Phosphate Buffered Saline (PBS) is a fundamental water-based salt solution. It maintains a stable pH and provides an isotonic environment, mimicking conditions in living organisms. Its balanced composition preserves biological material integrity and function, making it an important laboratory tool for maintaining physiological conditions.
Gathering Your Materials
Preparing 1 liter of 1x PBS requires specific chemical reagents and equipment. You will need Sodium chloride (NaCl), Potassium chloride (KCl), Disodium phosphate (Na2HPO4), and Monopotassium phosphate (KH2PO4). These salts establish isotonicity and buffering. NaCl and KCl contribute to the solution’s osmolarity, matching biological fluid concentrations. The phosphate salts (Na2HPO4 and KH2PO4) buffer the solution, resisting pH changes and maintaining it around 7.4.
Essential equipment includes a beaker or flask, a magnetic stir plate and stir bar, a pH meter for pH measurement and adjustment, a graduated cylinder for accurate volume measurements, and sterile bottles or containers for storage. Using deionized or distilled water is crucial to prevent contamination from impurities.
Precise Preparation Steps
To prepare 1 liter of 1x PBS, begin by adding approximately 800 milliliters of deionized water to a clean beaker or flask. Weigh out the following dry chemical reagents: 8.00 grams of Sodium chloride (NaCl), 0.20 grams of Potassium chloride (KCl), 1.44 grams of Disodium phosphate (Na2HPO4), and 0.24 grams of Monopotassium phosphate (KH2PO4). Add these weighed salts to the deionized water in the beaker.
Place the beaker on a magnetic stir plate and add a stir bar to facilitate the complete dissolution of the salts. Allow the solution to stir until all solid components have fully dissolved. Once dissolved, the next step involves adjusting the pH of the solution. Using a calibrated pH meter, measure the current pH of the PBS solution.
The target pH for 1x PBS is typically 7.4, which closely matches the physiological pH of many biological systems. If the measured pH deviates from 7.4, add small aliquots of hydrochloric acid (HCl) or sodium hydroxide (NaOH) to adjust it. For example, if the pH is too high, add dilute HCl drop by drop, while if it is too low, add dilute NaOH. Continue stirring and checking the pH until it reaches 7.4. Finally, transfer the solution to a 1-liter graduated cylinder and add additional deionized water until the total volume reaches 1 liter.
Ensuring Purity and Proper Storage
After preparation, verify the pH using a freshly calibrated pH meter to confirm it remains at 7.4, as slight deviations can impact experimental results. For sensitive applications, such as cell culture, sterilizing the PBS is often necessary to prevent microbial contamination. This can be achieved through autoclaving, using high-pressure steam typically at 121°C for 20 minutes.
Alternatively, filter sterilization is a common method, involving passing the solution through a sterile filter with a pore size of 0.22 micrometers (µm) or 0.2 µm. These pore sizes retain bacteria, ensuring sterility. Proper storage maintains PBS integrity and prolongs shelf life. Sterile 1x PBS can generally be stored at room temperature (15-30°C) for up to 12 months, provided it remains sealed and free from contamination. Observe the solution for any signs of deterioration, such as cloudiness or particulate matter, indicating contamination or precipitation; discard if observed.
Common Applications and Concentrated Forms
1x PBS is widely used in biological research due to its isotonic and non-toxic properties, making it suitable for direct contact with cells and tissues. It serves as a gentle washing solution for cells in culture, effectively removing media components or unbound reagents without causing cellular damage. Researchers also use PBS as a diluent for various reagents, antibodies, and biological samples, ensuring that the surrounding environment maintains a stable pH and osmotic balance during experiments. It plays a role in maintaining tissue viability during short-term handling and transport, and it is a common buffer in biochemical assays, including ELISA and Western blotting.
Beyond its immediate use, PBS is frequently prepared and stored in concentrated forms, such as 5x or 10x PBS. These concentrated stock solutions offer practical advantages, saving storage space and increasing convenience. A concentrated solution can be easily diluted to its 1x working concentration by adding the appropriate volume of deionized water, typically just before use. This approach streamlines laboratory workflows and helps ensure a consistent supply of fresh working solution.