1) Phosphate buffered saline, pH 7.2 to 7.4
Phosphate buffered saline (PBS) is often described in two forms, as incomplete or complete solutions. Both of these forms are available commercially. An incomplete solution of PBS contains no calcium or magnesium ions. A complete solution of PBS contains calcium and magnesium ions and is used for preparation of faecal suspensions and as diluent for viruses. The presence of calcium and magnesium ions stabilizes viruses, particularly poliovirus and other enteroviruses.
2) Solution A
NaCl 8.00 g
KCl 0.20 g
NA2HPO4 (anhydrous) 0.91 g
KH2PO4 0.12 g
Dissolve the salts in 600–800 ml distilled H2O. Make up to 1000 ml with distilled H2O and autoclave at 10 psi (70 kPa) for 15 minutes. This gives a working solution of incomplete PBS (i.e. no calcium or magnesium ions present).
3) Solution B
MgCl2.6H2O 0.10 g
Dissolve in 100 ml distilled H2O. Autoclave at 10 psi (70 kPa) for 15 minutes.
4) Solution C
CaCl2 0.10 g
Dissolve in 100 ml distilled H2O. Autoclave at 10 psi (70 kPa) for 15 minutes.
5) Working solution of complete PBS
The working solution of complete PBS contains 0.11 M NaCl. Add 1 part of Solution B and 1 part of Solution C to 8 parts of Solution A.
Alternatively, use commercially prepared tablets or powder following the manufacturer’s instructions for reconstitution and sterilization.
6) PBS with antibiotics
Dissolve 1x106 units crystalline penicillin G and 1g streptomycin sulfate in 100 ml sterile complete PBS, distribute into 5 ml volumes and store at -20°C. For use, add 5 ml of this stock solution to 500 ml complete PBS to give a final concentration of 100 units / ml penicillin and 100µg / ml streptomycin. Store refrigerated at 4 to 8°C for up to one week.
7) Other antibiotics
Penicillin and streptomycin are the antibiotics most commonly used in cell and routine virus culture work; they are also the least expensive. When used at the correct concentration and in conjunction with chloroform extraction, penicillin and streptomycin are usually capable of limiting the level of contaminating bacteria in the faecal suspensions. Occasionally, however, use of penicillin and streptomycin does not provide adequate protection and alternative antibiotics can be tried. Gentamicin is more expensive than penicillin and streptomycin, but it is inhibitory to a wider range of bacteria and it is autoclavable. Gentamicin should be used at a final concentration of 50µg/ml. Mycostatin may be used at 25 units/ml to counteract fungal and yeast contaminants; however, it is only fungistatic and not fungicidal, is rapidly inactivated at 36°C (usual cell culture incubation temperature) and some batches are slightly cytotoxic.