# Prepare dilution series
## Pick the right number of flasks
{% hint style="success" %}
## 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.
{% endhint %}
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
{% stepper %}
{% step %}
### 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](https://help.sbtinstruments.com/protocols/optimize-bioprocesses/identify-optimal-inoculum-size/measure-on-cryo-stock "mention").
{% endstep %}
{% step %}
### 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.
{% endstep %}
{% step %}
### Continue dilution series
Repeat step 2 for the subsequent 10-2 to 10-10 flasks.
{% endstep %}
{% step %}
### Incubate overnight
Incubate overnight in shaking incubator at the desired growth conditions. For this *E. coli* example we used 37 °C, 200 RPM.
{% endstep %}
{% endstepper %}
{% hint style="info" icon="turtle" %}
## 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.
{% endhint %}
## Summary
A tenfold dilution series has been prepared from the cryo stock for overnight incubation.\
Next step: [select-relevant-cultures](https://help.sbtinstruments.com/protocols/optimize-bioprocesses/identify-optimal-inoculum-size/select-relevant-cultures "mention").
