The overall goal of this experiment is to analyze bacterial growth precisely in a high throughput manner. Using this procedure, either multiple media or varied strains can be tested simultaneously. This method can help answer key questions in the systems biology and the microbiology fields such as, what determination factors of cell growths are.
The main advantages of this technique is that it allows researchers to precisely evaluate global parameters of maximal growth rate and maximal population density in growth curves of bacteria cells. Though this method can provide insight into e. Coli growth, it can also be applied to other micro-organisms such as, the growth assay and screening of environmental bacteria.
To begin, place five sterilized glass tubes with silicone rubber stoppers, pipettes and the target strains on a clean bench. Expose the mouth of the glass tube to a bunsen burner before opening the silicone rubber stopper. Expose the silicone rubber stopper to the flame after the tube is opened and then lightly place the cap back on the glass tube.
Next, use the electronic pipette and disposable serological pipette to add five milliliters of M63 to one of the glass tubes and 4.5 milliliters of M63 to other four tubes. Use the p200 tip to pick a colony and inoculate it in the glass tube containing five milliliters of M63. Vortex the tube to make a suspension.
Then, dilute the solution 10 fold by transferring 0.5 milliliters of this solution to one of the four tubes containing 4.5 milliliters of M63. Repeat this process for the remaining tubes. After sterilizing the mouths of the glass tubes and the silicone rubber stoppers, cap the tubes with the stoppers.
Then, place the five tubes in a pre-warmed shaking incubator at 37 degree celsius and shake it 200 rpm. Incubate the culture overnight or for 10 to 30 hours. Following incubation, add 1, 000 microliters of M63 to a disposable cuvette with the p1000.
Place the disposable cuvette in a spectrophotometer. Start the program at a fixed wavelength of 600 nanometers and measure the blank. Move the five glass tubes from the shaking incubator to the clean bench.
Discard M63 from the disposable cuvette and add 1, 000 microliters of culture to the same disposable cuvette with the p1000. Then, measure the optical turbidity of the cell culture. Next, add 250 microliters of sterilized 60%glycerol solution and 750 microliters of the selected cell culture to a 1.5 milliliter microtube and mix by pipetting.
Place the remaining nine microtubes in the microtube stand and dispense 100 microliters of the prepared mixture to each tube. Store the stocks in the deep freezer at 80 degrees celsius. To set up the microplate reader, first open the software, open protocols in the task manager and choose create new.
Then, select standard protocol. Open procedure and adjust the settings. Open set temperature and select, incubator on.
Then, set temperature to 37 degree celsius and gradient to zero degree celsius. Check preheat before continuing with the next step. Open start kinetic and set run time for 24 or 48 minutes, then, set the interval 30 minutes or one hour.
Open shake and set shake mode as linear. Check continuous shake and set frequency at 567 cpm. Next, open read, check absorbance, endpoint/kinetic and monochromators.
Set the wavelength to 600. Finally, click validate to confirm that the procedure is correct. And click save to save it as a new program for future use.
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 and task manager and choose the program. Click okay to start measuring. Finally, save the recording as a new experimental file for data analysis.
Shown here is an example of a reference table that was made before performing the experiment. A 96 well plate pictogram was drawn as an eight by 12 table to indicate the positions of the inoculated culture samples on the 96 well plate. The growth rate is calculated by applying the equation for all pairs of consecutive values of OD600.
The mean and the standard deviation of five consecutive growth rates were calculated to estimate the maximal growth rate. The growth rates of a total of 60 cell cultures are indicated as the displayed heat map. Degradation from white to red indicates the growth rates from low to high.
Once mastered, this technique can be done within a few minutes if it is performed properly and in order. After watching this video, you should have a good understanding of how to prepare and load samples to the microplate for highly precise measurements of bacterial growths in a high throughput manner. While attempting this procedure, it's important to remember to make a reference table before performing the experiment to avoid cell cultures of low density and to make sure the cells start growing within 24 hours.
Following this procedure, other methods like automation of experimental manipulation and the computational analysis can be performed in order to answer additional questions like what determination factors for population propagation and the dynamics are. After its development, this technique paved the way for researchers in the fields of systems biology and microbiology. To explore the fundamental principles in cell growth and to optimize compositions of culture media.
