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Background/aim: The consequences of fungal contamination of an organ cultured cornea, though exceptional, are often disastrous for the recipient. Consequently, eye banks often quarantine corneas for 10 days or more before passing them for grafting. This period, though detrimental to the endothelial cell density of the delivered cornea, is necessary to detect contamination using conventional microbiological methods. The authors previously validated the use of a pair of aerobic and anaerobic blood bottles for sensitive and rapid detection of bacteria. To allow a short quarantine period, it remained only to optimise detection of fungi. The authors aimed to compare sensitivity and rapidity of fungal contamination detection by three methods: blood bottles, Savoyard, and daily visual inspection of the organ culture medium. Methods: Four inocular (106, 1 102, 10 colony forming unit (CFU) per ml) of 11 fungi (Candida olbicans, C tropicalis, C globrata, Saccharomyces Ce reviS ice, Rhodotorula rubra, Ciyptococcus neoformans, Fusarium oxysporum, Aspergillus niger, A him igatus, A Ravus, Acremonium falciforme) were inoculated in a commercial organ culture medium containing a coloured pH indicator (CorneaMax, Eurobio, las Ulis, France). The real live fungal inoculum was verified immediately after inoculation. After 48 hours at 31°C, samples of the contaminated media were inoculated in three blood bottles: Bactec Aerobic/F, Bactec Mycosis IC/F, and Bactec Myco/F Lytic (Becton Dickinson, Le Pont de Claix, France), then placed in a Bactec 9240 rocking automat, and in four Sabouraud media (solid and liquid, 28°C and 37°C) with daily observation. Contaminated organ culture media were also checked daily for any change in turbidity and/or colour. Experiments were performed in triplicate. Results: Mycosis IC/F and Myco/F Lytic bottles were neither faster nor more sensitive than the aerobic bottle. The three methods were positive for all inocula, even the lowest (viable inoculum below 10 CFU/ml for each fungus). Contamination was detected within 24 hours by the aerobic bottles in 91 % (40/44), by Sabouraud in 98% (43/44) (no significant difference) and by visual inspection in 66% of cases (29/44) (p⩽0.001 with the Iwo others). Maximum times to detection were 46, 48 and 72 hours respectively. Conclusion: This study further counters the preconception that fungal contamination is hard to detect in corneal organ culture media. This study is the last step invalidating the use of a pair of blood bottles for the sterility testing of organ culture media, this time for fungi. Their use should make it possible to shorten microbiological quarantine and thus deliver corneas with higher endothelial cell density, without increasing the risk of recipient contamination. [ABSTRACT FROM AUTHOR]

Aim: ro examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant. Methods: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data. Results: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a "magnification correcting factor" to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non- adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism ("hexagonalify"). Conclusions: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system. [ABSTRACT FROM AUTHOR]

Background: Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage. However, the mechanisms underlying endothelial apoptosis are poorly understood. The response of a human corneal endothelial cell (HCEC) line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular modulators that participate in endothelial cell death. Methods: Immortalised HCECs were studied after 3, 6, 12, and 24 hours of incubation with 0.2 µM staurosporine. Cell shedding was monitored. Hoechst 33342 fluorescent DNA staining combined with propidium iodide was used for apoptosis/necrosis quantification and morphological examination. The caspase-3 active form was assessed using western blot, proteolytic activity detection, and immunocytochemistry. The cleaved form of poly(ADP-ribose) polymerase (PARP) was assessed using immunocytochemistry and western blot. The ultrastructural features of cells were screened after 12 hours with staurosporine or vehicle. Results: The specific apoptotic nature of staurosporine induced HCEC death was confirmed. The ultrastructural features of staurosporine treated cells were typical of apoptosis. HCEC shedding and DNA condensation increased with time. Caspase-3 activity was detected as early as 3 hours after exposure with staurosporine, peaking at 12 hours of incubation. The presence of cleaved PARP after 3 hours confirmed caspase-3 activation. Conclusions: These data suggest strongly that HCEC cell death induced by staurosporine is apoptosis. The main consequence of HCEC apoptosis is shedding. Staurosporine induced apoptosis of endothelial cells involves activation of caspase-3, and could be a useful model to study strategies of cell death inhibition. [ABSTRACT FROM AUTHOR]

Aims: To test the bactericidal activity of standard organ culture medium, and to compare the sensitivity and rapidity of blood culture bottles with conventional microbiological methods for detection of bacteria and fungi inoculated in a standard cornea organ culture medium. Methods: The bactericidal activity of contaminated standard organ culture medium containing 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B was evaluated after 48 hours of incubation at 31°C with five inocula of 14 bacteria. Two yeasts (Candida spp) and one Aspergillus were also tested. Contaminated media were then inoculated in three blood bottles (aerobic, anaerobic, fungal) placed in a Bactec 9240 automat; three conventional microbiological broths were the control. Changes in colour of organ culture medium and growth on conventional broth were screened daily by visual inspection. The sensitivity and rapidity of detection of contamination were compared between the three methods: blood bottle, conventional, and visual. Results: Organ culture medium eradicated five bacteria irrespective of the starting inoculums: Streptococcus pneumoniae, Branhamella catarrhalis, Escherichia coli, Propionibacterium acnes, and Haemophilus influenzae. For micro-organisms where the medium was ineffective or bactericidal only (methicillin resistant Staphylococcus aureus, methicillin sensitive Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus subtilis, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, Candida kruzei, Aspergillus fumigatus), the blood bottle, conventional, and visual methods detected microbial growth in 100%, 76.5%, and 70% of cases respectively. Mean detection time using blood bottles was 15.1 hours (SD 13.8, range 2-52). In cases of detection by the blood bottle method and the conventional method, the former was always faster: 95.5% against 65.2% detection... [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about controlled study of the influence of storage medium type on endothelial assessment during corned organ culture.

A letter to the editor is presented concerning the use of in vivo Topcon SP2000P specular microscope versus ex vivo sambacorneo eye bank analyser.

Examines the dynamics of corneal endothelial cell death in organ culture. Trypan blue staining procedure; Time spent outside the organ culture medium before fixing in formaldehyde.