Prepare dilution series

Pick the right number of flasks

Choose the right number of flasks

If the concentration in the cryo stock is lower than 1 × 10⁸, using 10 flasks is excessive. As a general guideline, add 2 to the exponent. In this example, the number of flasks would be 8 + 2 = 10.

In the present example, the concentration in the cryo vial is 9.6 × 10⁸ cells/mL. As shown in the table, diluting beyond the 10^-10 flask is unnecessary, as higher dilutions likely contain fewer than one cell. Thus, the growth medium will likely remain sterile after overnight incubation. This is why we choose 10 flasks for the dilution series.

Flask	Cells per mL	Cells per 10 mL
10-1	9.6 × 107	960 000 000
10-2	9.6 × 106	96 000 000
10-3	9.6 × 105	9 600 000
10-4	9.6 × 104	960 000
10-5	9.6 × 103	96 000
10-6	9.6 × 102	9 600
10-7	9.6 × 101	960
10-8	9.6 × 100	96
10-9	9.6 × 10-1	~10
10-10	9.6 × 10-2	~1

Introduction to workflow

The dilution series is prepared as a 1:10 dilution series directly in shake flasks.

High-level view of OptiSeeding: Dilution series are prepared directly in shake flasks and then incubated overnight.

For each shake flask 1 mL of the previous dilution (or cryo stock) is transferred to 9 mL of growth medium. The flask is capped and the bacteria are dispersed by figure 8 movements.

Step by step

1

Vortex the cryo stock for a single-cell suspension

Briefly vortex the cryo stock for 10 seconds at maximum RPM. This assumes that the cryo stock was already thoroughly vortexed in the previous step Measure on cryo stock.

2

Prepare first 1:10 dilution

Transfer 1 mL cryo stock to 9 mL growth medium.

Use long, sterile pipette tips to avoid contamination from the non-sterile pipette.

Cap flask and make figure 8 motion to distribute the cells evenly.

3

Continue dilution series

Repeat step 2 for the subsequent 10^-2 to 10^-10 flasks.

4

Incubate overnight

Incubate overnight in shaking incubator at the desired growth conditions. For this E. coli example we used 37 °C, 200 RPM.

Do your bacteria have slow generation time?

The workflow presented here works for bacteria with a generation time of ≤ 30 min. Adjustments may be necessary when working for slow-growing bacteria:

• Increase incubation time.

• Use 1:5 dilutions when preparing the shake flask dilution series.

Summary

A tenfold dilution series has been prepared from the cryo stock for overnight incubation. Next step: Select relevant cultures.