In this study, a detection method for Bacillus atrophaeus spores encapsulated in blow-fill-seal (BFS) containers made from extruded copolymer was developed and validated. Bacillus atrophaeus spores, known for their high thermal resistance, serve as model organisms for evaluating sterilization efficacy in polymer extrusion processes. A method for quantifying viable spores post-extrusion was established. The usually used low density polyethylene (LDPE) 3020D was replaced by comparable Styrolux® 656C styrene-butadiene copolymers (SBC) that could be dissolved at room temperature using toluene. Finally, SBC samples could be dissolved in toluene and B. atrophaeus spores were added to check the recovery rate. A standard filtration was developed – which did not reduce the bacterial spore count – using a Milliflex Plus Pump Single Head Kit with PVDF filters including rinsing with ethanol and NaCl-peptone buffer to reactivate bacterial spores.
All positive controls showed a good recovery rate while the treatment with toluene alone yielded a recovery rate of 30% to 52%. This reduction decreased when the SBC was added to the solvent toluene giving a recovery rate of 57%. The method developed thus yielded an acceptable recovery of bacterial spores of B. atrophaeus with the copolymer Styrolux® 656C and can therefore be used to enumerate bacterial spores in BFS-manufactured containers to evaluate the efficacy of the extrusion process in eliminating microorganisms, allowing determination of the corresponding killing kinetics.