PHOSPHATE BUFFER (PBS): RATIONALE, PREPARATION AND USES - BIOLOGY - 2025

The phosphate buffer, phosphate buffered saline or BPS is a buffer isotonic, whose function is to maintain the pH and osmotic pressure as close to the natural biological environment (physiological). The acronym PBS stands for phosphate buffered saline.

PH and osmolarity are two very important aspects that must be controlled in certain laboratory protocols. In the case of pH, it is essential that it be controlled, especially in biochemical reactions, since these may vary or not be carried out if the reactants are at an inappropriate pH.

PBS phosphate buffered saline at two different pH's. Source: Pixinio.com

Meanwhile, the control of osmolarity is essential especially when working with living cells, since the plasma membranes of the cells react according to the concentration of solutes in which they are found.

If the cells are transferred to a hypertonic medium they will become dehydrated, since the water gradient will be transported to the side where there is a higher concentration of solutes. If, on the other hand, cells are placed in a hypotonic medium, the cells will absorb liquid until they are lysed.

That is why PBS buffer is used for laboratory protocols that require the maintenance of cells in vitro, in this way the cells will not be damaged.

PBS is made up of a combination of salts, such as sodium chloride, sodium phosphate, potassium chloride, and potassium phosphate. The composition of the PBS can vary depending on the protocol.

Basis

Basically the function of the phosphate buffer is to maintain a constant physiological pH together with an electrolyte concentration similar to that found inside the body.

In this environment, cells are able to remain stable, as physiological conditions are simulated as much as possible.

Other compounds can be added to the original PBS formulation if necessary, for example the addition of EDTA to the buffer is useful for washing red blood cells in the cross-incompatibility test.

EDTA prevents the fraction of complement C1 present in the serum from being cleaved and lysed to the red blood cells, that is, it avoids false incompatibility results. In addition, EDTA helps to dissociate cells.

Preparation

The quantity of salts that must be weighed for the preparation of the PBS phosphate buffered saline will depend on the quantity that must be prepared:

-Phosphate buffered saline stock solution (10X PBS)

For one liter of solution:

To weigh:

80.6 g of NaCl,

2.2 g of KCl, 11.5 g of Na ₂ HPO ₄, 2.0 g KH ₂ HPO ₄

Preparation technique

Place the heavy salts in a beaker, add enough water (80%) and mix on the stirring plate with magnetic bar until the salts are dissolved.

Preparation of PBS using a stirring plate with a magnetic bar. Source: User: Ruhrfisch, Wikipedia. com

Filter to remove undissolved particles. Use filters with 0.45 µm pores. Autoclave and distribute aseptically in a laminar flow hood in glass jars with lids.

The 10X solution (concentrated) does not adjust the pH. The adjustment is made once diluted to the 1X PBS buffer concentration, (1:10 dilution).

-Buffer phosphate saline (1X PBS)

1X PBS can be prepared directly, weighing the corresponding amounts of each salt, or it can be prepared by diluting the stock solution (1:10) with sterile distilled water.

-To prepare directly one liter of 1X PBS phosphate buffered saline, weigh:

8.06 g of NaCl, 0.22 g of KCl, 1.15 g of Na ₂ HPO ₄, 0.20 g of KH ₂ HPO ₄

Preparation technique

Proceed as explained in the concentrated solution. Subsequently, the pH must be adjusted. To do this, measure the pH and depending on the result use acid (HCl) or base (NaOH) to lower or raise the pH respectively as necessary, until it is 7.4.

The acid or base are added drop by drop while monitoring the pH value of the solution using a pH meter. Filter, autoclave, and aseptically distribute into conical tubes or jars as needed.

-To prepare 1X PBS from the 10X stock solution:

Make a 1:10 dilution. For example, to prepare 1 liter of 1X PBS, measure out 100 ml of the stock solution and add 700 ml of sterile distilled water. Adjust the pH and fill the amount of water up to 1000 ml.

The prepared PBS buffer is colorless and clear.

Daily PBS can be stored at room temperature and the rest in the refrigerator.

Solutions for pH adjustment

HCl

For 100 ml of 1 molar HCL (hydrochloric acid).

