Phosphate Buffered Saline (PBS) | Composition, Preparation, and Uses in Microbiology

Phosphate Buffered Saline (PBS) is a widely used buffer solution in biological and pharmaceutical laboratories. It provides a stable environment that mimics the osmolarity and ion concentration of the human body, making it ideal for maintaining the integrity of cells and biological molecules during experiments. In microbiology and biotechnology, PBS plays a crucial role in washing cells, diluting substances, and serving as a base for reagent preparation.

What is Phosphate Buffered Saline (PBS)?

Phosphate Buffered Saline is an isotonic, non-toxic solution used in various biological applications. It maintains a constant pH, generally around 7.2 to 7.4, and provides essential ions such as sodium and potassium for maintaining physiological balance. It is primarily composed of sodium chloride (NaCl), potassium chloride (KCl), disodium phosphate (Na₂HPO₄), and monopotassium phosphate (KH₂PO₄).

Purpose of PBS in Microbiology

The main purpose of using PBS is to maintain a stable pH and osmotic balance during biological and microbiological processes. It helps prevent cell lysis or distortion caused by changes in ionic strength or pH, ensuring the accuracy of experimental results.

Composition of Phosphate Buffered Saline

Typical PBS (1X) contains the following components:

Component	Concentration (1X PBS)	Chemical Formula
Sodium Chloride	137 mM (8.0 g/L)	NaCl
Potassium Chloride	2.7 mM (0.2 g/L)	KCl
Disodium Phosphate (Anhydrous)	10 mM (1.44 g/L)	Na2HPO4
Monopotassium Phosphate	1.8 mM (0.24 g/L)	KH2PO4

Final pH: 7.2 – 7.4 (adjusted at 25°C)

Osmolarity: Approximately 290 mOsm/L (isotonic with human body fluids)

Types of PBS Solutions

Depending on laboratory requirements, PBS can be prepared in different forms:

• 1X PBS: Working solution used for washing and cell suspension.
• 10X PBS: Concentrated stock solution that can be diluted 10 times with distilled water to make 1X PBS.
• PBS with Calcium and Magnesium: Used when divalent cations are required for enzyme activity or cell adhesion.
• PBS without Calcium and Magnesium: Used for cell washing or detachment to prevent unwanted enzyme activation.

Preparation of Phosphate Buffered Saline (1X PBS)

Here is the step-by-step method to prepare 1 liter of 1X PBS:

Reagents Required:

• NaCl – 8.0 g
• KCl – 0.2 g
• Na₂HPO₄ – 1.44 g
• KH₂PO₄ – 0.24 g
• Distilled or deionized water – up to 1 liter

Preparation Steps:

1. Dissolve all salts in about 800 mL of distilled water using a magnetic stirrer.
2. Check the pH of the solution using a calibrated pH meter.
3. Adjust the pH to 7.2 – 7.4 using 1 N HCl or 1 N NaOH as required.
4. Make up the final volume to 1000 mL with distilled water.
5. Filter the solution through a 0.22 µm membrane filter for sterilization or autoclave at 121°C for 15 minutes.
6. Store the PBS at room temperature or 2–8°C for long-term use.

Alternative PBS Concentrations

You can prepare PBS in different strengths as required:

• 10X PBS Stock: Multiply all reagent quantities by 10 (e.g., 80 g NaCl per liter).
• 0.5X PBS: Use half of the standard reagent concentration for gentle washing.

Applications of Phosphate Buffered Saline in Microbiology

PBS is used in a wide variety of microbiological and biochemical applications, including:

• Cell Washing: To remove serum, media, or other impurities during cell culture.
• Reagent Dilution: Used as a diluent for enzymes, antibodies, and other reagents.
• Immunohistochemistry and ELISA: Used for washing microplates or slides to remove unbound substances.
• Microbial Suspension: Used for resuspending bacterial or fungal cells for testing.
• pH Maintenance: Maintains a stable environment for biochemical reactions.
• DNA/RNA Experiments: Provides a salt-balanced buffer for nucleic acid handling.

Advantages of PBS Buffer

• Maintains physiological pH similar to body fluids.
• Non-toxic to cells and microorganisms.
• Compatible with most biological assays.
• Easy to prepare and inexpensive.
• Suitable for both sterile and non-sterile laboratory use.

Storage and Stability

• Room Temperature: Stable for 2–3 months.
• Refrigerated (2–8°C): Stable for up to 6 months.
• Always check for contamination or precipitation before use.
• Sterile PBS should be stored in aseptic, sealed containers.

Precautions

• Ensure accurate pH adjustment for optimal buffering capacity.
• Use analytical-grade chemicals and deionized water for preparation.
• Do not use contaminated or expired PBS for critical assays.
• Label the bottle clearly with preparation date and concentration.

Applications in Pharmaceutical Microbiology

In pharmaceutical laboratories, PBS plays an important role in quality control, microbial limit testing, and sterility testing procedures. It is used for sample dilution, microbial recovery, and preparation of inoculum suspensions. It also acts as a rinse solution in sterility test isolators and filtration systems.

Conclusion

Phosphate Buffered Saline (PBS) is a fundamental laboratory reagent that ensures pH stability and isotonicity during biological and microbiological procedures. Its simplicity, safety, and wide range of applications make it indispensable in pharmaceutical microbiology, biotechnology, and cell culture laboratories. Following proper preparation and storage guidelines ensures reliable and reproducible results across experiments.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
📱 Mobile: 09505626106

Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.