Phosphate-Buffered Saline (PBS) is a water-based salt solution widely used in biological research to provide a stable environment for cells and biomolecules. Its primary function is to maintain the solution’s pH and osmotic pressure, ensuring conditions that closely mimic the natural environment of cells within the body. This isotonic quality prevents cells from shrinking or swelling during procedures like washing, dilution, or transport. This procedure details the standard recipe and preparation for the one-times (1X) working solution, the concentration most frequently used in the laboratory.
Essential Components and Target Concentrations
The formulation of 1X PBS relies on four specific salts dissolved in high-purity water, typically distilled or deionized water, to ensure accuracy and prevent contamination. Sodium chloride (NaCl) and potassium chloride (KCl) provide the necessary ionic strength and salinity. Disodium phosphate (\(text{Na}_2text{HPO}_4\)) and monopotassium phosphate (\(text{KH}_2text{PO}_4\)) form the buffering system, which resists changes in pH, keeping the solution near neutrality.
For a standard one-liter batch of 1X PBS, the target molar concentrations are established to achieve the desired isotonic environment. The solution aims for approximately 137 millimolar (mM) NaCl and 2.7 mM KCl. The phosphate buffer component is set at 10 mM \(text{Na}_2text{HPO}_4\) and 1.8 mM \(text{KH}_2text{PO}_4\). To achieve these concentrations for one liter, approximately 8.0 grams of NaCl, 0.2 grams of KCl, 1.44 grams of \(text{Na}_2text{HPO}_4\), and 0.24 grams of \(text{KH}_2text{PO}_4\) are required.
Detailed Preparation Procedure for 1X PBS
Preparation begins by accurately weighing each of the four dry salt components using a precision balance. These salts should be added to a suitable container containing approximately 800 milliliters of the high-purity water. The mixture is then gently stirred, often using a magnetic stirrer, until all the powders are completely dissolved, ensuring the components are fully incorporated before the final adjustments.
The next step involves calibrating the \(text{pH}\) of the dissolving solution to the target value of 7.4, which is the standard for most biological applications. A calibrated \(text{pH}\) meter is used to monitor the value as small amounts of a strong acid, like hydrochloric acid (HCl), or a strong base, like sodium hydroxide (NaOH), are carefully added. Accurate \(text{pH}\) is paramount because the function of the phosphate buffer is dependent on the precise ratio of the two phosphate salts at this specific \(text{pH}\). Once the \(text{pH}\) is confirmed to be 7.4, the solution is transferred to a graduated cylinder or volumetric flask, and additional water is added until the total volume reaches one liter.
Verification and Safe Storage
After the 1X PBS solution is prepared, a final verification of the \(text{pH}\) should be performed to ensure the solution meets the 7.4 target. For applications involving sensitive biological samples, the osmolarity, which reflects the total solute concentration, is also sometimes checked to ensure it is isotonic. If the solution appears cloudy or has visible particulate matter, it should be discarded, as this may indicate contamination or improper dissolution.
For applications involving cell culture, microbiology, or other sterile procedures, the 1X PBS must undergo a sterilization process to eliminate microbial contamination. The two most common methods are passing the solution through a 0.22-micrometer filter or autoclaving it, which involves using high-pressure steam. Once sterilized, the 1X PBS is stable and can be stored at room temperature (15°C to 30°C) for up to a year, especially if kept in a clean, closed container. To minimize the risk of microbial growth over extended periods, some laboratories opt to store the solution at 4°C.