Measure 91 mL of distilled water and place it in a 250-mL beaker.

Measure 8.62 mL of concentrated HCl and slowly add it to the beaker containing the water (never do it the other way around). Take the appropriate biosafety measures when handling strong acids (highly corrosive substance).

Mix for 5 minutes preferably using a stirring plate with a magnetic bar inside the glass. Transfer to a 100 ml flask and make up to 100 ml with distilled H ₂ O.

NaOH

For 100 ml of NaOH (sodium hydroxide) 10 molar.

Measure 40 mL of distilled water and place it in a 250-mL beaker. Measure 40 g of NaOH and add to the water. Mix using a stirring plate with a magnetic bar inside the glass.

Transfer to a 100 ml volumetric flask and make up to the mark with distilled water. Comply with biosafety regulations, because this reaction is exothermic (it releases energy in the form of heat).

If you want to prepare other amounts of phosphate saline solution, you can consult the following table:

Source: «Laboratory of Viral and Human Genomics, UASLP School of Medicine»

Applications

It is primarily used in cell biology, immunology, immunohistochemistry, bacteriology, virology, and research laboratories.

It is ideal for cell washing by centrifugation (red blood cells), cell monolayer washes, in spectroscopic ellipsometry techniques, in the quantification of bacterial biofilms, in the maintenance of cell cultures for viruses, as a washing solution in the indirect immunofluorescence technique and in techniques for the characterization of monoclonal antibodies.

It is also used to transport cells or tissues, as a diluent for cell counting, the preparation of cellular enzymes (trypsin), as a diluent for the biomolecule desiccation method and to prepare other reagents.

On the other hand, Martin et al. In 2006 demonstrated that PBS is useful in forensic science laboratories, specifically in the recovery of sperm from vaginal smears, or in the recovery of vaginal cells from penile smears. In this way it can be established if there has been a sexual relationship.

Limitations

-Some PBS buffers contain a substance called sodium azide as a preservative. This compound can generate explosive substances if it comes in contact with lead or copper. For this reason, special care must be taken when disposing of this reagent down the drain. If it is discarded in this way, plenty of water must be added to dilute it as much as possible.

-Zinc should not be added to the phosphate buffer, since it causes some salts to precipitate.

-Wangen and collaborators in 2018 determined that the use of PBS was not suitable for washing primary cells of acute myeloid leukemia (AML) extracted from peripheral blood, due to the fact that many cells are lost by lysis, with a great decrease in the material protein.

Therefore, they determined that primary AML cells should not be washed with PBS after storage in liquid nitrogen.

References

1. Coll J. (1993). Diagnostic techniques in virology. Ed Díaz de Santos. 360 pg
2. Rodríguez M, Ortiz T. Cell culture. Change of Medium. Department of Normal and Pathological Cytology and Histology University of Seville. Available at personal.us.es
3. Preparation of Phosphate Buffered Saline (PBS). (2008). Standard Operating Procedures (SOPs) Human and Viral Genomics Laboratory UASLP School of Medicine. Available at: genomica.uaslp.mx
4. "Phosphate Buffer Saline." Wikipedia, The Free Encyclopedia. 3 Apr 2019, 19:36 UTC. 13 Apr 2019, 02:57 en.wikipedia.org.
5. Pietrasanta L, Von-Bilderling C. Topics in molecular biophysics. Available at: users.df.uba.ar
6. Rediar. Manual. PBS + EDTA. Available at: felsan.com.ar
7. Martin NC, Pirie AA, Ford LV, Callaghan CL, McTurk K, Lucy D, Scrimger DG. The use of phosphate buffered saline for the recovery of cells and spermatozoa from swabs. Sci Justice. 2006; 46 (3): 179-84. Available in: ncbi.nlm.nih.gov
8. Wangen R, Aasebø E, Trentani A, et al. Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome. Int J Mol Sci. 2018; 19 (1): 296. Available in: ncbi.nlm.nih.gov
9. Martínez R, Gragera R. (2008). Theoretical and practical foundations of histochemistry. Superior Council of Scientific Investigations. Madrid. Available at: books.google.co.ve