# Osmotic pressure induced by salt

##### Success Goal

Environmental salt content is one of several factors that influence the growth rates of yeast. Teachers can take many directions for this experiment, like introducing osmotic pressures (hypotonicity and hypertonicity of a cell) or discussing the effectiveness of salt as a food curing agent.

## Requirements​

• At least two available Pioreactors
• Dry baker's yeast
• YPD broth mix, or any other media mix
• Table salt
• Scale (accurate to 0.1)

## Steps​

1. Prepare a sterile stock of 50g YPD / 1L distilled water. Aim to make 20ml × the number of Pioreactors you are using, as each Pioreactor will require about 15mL of media.
2. Weigh table salt to add to your media. This amount can be calculated (as per the example below), or decided by the teacher beforehand.
3. Once stock is cool, divide the stock into the sterilized vials and add salt. You can mix the salt with gentle shaking or by using the Stirring function on the Pioreactor.
4. When the salt is diluted into the media, inoculate the stock with a very small amount of baker's yeast using best practices to avoid other contamination.
• A yeast stock solution can be made by diluting a small amount of yeast in 15mL of YPD stock media, then a drop of this stock solution can be added to your vials. Add 3-4 drops of this yeast slurry.
5. Wipe the vials and place them in the Pioreactor.
6. Visit pioreactor.local and start a new experiment.
7. On the left menu, select the Pioreactors page. Add any additional Pioreactors that you would like to use (more information here). Select Manage all Pioreactors, and start Stirring activity and OD reading activity.
• Optional: you can start the Temperature automation activity set on an optimal temperature.
8. Confirm that everything looks normal on the Overview page (ex: receiving optical density signal).
9. Back on the Pioreactors page, select Manage all Pioreactors and start Growth rate. It will take a minute for results to begin showing up.
10. Optional: you can change the names of the Pioreactor in the UI to display the target temperature.
11. Students can watch growth progress on the Overview page.
12. Within 24 hours, students can observe the growth curves of yeast under various salt stresses.

## Example​

We replicated this experiment using five Pioreactors and vials containing 15 mL of sterile YPD stock and salt concentrations of 0%, 1.5%, 3%, 4.5%, and 6% w/v. A sample calculation is as follows:

$\text {w/v \%}= \frac {\text{mass}}{\text {volume}}$

To create a salt concentration of 3% w/v for 15 mL of media:

$0.03 = \frac {\text{mass}}{15 mL}$
$\text {mass} = 0.45 g$

Therefore, 0.45 grams of table salt is needed for 3% w/v of salt. To each vial, 0g, 0.225g, 0.45g, 0.675g and 0.9g of table salt were added, repectively. They were then innoculated with one drop of rehydrated active yeast (0.500 g of dry yeast in a 15 mL YPD broth vial). The Temperature automation was set on Stable at 32.5°C.

### Results​

After approximately 26 hours, the following implied growth rates were recorded:

We can summarize our results in the following table to highlight the non-linearity of our data:

Salt percent (w/v)Peak growth rate (h⁻¹)Time to reach stationary phase
0% (control)0.788 hours
1.5%0.678.3 hours
3%0.4911.5 hours
4.5%0.2917 hours
6%0.2224 hours

The highest growth was recorded in the vial at 0% salt w/v (no salt stress), at approximately 0.78 h⁻¹. We conclude that the phases of growth (lag, log phase) occured quickest with no salt stress and the stationary phase was reached in the smallest time frame of about 8 hours.

As the salt percentage increases, the peak growth rate decreases and the time to reach the stationary phase was extended. These time increases are non-linear; between 0% and 1.5% salt, the stationary phase was reached within an increment of half an hour however 4.5% and 6% salt had a large increment of 7 hours (from 17 to 24 hours).

Teachers can consider expanding on this experiment. Suggestions include:

• performing the experiment over a longer period of time, or with higher salt percentages; papers commonly go up to 10%.
• exploring pretreated cells (by growing one culture in a saltier environment, then comparing a non-treated culture to the treated culture under the same conditions).
• considering different species and how they react to the same salt conditions.