的基础上应用不同的方法。正如我们在这项研究中所做的那样，检查多种技术之间的一致性的努力是必要的，以避免像文献中那样报告特定细菌物种或菌株的倍增时间的可变值。
