Making PBS (phosphate-buffered saline) requires dissolving four salts in distilled water and adjusting the pH to 7.4. The standard 1X recipe calls for 8 g sodium chloride, 1.44 g dibasic sodium phosphate, 0.24 g monobasic potassium phosphate, and 0.2 g potassium chloride per liter. You can prepare it directly at working strength or make a 10X concentrated stock for convenience.
1X PBS Recipe (1 Liter)
Start with 800 mL of distilled water in a clean bottle or beaker. Weigh out and add the following salts:
- NaCl: 8 g
- Na₂HPO₄ (dibasic sodium phosphate): 1.44 g
- KCl (potassium chloride): 0.2 g
- KH₂PO₄ (monobasic potassium phosphate): 0.24 g
Stir until all salts dissolve completely. Check the pH and adjust to 7.4 (or 7.2, depending on your application) using small additions of 1 M NaOH or 1 M HCl. Then bring the total volume to 1 liter with distilled water. Starting with only 800 mL gives you room to adjust pH and top off without overshooting your final volume.
The final molar concentrations are approximately 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.47 mM KH₂PO₄.
10X Concentrated Stock Recipe (1 Liter)
A 10X stock saves time when you use PBS frequently. You simply dilute it 1:10 with distilled water whenever you need a working solution. For 1 liter of 10X PBS, combine:
- NaCl: 80 g
- Na₂HPO₄·2H₂O (dibasic sodium phosphate dihydrate): 17.8 g
- KH₂PO₄: 2.4 g
- KCl: 2 g
Dissolve everything in roughly 800 mL of distilled water, then bring the volume to 1 liter. The pH of 10X stock will sit around 6.8, which looks low. Don’t worry about adjusting it at this stage. Once you dilute it to 1X, the pH shifts up to approximately 7.4 on its own.
Note the sodium phosphate form: the 10X recipe above uses the dihydrate (Na₂HPO₄·2H₂O), which weighs more per mole than anhydrous Na₂HPO₄. If your bottle says “anhydrous” or “heptahydrate,” the gram amounts will differ. Always check which form you have on the shelf and recalculate if needed.
Adjusting the pH
The target pH for most applications is 7.4, though some protocols call for 7.2. Use either 1 M NaOH to raise the pH or 1 M HCl (some protocols use 3 M HCl) to lower it. Add small amounts at a time while stirring and re-reading the meter. Overshooting and then back-titrating adds unnecessary salt to your solution, which can subtly change the ionic strength. It’s better to approach your target slowly from one direction.
Phosphate buffers have a slight temperature dependence. PBS adjusted to pH 7.4 at room temperature (25°C) will read a bit lower if you measure it cold, dropping roughly half a pH unit between room temperature and freezing. For most routine lab work this is negligible, but it matters if you’re doing pH-sensitive experiments at 4°C or 37°C. Calibrate your pH meter at the temperature you plan to use the buffer.
Sterilization
PBS can be sterilized by autoclaving on a liquid cycle at 121°C for 15 to 20 minutes. This is the most common method for lab-prepared PBS and works well for most applications.
If your PBS contains heat-sensitive additives, filter-sterilize it instead by passing it through a 0.22 µm syringe filter or bottle-top filter into a sterile container. Either method produces a sterile solution suitable for cell culture and other sensitive work.
Storage and Shelf Life
Autoclaved 1X PBS stores at room temperature and remains stable for months. A 10X stock kept at room temperature is stable for up to three years. There’s no need to refrigerate standard PBS unless your lab protocol specifically requires it. Keep the cap tight to prevent evaporation, which would concentrate the salts and shift your ionic strength over time.
If you notice crystals or precipitate forming in a 10X stock, especially in cooler rooms, warm the bottle gently and swirl until everything redissolves. The high salt concentration in 10X solutions can come out of solution at lower temperatures, but this reverses easily with gentle warming.
When to Add Calcium and Magnesium
Standard PBS does not contain calcium or magnesium. Some applications, particularly tissue dissection, immunostaining, and perfusion, call for PBS supplemented with both calcium and magnesium ions. These divalent cations support cell adhesion and help maintain tissue integrity during handling. This version is often sold commercially as DPBS with calcium and magnesium.
For applications like cell dissociation or passaging, you typically want PBS without calcium and magnesium, since removing those ions helps cells detach. Keep in mind that adding calcium to phosphate-containing solutions can cause calcium-phosphate microprecipitates to form, especially at higher concentrations. If you see unexpected cloudiness after adding calcium, that’s likely what’s happening. A calcium chelator like EDTA will dissolve these precipitates.
Common Mistakes to Avoid
The most frequent error is using the wrong form of sodium phosphate. Anhydrous, dihydrate, and heptahydrate versions of Na₂HPO₄ all have different molecular weights. A recipe written for one form will give the wrong phosphate concentration if you substitute another without recalculating. The 1X recipe above (1.44 g) uses anhydrous Na₂HPO₄. The 10X recipe (17.8 g) uses the dihydrate. Check your reagent bottle’s label carefully.
Another common issue is adjusting the pH of a 10X stock to 7.4 before dilution. The concentrated stock is supposed to be around pH 6.8. If you force it to 7.4 at 10X concentration, your 1X working solution will end up too alkaline. Only adjust pH after diluting to final working concentration, or trust that the 10X stock will self-correct upon dilution.
Finally, always use distilled or deionized water. Tap water contains variable amounts of calcium, magnesium, and other ions that will change the buffer’s composition and can cause precipitation of phosphate salts.