This article details a protocol for quantifying bacterial growth dynamics using optical density measurements. The method allows for high-throughput analysis by utilizing a microplate reader to monitor growth over time.
Begin with bacteria preserved in a glycerol stock, previously grown to the early exponential phase to ensure uniform physiological states and reproducible growth.
Add growth media and vortex to evenly resuspend the bacteria.
Serially dilute the suspension using fresh media to obtain a range of starting cell densities.
Load the samples into the inner wells of a multi-well plate, leaving the edge wells filled with blank media to minimize evaporation-related variability.
Place the plate in a microplate reader set to maintain constant shaking and a temperature optimal for bacterial growth.
As the bacteria multiply, the optical density or OD of the culture increases.
Measure the OD at fixed intervals.
Subtract the initial OD values obtained from the blank wells to correct for background absorbance.
Then, calculate the growth rate from consecutive OD values measured at defined time intervals, enabling high-throughput and precise quantification of bacterial growth dynamics.
For real time recording of growth, add approximately 25 milliliters of M63 to a sterilized reagent reservoir. Then, add 900 microliters of M63 to the microtubes in preparation for making serial dilutions. Next, add 900 microliters of M63 to the thawed glycerol stock and vortex.
Transfer 100 microliters of the 10 fold dilution to another microtube containing 900 microliters of M63 and vortex. Repeat this step until the desired number of dilutions are achieved. Now, fill the wells at the edge of a sterilized 96 well flat bottom microplate with 200 microliters of M63, using an eight channel pipette.
Load 200 microliters of each diluted sample to the microplate wells according to the reference table. To avoid allocational layers due to the heating and the seeding efficiency, never use the wells at edge of the microplate for your samples and load the same sample into multiple wells at varied locations on the microplate. Place the 96 well microplate onto the plate reader.
Open Read Now in Task Manager and choose the program. Click Okay to start measuring. Finally, save the recording as a new experimental file for data analysis.
繁體中文
