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The contamination of ophthalmic solutions in ophthalmic practices remains an important cause of a myriad of secondary eye infections and a source of aggravation of ocular disorders such as corneal ulcers and keratitis. The aim of this study was to investigate the possible microbial contamination of fluorescein sodium dye solutions used in eye clinics in Ghana.Fluorescein sodium solutions were collected from various eye clinics in Ghana. Twenty-one samples of multiple-use fluorescein ophthalmic solutions were collected from various regions in Ghana. Eighteen unopened bottles yet to be used were also collected to serve as controls from the same facilities. The solutions were inoculated in different culture plates (blood agar, MacConkey agar, Sabouraud dextrose agar and plate count agar). The resulting microbial growth was identified using standard microbial identification techniques. Susceptibility tests were performed to ascertain the clinical importance of the organisms identified.Positive cultures were recorded for all 21 multiple-use bottles (in-use) collected, but there were no positive cultures for the unopened bottles (yet to be used). Six different genera of bacteria were identified from fluorescein solutions, including resistant strains of Staphylococci spp., Bacillus spp., Klebsiella spp., Pseudomonas spp., Haemophilus spp. and Bordetella spp. Pseudomonas spp. were the most common bacterial contaminants. For fungi contaminations, Aspergillus spp., Penicillium spp. and Cladosporium spp. were isolated. The most common fungal contaminants were Aspergillus spp.Multiple-use bottles of fluorescein solution used in eye clinics in Ghana were contaminated with clinically important strains of bacteria and fungi.