Sterilisation by filtration

Pronase is recommended for primary cultures as it gives a better single cell suspension than trypsin (Gwatkin, 1973). It is not always as good with cell lines. An 0.25% solution is made by dissolving 1.25 g standard B grade pronase (Calbiochem Corp.) in 500 ml PBS-A at room temperature. After 30 min the cloudy solution is centrifuged (100 g, 10 min) and sterilised by filtration as for trypsin. 

Powdered medium should be reconstituted at room temperature as per maufacturers instructions, which leads to 1 X medium. It is important not to leave traces of powder undissolved. Bicarbonate is added separately and the pH adjusted to about 7.1 before sterilisation by filtration. Before use, glutamine should be added, if not already present, together with serum (usually to 10%, as described in Appendix 1). 

A stock of the Hoechst 33258 bisbenzamid fluorochrome solution is made by dissolving 5 mg in 100 ml PBS-A (Appendix 1) using a magnetic stirrer. It must be free of bacterial contamination and should be sterilised by filtration through a 0.22 (im membrane and should be stored in the dark at 4°C. It should be diluted 100-fold with filtered PBS-A for use. 

Sterilise by filtration using a 0.22 jum membrane filter and store as aliquots of 5 ml at room temperature. 

Sterilisation by filtration should only be employed when heat sterilisation cannot be applied because of its detrimental effect on the active ingredients. 

Solutions or liquids can be sterilised by filtration through a sterile filter of nominal pore size of 0.22 micron (or less), or with at least equivalent micro-organism retaining properties, into a previously sterilised container. Such filters can remove bacteria and moulds but not all viruses or mycoplasmas. 

Sterilisation by filtration differs from the above methods as it removes cells rather than destroys them. It also removes particles and can be used for clarification, carrying out both functions simultaneously. Filtration can be used for both heat sensitive liquids and air supplies to aseptic areas. 

Current sterilisation processes are generally not well adapted to polymers, except in the cases of water-soluble polymers and colloids, which can be sterilised by filtration in solution. The simplest process is autoclaving with steam at 120 °C for 20 min. This process can be detrimental to devices that include polymers with thermomechanical properties not compatible with the temperature used in the sterilisation process. 

Alginate solutions can be sterilised by filtration. However, sterilisation of the solid alginate materials is not straight forward. Both heat and classical y-irradiation are deleterious to the material, may cause significant breakage in the alginate chains. Sterilisation in electron accelerators for short times (e.g. 1 min) may cause less damage to the material. 

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