Protocol

1. Prepare as many 500 mL bottles as the number of different solutions to make.

2. Place 50 mL of 10x ACSF without Mg or Ca in each beaker.

3. Weigh and add the desired amount, as indicated below, of magnesium sulfate and calcium chloride in each beaker. Use a small weigh boat and the more precise scale. To add the crystals to the solution in the beaker, add small amounts of bottled DI water into the weigh boat and carefully pour directly into the solution in the beaker without touching the rim. Wash the weigh boat for at least 3 times and pour out from different vertices of the weigh boat.

4. Add bottled DI water up to around 300 mL and make sure everything is dissolved.

5. Add 50 mL of 10x bicarbonate, then add bottled DI water up to around 400 mL

6. Use the osmometer and bottled DI water to achieve an osmolality in the range 295-300 mmol/kg. Add a clean stir bar and stir for 1 minute before taking the first measurement. For subsequent measurements, stir for at least 30 seconds after adding water. Take 2 stable readings for the final solution

Note: If bicarbonate is added before calcium and magnesium are complete dissolved, CaCO₃ might precipitate out:

\[ \text{Ca}(\text{HCO}_3)_2 (aq) \rightarrow \text{CO}_2 (g) + \text{H}_2\text{O} (l) + \text{CaCO}_3 (s) \]

This is irreversible because CO₂ would leave the solution quickly! Therefore, if the solution is cloudy, throw it out and remake!

Solution 1

Solution 2

Solution 3

Solution 4