Increased sensitivity of microbiological testing of cornea organ culture medium by additional resin treatment

ObjectiveThis validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC TM blood culture bottles.Methods and analysis10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium. In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles. In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTECTMvials (positive control). All samples were incubated in the automated BACTECTMblood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liqiud chromatography.ResultsAfter 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated. In group A , all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls. In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis.ConlclusionThe use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTECTMPlus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

Preservation of Preloaded DMEK Lenticules in Dextran and Non-Dextran-Based Organ Culture Medium

Purpose.To determine the optimum preservation conditions for preloading DMEK lenticules using organ culture system.Methods.8.5 mm DMEK lenticules were stripped and preserved with endothelium flap-in for 4 days at RT in an IOL cartridge that was blocked with rubber stoppers from each end. In C1, tissues were collected from tissue culture medium (TCM) and preserved in TCM. In C2, tissues were collected from transport medium (TCM + 6% dextran T500) (TM) and preserved in TM. In C3, tissues were collected from TCM and preserved in TM. Mortality, glucose uptake, histological staining, tight junctions and cell apoptosis were studied post-preservation.Results.Mortality in C1, C2, and C3 were 49.40%, 8.53%, and 27.74%, with 40.7%, 13%, and 41.8% uncovered areas. Glucose uptake (mg/mL) was 0.32, 0.43, and 0.56 in C1, C2, and C3. PAS staining showed presence of DM and endothelium in C2 but not in C1 and with fewer cells in C3. ZO-1 was expressed in all the conditions. Polymorphism was higher in C1 and C3. Mild apoptosis was observed in C3.Conclusions.Dextran may play an important role in preserving the endothelial cells before and after stripping for trifolded (endothelium-in) preloaded DMEK lenticules.