Purpose:

We think that our experimental pipeline for measuring salt tolerance is close to finalized, and this document does some exploration of whether we should make any changes to the analysis before we are confident in the technique.

<aside> 🟢

Main conclusions:

• Switch to quantifying salt tolerance relative to a control strain
• Switch to incorporating the control strain on to every plate, and run many plates </aside>

Experimental setup:

Our plate deck can hold everything we need to set up 4 plates at once, so we’ve mostly been doing experiments in sets of 4 plates.

The overall protocol is:

1. For each strain, seed 4 replicate overnights from glycerol stocks, grow overnight at high-speed shaking.
2. The next day, normalize ODs and seed into salt plates.
3. Grow for 24 hours at high-speed shaking, measure final OD.

Current plate setup:

We do one strain per plate, with 4 replicates per plate:

If we’re testing 4 strains, we make 4 plates, each of which contains 1 strain. So one plate is for the negative control, then each experimental stain gets its own plate, so an experiment will look like this:

Proposed future plate setup:

Put every strain on every plate, as such:

So we have 4 plates that are effectively replicates of each other. Yup, that is an improvement we will make.

IC50 calculation