A New Approach for Sterility Testing of Corneal Organ Culture

Abstract: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to “Method Suitability Test” defined by the European Pharmacopoeia, using blood culture bottles and the automated BACTEC system (BD), after removal of antibiotics with the new medical device RESEP (AL.CHI.MI.A. Srl, Italy).
10-100 CFU of Staphylococcus Aureus, Pseudomonas Aeruginosa, Candida Albicans, Bacillus Subtilis, Aspergillus Niger, Clostridium Sporogenes, Enterobacter Cloacae and Staphylococcus Epidermidis were inoculated in 9 ml cornea organ culture medium (MEM with 2% fetal calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml), Biochrom). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC plus vials (BD) and incubated until a positive reading or for at least 14 days at 36 ± 1°C. Untreated medium, positive and negative control samples were also tested. The elimination of antibiotics from the medium by RESEP was determined by HPLC.
The antibiotics were completely eliminated after 20 min. of treatment with RESEP at room temperature. In RESEP treated samples, the growth of all microorganisms was detected within 3 days, and showed no delay compared to the positive control samples. In contrast, the untreated medium samples showed no repeatable results of Staphylococcus Aureus, Enterobacter Cloacae, Candida Albicans and Bacillus Subtilis.
The microbiological testing of cornea organ culture medium with the automated BACTEC blood culture system using RESEP for the removal of antibiotics, could be successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).