Enhanced Reproducibility and Precision of High-Throughput Quantification of Bacterial Growth Data Using a Microplate Reader
Summary
Here, a high-throughput protocol is presented to measure growth data, including growth curves, growth rate, and maximum growth rate. The protocol was verified and validated using two biofilm-producing bacteria. The results and approach applied in this study can be expanded to other high-throughput protocols using microplate readers.
Abstract
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−1 ± 0.03 h−1 and 0.85 h−1 ± 0.025 h−1, 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.
Introduction
Developing interest in multi-omics manipulation, including mechanism and metabolic studies of bacteria, emphasizes the importance of high-throughput and automated methods such as recording growth data1,2. Growth data comprising kinetic parameters, such as maximum growth rates, can help characterize bacterial responses to different physical, chemical, and antibacterial conditions. Growth rate data are a standard response variable utilized to uncover potential genotype-phenotypes linkages1 or indicate the microbial safety and shelf life of food produce3,4. Techniques such as adaptive laboratory evolution5,6,7, genome-wide screening, certain chemical assays8, and various forward genetic screens9 rely on growth rates to evaluate the results.
Optical density (OD) measurements of bacterial cultures are a standard microbiological method to monitor bacterial growth. OD measurements are often recorded at a wavelength of 600 nm, relying on light scattering and the cell density10,11. The Beer-Lambert law explains the OD values' dependency on the concentration (i.e., cell density, cell number), path length, and absorptivity coefficient. The geometry and optical system of a spectrophotometer influence the OD readings11. Classical methods of OD measurements can be very time- and labor-intensive, and the data can carry a variety of human errors. In this protocol, a microplate reader is used to decrease the analyst time12,13 and the chance of biological contamination. High-throughput analysis using microplate readers is broadly applied in different microbiology areas, such as screening biofilm-producing bacteria14,15, bacterial growth inhibition16, yeast cell growth17, the determination of antifungal susceptibility18, and toxicity screening of nanomaterials19.
A few researchers have published bacterial growth rate protocols using a microplate reader12,20,21. However, a thorough protocol that examines the reliability of collected data has not been fully established. It is reported that factors such as the type of species22,23,24 and sealing tapes impact the repeatability due to the oxygen transfer inadequacy in a 96-well plate25,26. Delaney et al. reported large clusters of Methylorubrum extorquens (wild-type strain) in the growth medium when using a microplate reader, which caused extremely noisy growth data24. The issue was resolved by removing the genes associated with biofilm production24. Due to the secretion of extracellular polymeric substances, biofilm-producing bacteria have a greater affinity to coalesce together and create cell clusters. Therefore, it is more challenging to monitor their growth using light scattering techniques (e.g., spectrophotometers and microplate readers).
This protocol aims to establish steps to obtain reproducible data in a high-throughput method using a microplate reader. Bacillus mycoides and Paenibacillus tundrae were used due to their fast growth and biofilm-producing ability, which are traditionally challenging in manual and automated approaches. Factors such as (i) pathlength correction, (ii) condensation on the lid, (iii) inoculum size, (iv) sampling time interval, and (v) spatial bias were investigated to assess the reliability and reproducibility of the data. This protocol presents steps for accurately monitoring bacterial growth and measuring specific growth rates using a microplate reader.
Protocol
Representative Results
Discussion
Microplate readers allow for obtaining consistent and repeatable growth rates. This technology minimizes human error and enables high-throughput sampling. The small amount of culture required per sample makes this approach an attractive, low-cost alternative to cell counts using flasks or test tubes. Microplate readers allow a large sample size, increasing the statistical power and subsequently facilitating reliable growth rate calculations while keeping costs and labor low.
This article prese…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by the Natural Sciences and Engineering Research Council (NSERC) / Halifax Water Industrial Research Chair in Water Quality and Treatment (Grant No. IRCPJ 349838-16). The team of authors also would like to acknowledge the help of Anita Taylor in reviewing this article.
Materials
| Centrifuge | Eppendorf | 5810 R | |
| Centrifuge tubes – 15 mL | ThermoFisher- Scientific | 339650 | Sterile |
| Centriguge tubes – 50 mL | ThermoFisher- Scientific | 339652 | Sterile |
| Disposable inoculating loop , 10 µL | Cole-Parmer | UZ-06231-08 | Sterile |
| Erlenmeyer flasks – 250 mL | Cole-Parmer | UZ-34502-59 | Glass |
| Isopropanol | ThermoFisher- Scientific | 396982500 | ≥99.0 |
| Phosphate Buffer Saline | Sigma-Aldrich | P4417 | |
| Pipett tips 1,000 µL | ThermoFisher- Scientific | UZ-25001-76 | |
| Pipett tips 10 mL | ThermoFisher- Scientific | UZ-25001-83 | |
| Pipett tips 200 µL | ThermoFisher- Scientific | UZ-25001-85 | |
| Pipett tips 5 mL | ThermoFisher- Scientific | UZ-25001-80 | |
| Pipettor 1,000 µL | Cole-Parmer | UZ-07909-11 | |
| Pipettor 10 mL | Cole-Parmer | UZ-07909-15 | |
| Pipettor 200 µL | Cole-Parmer | UZ-07909-09 | |
| Pipettor 5 mL | Cole-Parmer | UZ-07859-30 | |
| Tryptic Soy Broth | Millipore | 22091 | Suitable for microbiology |
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