Preparation of Phosphate Buffered Saline (PBS) - General Buffer Method
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Preparation of Phosphate Buffered Saline (PBS) - General Buffer Method
Overview
The protocol describes a generalized method for the preparation, sterilization, and storage of phosphate buffered saline. The pH is generally adjusted to 7.4. There are many variant recipes for PBS that include different components or are adjusted to a different pH.
Procedure
1. Measure a volume of 800 ml of ddH2O with a graduated cylinder and transfer to an Erhlenmyer flask.
2. Add a magnetic stir bar to the Erhlenmyer flask and place the flask on a magnetic stir plate (see Hint #1). Adjust the speed of the magnetic stir bar so that oxygen is not introduced into the solution while it is rapidly mixed.
3. Transfer to the flask:
8 g of NaCl, 0.2 g KCl
1.44 g of Na2HPO4
0.25 g of KH2PO4
4. Allow the solutes to dissolve for 3 to 5 min.
5. Ensure that there are no remaining particles of undissolved salts in the solution before adjusting the pH. If particles are present, continue stirring vigorously (see Hint #2).
6. Reduce the speed of the magnetic stir bar so that the solution is gently mixing.
7. Ensure that the pH meter has been properly calibrated (see Protocol ID# 2160) and rinse the pH probe with ddH2O. Remove the excess water from the probe tip (without touching the probe tip) with a clean paper towel. Place the pH probe into the solution.
8. Slowly add 1 M HCl dropwise with a transfer pipette and allow the HCl to fully dissolve into the solution.
9. Stop stirring the solution (see Hint #3).
10. Measure the pH with the pH meter.
11. Repeat Steps #8 through #10 until the pH of the solution is 7.4.
12. Pour the solution into a fresh graduated cylinder and adjust the final volume to 1 liter with ddH2O.
13. Transfer the solution to a container that is suitable for autoclaving at 15 lb/in2.
14. Autoclave the solution on liquid cycle at 15 lb/in2 for 20 min (see Hint #4).
15. Allow the solution to cool to room temperature.
16. Store the PBS solution at room temperature. The PBS Solution is sterile; when using the PBS Solution, ensure that sterile techniques are employed.
Solutions
PBS
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
137 mM Sodium Chloride (NaCl)
2.7 mM Potassium Chloride (KCl)
1.4 mM Potassium Phosphate, Monobasic (KH2PO4)
See protocol for preparation procedure 
1 M HCl
BioReagents and Chemicals
Hydrochloric Acid
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Protocol Hints
1. The mixing apparatus should be located close to a pH meter to allow easy measurement of the pH.
2. It is important that all of the salt is dissolved into the solution prior to adjusting the pH. If some of the salt is undissoved when the pH of the solution is adjusted, the pH of the solution will drift as the salt dissolves.
3. To achieve an accurate pH reading, do not stir the solution when measuring the pH. If the solution is stirring rapidly, the pH will be incorrectly measured.
4. Ensure that the autoclave is properly programmed before initiating. Refer to the manufacturer's instructions when operating the autoclave.
