Phosphate-Buffered Saline or PBS Solution

How To Prepare Phosphate-Buffered Saline Solution

Phosphate-buffered saline or PBS is isotonic to human body fluids.
Phosphate-buffered saline or PBS is isotonic to human body fluids. Eugenio Marongiu / Getty Images

 PBS or phosphate-buffered saline is a buffer solution that is particularly valuable because it mimic the ion concentration, osmolarity, and pH of human body fluids. In other words, it's isotonic to human solutions, so it's less likely to cause cell damage, toxicity, or unwanted precipitation in biological, medical, or biochemical research.

PBS Chemical Composition

There are several recipes to prepare PBS solution.

The essential solution contains water, sodium hydrogen phosphate, and sodium chloride. Some preparations contain potassium chloride and potassium dihydrogen phosphate. EDTA may also be added in cellular preparation to prevent clumping.

Phosphate-buffered saline is not ideal for use in solutions that contain divalent cations (Fe2+, Zn2+) because precipitation may occur. However, some PBS solutions do contain calcium or magnesium. Also, keep in mind phosphate may inhibit enzymatic reactions. Be particularly aware of this potential disadvantage when working with DNA. While PBS is excellent for physiological science, be aware the phosphate in a PBS-buffered sample may precipitate if the sample is mixed with ethanol.

A typical chemical composition of 1X PBS has a final concentration of 10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl. Here's the final concentration of reagents in the solution:

SaltConcentration (mmol/L)Concentration (g/L)
  NaCl  1378.0
  KCl  2.70.2
  Na2HPO4  101.42
  KH2PO4  1.80.24

Protocol for Making Phosphate-Buffered Saline

Depending on your purpose, you may prepare 1X, 5X, or 10X PBS. Many people simply purchase PBS buffer tablets, dissolve them in distilled water, and adjust the pH as needed with hydrochloric acid or sodium hydroxide. However, it's easy to make the solution from scratch.

Here are recipes for 1X and 10X phosphate-buffered saline:

ReagentAmount
to add (1×)
Final concentration (1×)Amount to add (10×)Final concentration (10×)
NaCl   8 g137 mM  80 g 1.37 M
KCl 0.2 g 2.7 mM   2 g 27 mM
Na2HPO4 1.44 g 10 mM14.4 g100 mM
KH2PO4 0.24 g 1.8 mM 2.4 g 18 mM
Optional:
CaCl2•2H2O0.133 g 1 mM1.33 g 10 mM
MgCl2•6H2O 0.10 g 0.5 mM 1.0 g 5 mM

 

  1. Dissolve the reagent salts in 800 ml distilled water.
  2. Adjust the pH to the desired level with hydrochloric acid. Usually this is 7.4 or 7.2. Use a pH meter to measure the pH, not pH paper or other imprecise technique.
  3. Add distilled water to achieve a final volume of 1 liter.

Sterilization and Storage of PBS Solution

Sterilization isn't necessary for some applications, but if your are sterilizing it, dispense the solution into aliquots and autoclave for 20 minutes at 15 psi (1.05 kg/cm2) or use filter sterilization.

Phosphate-buffered saline may be stored at room temperature. It may also be refrigerated, but 5X and 10X solution may precipitate when cooled. If you must chill a concentrated solution, first store it at room temperature until you are certain the salts have completely dissolved. If precipitation does occur, warming the temperature will bring them back into solution.

Shelf life of refrigerated solution is 1 month.

Diluting a 10X Solution to Make 1X PBS

10X is a concentrated or stock solution, which may be diluted to make a 1X or normal solution. A 5X solution must be diluted 5 times to make a normal dilution, while a 10X solution must be diluted 10 times.

To prepare a 1 liter working solution of 1X PBS from a 10X PBS solution, add 100 ml of the 10X solution to 900 ml of water. This only changes the concentration of the solution, not the gram or molar amount of the reagents. The pH should be unaffected.