Determining the minimal concentration of a substance-whether a compound used to inhibit cell growth or a growth-limiting nutrient-can be an arduous process. Carrying out the experiment in flasks or tubes and estimating cell density in a single-sample spectrophotometer has been a routine method of choice due to the ease of measurement and standardized conversions from optical density measurements to cell concentrations. However, when dealing with dozens or more samples, several challenges arise, including increased processing time, increased risk of contamination through repeated sampling, and an increased risk of confounding one sample for another. The protocol described here details a rapid method to estimate cell concentrations for such experiments using a microplate spectrophotometer. Using a microplate spectrophotometer to measure optical density of many cultures can be automated for high throughput and benefits from a reduced risk of contamination. Since one of the caveats of a microplate spectrophotometer is its low saturation limit, we further describe how to convert optical density readings of the microplate spectrophotometer into its single-sample spectrophotometric equivalent. To further illustrate the applicability of this protocol, we compare OD₆₀₀ readings generated using a microplate spectrophotometer and a single-sample spectrophotometer for different nutrient starvations and show that the results are comparable. Overall, this method reduces the required resources, reduces the risk of contamination, and allows for faster processing of samples. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generating an equation to convert microplate spectrophotometer readings to single-sample spectrophotometric values Basic Protocol 2: Evaluating growth-limiting nutrient concentrations using a microplate spectrophotometer.

• PMID: 37166239
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Lewis AG, Gibney PA

Primary Lit For

Additional Lit For

Review For