Choose best seeding ratio

The measurements are now completed and it is time to choose the inoculation ratio that results in the best https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md. In this 11-hour E. coli example, the 10^-5 cryo stock dilution produces a culture that has likely just arrived at stationary stage. The 10^-4 cryo stock dilution has a similar cell concentration but is less suitable due to being in the stationary phase longer. It therefore likely contains a higher proportion of cells that have lost culturability.

The inoculation ratio is only valid for the given cryo stock and growth parameters.

Conduct a new optimization experiment if there are any changes to cryo stocks, vessel type, broth, or incubation time.

How to prepare high dilutions of the cryo stock

In the present example, a 10^-5 dilution resulted in the best https://github.com/sbtinstruments/help--mpd/blob/main/encyclopedia/bacterial-growth/high-quality-culture.md. This ratio is equivalent to a 1:100,000 dilution of the cryo stock, i.e substantially higher dilution than the typical 1:100 volumetric seeding ratio.

For future 11-hour overnight cultures, this would correspond to adding just 1 µL cryo stock to 100 mL (100,000 µL) growth medium. Because it it tricky to accurately pipette 1 µL we recommend that you first prepare a 1:100 dilution and then dilute this 1:1 000 for the final 1:100,000 dilution:

1. Prepare 1:100 dilution: Transfer 0.1 mL cryo stock to 9.9 mL sterile growth medium

2. Prepare 1:100,000 dilution: Transfer 0.1 mL 1:100 dilution to ~100 mL sterile growth medium in shake flask

Summary and next steps

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