Get high-quality overnight cultures from cryo stocks
This is a practical how-to guide to identify a suitable seeding ratio that ensures a high-quality overnight culture after the desired incubation time.
Introduction
In the encyclopedia section Overnight cultures we showed that overseeding may result in low-quality cultures. Below we detail how to easily identify the optimal volumetric inoculation ratio that results in a high-quality overnight culture from a cryo stock. In this experiment, we conduct a screening by performing 1:10 dilutions directly in shake flasks. These are incubated overnight and characterized using BactoBox® measurements. This process helps identify cultures with high cell concentrations while minimizing prolonged exposure to stressful stationary phase conditions.
General notes and recommendations
Start with a broad dilution series with a total of 9 shake flasks covering a volumetric seeding from 10-2 to 10-10 (v/v). Note that 10-2 is equivalent to 1% (v/v). Though it may appear excessive, our experience shows that a broad series is beneficial for the initial screening experiment. For future fine-tuning, you may consider using a narrower dilution series.
The below protocol will work for fast-growing bacteria with a generation time of less than 30 min. Adaptations to incubation times and dilution factors may be necessary for slow-growing bacteria.
For E. coli, use LB medium at a concentration of 25 g/L. We recommend filtering the fully dissolved medium through a 0.2-micron filter prior to autoclaving to remove any particulate matter from the cultivation broth.
When preparing cultivation media, it is standard to autoclave the medium directly in shake flasks. However, this can lead to evaporation and a reduced final volume, affecting the accuracy of overnight dilution series. Instead, sterilize empty shake flasks and, once cooled, add the medium using a serological pipette.
Check if the culture medium contains bacteria-like background that may confound the data interpretation: Prior to the experiment, Measure a 1:100 dilution of the non-inoculated culture medium on BactoBox®.
If the results shows >30,000 cells/mL, then it is beneficial to filter the medium through 0.2 µm syringe/vacuum filter prior to autoclaving.
If the results shows <30,000 cells/mL, then there is no need to filter the medium.
Use the BacTotal_v2024-10 for measurements. This ruleset applies to most bacteria but does not differentiate between live and dead cells.
Note that a new optimization experiment must be done if any changes are made with respect to cryo stocks, vessel type, broth, and incubation time.
Prepare flasks and media
Prepare 1 L culture medium in a bluecap flask as recommended by the manufacturer. Ensure that the powder is fully dissolved by magnetic stirrer before proceeding to the next step.
Optional: Remove particulates from culture medium by 0.2 µm filters if necessary. See the above recommendations for more information.
Sterilize the bluecap flask containing the cultivation medium and 9x 250 mL Erlenmeyer baffled flasks with vented caps. Follow the medium manufacturer's autoclaving instructions: typically 121 °C, ~15 psi for 15 minutes.
Once the flasks and cultivation medium have cooled, proceed with the following transfers:
1x flask for 1% (v/v) dilution: Transfer 49.5 mL broth using a serological pipette.
8x flask for dilutions: Transfer 45 mL broth using a serological pipette.
Prepare dilution series for overnight incubation
Label the flasks from 10-2 to 10-10 as shown in the below illustration.
Allow the cryo stock to thaw at room temp, vortex thoroughly and prepare the first 10-2 (v/v) dilution by transferring 0.5 mL cryo stock to 49.5 mL medium.
Cap flask and swirl for 10 seconds to disperse the cells in the broth.
Prepare a 1:10 dilution directly in the next flask by transferring 5 mL of the preceding flask to the 45 mL culture medium.
Repeat steps 3 and 4 until the entire dilution series has been prepared.
Incubate overnight in shaking incubator at the desired growth conditions, e.g. 37 °C, 200 RPM for E. coli.
Measure bacterial concentration in overnight cultures
After the desired incubation time, identify the flasks that appear turbid to the naked eye .
Start BactoBox® measurements on these flasks from the flask with the highest dilution factor, i.e. the rightmost slightly turbid flask in the illustration above.
Use a serological pipette to extract 5 mL sample to a 15 mL centrifuge tube
Disaggregate cells by vortexing. Usually 30 seconds of vortexing at max speed is sufficient, but some bacteria require more aggressive sample workup. See the section Sample preparationfor more details.
Dilute 1:100 in BactoBox® diluent by transferring 101 µL sample to 10 mL diluent.
Vortex 10 seconds at max speed and measure on BactoBox®.
If the result is within 30,000 - 5,000,000 note down the value. If outside this range, see the section Hit the right concentration for how to get a value within the accepted range.
Work your way up the dilution series until the determined cells/mL concentration is similar to the preceding flask (e.g. within 50%).
Note that it will likely be necessary to dilute the samples from higher-concentration flasks more prior to BactoBox® analyses. A 1:10,000 (10⁻⁴) dilution factor is typically sufficient. To achieve a 1:10,000 dilution, perform two consecutive 1:100 dilutions. For additional information, refer to Hit the right concentration.
Use the obtained results to identify the culture that has a high bacterial concentration, but is yet to reach stationary stage.
In the below bar chart example the best culture would be the flask with the green cap seeded at an initial volumetric dilution down to 10-7 (v/v).
In the example below - for the given batch of cryo stocks, vessel type, broth, and incubation time - the optimal inoculation ratio would be 10-7 (v/v). This corresponds to as little as 0.005 µL cryo stock to 50 mL of broth. Since 0.005 µL is impossible to add by standard pipetting, it is best to do a small-volume 1:10 serial dilution of the cryo stock down to 10-5. Then add 0.5 mL diluted cryo stock to 49.5 mL broth and start the overnight incubation.
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