Sterility testing of tissue samples according to European Pharmacopoeia

Purpose: The aim of this study was to validate the sterility testing of tissue samples according to the “Method suitability test” defined by the European Pharmacopoeia (EP, chapter 2.6.1), using the MEB buffer (AL.CHI.MI.A. S.r.l.) for extraction of microorganisms from tissue samples and RESEP (AL.CHI.MI.A. S.r.l.) for elimination of antimicrobials before direct inoculation of growth media.

Materials and methods: Samples consisting of one gram of sterile porcine aortic valve were immersed in BASE.128 (AL.CHI.MI.A. S.r.l.) at 4°C for 24 h to simulate the tissue decontamination process with an antibiotic cocktail. The samples were then contaminated with 10-100 cfu of EP reference strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes), and introduced in a vial containing MEB extraction buffer and stirred at room temperature for 20 min in order to extract microorganisms from the tissues. The buffer was then treated with RESEP syringe for removal of antimicrobial residues and inoculated in Tryptic Soy Broth (TSB) or Thioglycolate (TG). The turbidity of the growth media was determined visually after 5 days of incubation at 22°C (TSB) or 33°C (TG).

Results: MEB buffer extracted the whole 10-100 cfu of all tested microorganisms from aortic valve samples. All microorganisms showed growth in TG or TSB media after RESEP treatment indicating vitality and absence of BASE.128 antimicrobial residues. Turbidity of growth media was detected within 5 days after inoculation in all tested conditions.

Conclusions: The sterility test of the tissue samples, including the extraction of microbial contaminants from tissues using MEB buffer and removal of antimicrobials using RESEP, before direct inoculation, was successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).