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Henning Thomasen, Klaus-Peter Steuhl, Daniel Meller; Evaluation of a new protocol for sterility controls of corneal culture medium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3140.
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To ensure a safe application of corneal transplants careful testing for microbial contamination is essential. Since direct testing of the tissue is not feasible in clinical practice tests are performed on the antibiotics containing culture medium leaving the problem those antibiotics might compromise the test results. In this study a protocol for the application of the automated BacT/Alert system for sterility testing of corneal cell culture medium was examined.
About five milliliters of corneal culture medium in combination with the enzyme penicillinase were injected in BacT/Alert FA plus and FN plus resin bottles for clinical use or i-FA plus resign bottles for industrial use (n=4) and incubated at room temperature for one hour. About 10 to 100 colony forming units of different Bacterial and fungal test strains were applied. Strains were chosen on the basis of the European Pharmacopaea monograph 2.6.27 (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, Propioniobacterium acnes and Clostridium sporogenes). Bottles contained balanced salt solution in addition to the enzyme and the organism served as growth controls (n=2) while bottles with medium lacking germs served as negative controls. The Bottles were incubated at 32°C (anaerobic bacteria) or 22°C (aerobic bacteria and fungi) for a period of 14 days. The time to detection (TTD) was monitored for each bottle.
Growth of the test strains except Aspergillus brasiliensis was detected in the FA and FN Plus bottles. The TTD for the strains was 44 + 1.5h (P. aeruginosa), 57.7 + 2.2h (B. subtilis), 56 + 1h (S. aureus), 26.3 + 1h (C. sporogenes), 223 + 4.6h (P.acnes), 64.4 + 10. A. brasiliensis was detected in i-FA Plus bottles intended for industrial use with a TTD of 94.9 + 3.7h.
The application of BacT/Alert FA Plus and FN Plus resin bottles in combination with the penicillin degrading enzyme penicillinase is able to detect small scale microbial contamination with a variety of microorganisms in corneal culture medium. For detection of Aspergillus brasiliensis in the medium the (i-) FAN Plus bottles should be used.
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