Phosphate-Buffered Saline (PBS) is a salt solution used extensively across biological research and medical diagnostics. It mimics the ion concentration and osmolarity found within the human body, making it a non-toxic, isotonic medium for cells and tissues. The designation “10x” indicates a concentrated stock, ten times stronger than the standard 1x working concentration. Preparing a concentrated stock allows researchers to save time and space by making large batches that can be diluted as needed for various applications.
Essential Ingredients and Their Purpose
The functional integrity of 10x PBS relies on a precise balance of four dissolved inorganic salts. These include two sodium-based salts, Sodium Chloride (\(\text{NaCl}\)) and Disodium Phosphate (\(\text{Na}_2\text{HPO}_4\)), and two potassium-based salts, Potassium Chloride (\(\text{KCl}\)) and Monopotassium Phosphate (\(\text{KH}_2\text{PO}_4\)). The combination of the monobasic (\(\text{KH}_2\text{PO}_4\)) and dibasic (\(\text{Na}_2\text{HPO}_4\)) phosphate salts creates the solution’s buffer system.
This phosphate pair provides the solution with its ability to resist changes in pH when small amounts of acid or base are introduced. \(\text{NaCl}\) and \(\text{KCl}\) are present in higher concentrations and primarily maintain the solution’s osmolarity. The balanced salt concentration in the 1x working solution mimics the cytoplasm of most cells, preventing water movement and preserving cellular structural integrity during experiments.
Step-by-Step Recipe for 10x PBS Stock
Preparing 10x PBS stock requires accurate weighing and measurement. For a standard 1 Liter volume of 10x PBS, the following salts are typically measured:
- 80 grams of Sodium Chloride (\(\text{NaCl}\))
- 2.0 grams of Potassium Chloride (\(\text{KCl}\))
- 14.4 grams of Disodium Phosphate (\(\text{Na}_2\text{HPO}_4\))
- 2.4 grams of Monopotassium Phosphate (\(\text{KH}_2\text{PO}_4\))
Confirm the exact hydrate form of the salts, as this can alter the required weight.
Transfer the weighed salts to a clean beaker or flask. Begin preparation by adding approximately 800 milliliters of high-quality water, such as deionized or ultrapure water. Do not add the full 1 Liter yet, as the final volume adjustment must occur after the pH is set. Stir the mixture using a magnetic stirrer until all the salts are completely dissolved.
Once the salts are fully in solution, measure and adjust the pH. The target pH for the final 1x working solution is typically 7.4, mimicking the physiological pH of mammalian blood. The 10x stock solution itself often measures around pH 6.8, which shifts to 7.4 upon dilution to the 1x concentration.
If the 10x solution’s pH needs adjustment, use a small volume of concentrated acid, such as hydrochloric acid (\(\text{HCl}\)), or a concentrated base, like sodium hydroxide (\(\text{NaOH}\)). Perform this adjustment slowly using a calibrated \(\text{pH}\) meter to ensure accuracy, as buffering capacity depends on the correct phosphate ratio. After the \(\text{pH}\) is finalized, add the remaining water to bring the total volume of the 10x PBS stock solution precisely to 1 Liter.
Dilution and Proper Storage Methods
The 10x PBS stock solution must be diluted before use in biological experiments. To prepare the standard 1x working solution, combine one part of the 10x stock with nine parts of deionized or ultrapure water (a tenfold dilution). For example, 100 milliliters of 10x stock plus 900 milliliters of water yields 1 Liter of 1x PBS.
Dilution reduces the concentration of all four salts to their working levels, making the solution isotonic and buffered to pH 7.4. The 10x stock solution is highly stable and can be stored at room temperature, remaining viable for at least one year. Storing the concentrated stock in a refrigerator is discouraged because the high salt concentration can lead to precipitation (white crystals).
If precipitation occurs in the 10x stock, redissolve the crystals by gently warming the solution back to room temperature and stirring. For sensitive biological applications, the 1x working solution should be sterilized to prevent microbial contamination. Sterilization is commonly achieved by passing the solution through a 0.22-micrometer filter or by autoclaving it for 20 minutes at \(121^{\circ}\text{C}\). The sterilized 1x PBS should be stored at \(4^{\circ}\text{C}\) to maintain stability, though its shelf life is shorter than the 10x stock.