Lens Case Study

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Lens cases were collected and processed for microbial investigation as soon as possible or stored at -4oC if the cases could not have processed within 24 hours. Lens holders and the case well were swabbed with a sterile cotton swab. The swab was placed in 2ml of phosphate buffer (PBS; pH 7.4 NaCl 8 g 1-1, KCl 0.2 g 1-1, Na2HPO4 1.15 g 1-1, KH2PO4 0.2 g 1-1 pH 7.2) and was vortexed at 2500 rpm for 10-20 sec. Four hundred microliters of the PBS was inoculated into chocolate agar plates and Sabouraud dextrose agar plate. Chocolate agar plates were incubated at 37oC for 24 hrs, 48 hrs and 96 hrs under aerobic, microaerophilic and anaerobic conditions. The Sabouraud dextrose agar plate was incubated at room temperature for 1 week for fungal identification. A total colony count was…show more content…
After cell lysis and DNA extraction, 100µl of DNA was stored at -30oC prior to subsequent 16S rRNA PCR and gene sequencing. Universal primers 27F; AGAGTTTGATCCTGGCTCAG and 1492R; GGTTACCTTGTTACGACTT were used to target the 16S rRNA gene. PCR amplification was performed using a thermocycler with the following program: 25 cycles for 5 mins at 94oC, 30 secs at 94oC, 30 secs at 56oC and 90 secs at 72oC. The final elongation step was performed for 10 mins at 72oC. PCR products were analysed by agarose gel electrophoresis (Figure 1) to ensure the appropriate size of the amplification product. The PCR procedures were repeated in the similar manner except adding forward and reverse primer separately. Post PCR products were cleaned using Safodex G-50per the manufacturer’s instructions. Subsequently, the filtered PCR product was collected in fresh microtiter plates and heated at 90oC for 30 mins. The PCR product was submitted to the Ramaciotti DNA Sequencing Analysis Unit, at the School of Biotechnology and Biomolecular Sciences, UNSW for DNA
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