Vibrio Parahaemolyticus Case Study

III.MATERIALS AND METHODS
3.1. Collection and confirmation of Vibrio parahaemolyticus isolate

Vibrio parahaemolyticus isolate was obtained from Cochin University of Science and Technology (Dr. I.S. Bright Singh) and grown in 1.5% TSB with 2% NaCl. A loop of the enriched culture was streaked onto thiosulphate-citrate-bile salt sucrose agar used for the selective isolation of vibrio strains (TCBS agar ).After 18-24hr incubation at 370c the cultures giving pure green colonies were randomly selected Vibrio parahaemolyticus colonies are green or blue green on the agar due to sucrose fermentation. The isolate was confirmed by PCR performed according to (Kim et al., 1999).and also checked for virulence and human ..........using tdh and trh primers
parahaemolyticus were grown on chitin flakes same as previous experiment and biofilm cells for each day (1, 2, 3, 4, 5, 6 and 7th day). Before bacterial biofilm harvest, loosely associated planktonic cells were removed by three time gentle stirring in 5x PBS. The chitin flakes are then suspended in 10ml 5x PBS .Immediately transferred to ice to maintain the low temperature in the tube containing biofilm. Then the culture tubes containing 0.3% chitin flakes with biofilm are agitated for 5 min on a cyclomixer todislodge the biofilm and the supernatent was collected Immediately transferred to fresh RNaes free round bottom tube. Centrifuged at 2000 rpm at 40c to settle down the small accidental chitin flakes. To reduce contaminating genomic DNA from RNA, proper RNA isolation and cDNA synthesis procedures need to follow .The supernatant biofilm mass was pelleted after centrifugation at 18,400 g for 20 min at 40c . The supernatant is discarded and double volume (1ml) of RNA protect bacterial reagent (qiagen) was added to each pellet and re-suspended and transferred to 1ml tube. The mixture was mixed immediately by vortexing for 5 s and then it was incubated at room temperature for 15 minutes. Again centrifuged at 18,400g for 10min at 40c and the pellet obtained, of which The supernatant RNA protect was discarded and residual supernatant was removed by gently dabbing the inverted tube onto a paper towel. A mixture of 200 μl TE buffer (30 mM Tris-Cl, 1 mM EDTA) containing lysosome (15mg/ml) and 20 μl Qiagen proteinase K was prepared and then added to the pellet which was mixed by vortexing for 10 s thereafter, and the pellet is used for total RNA was extracted from biofilms using the RNeasy Protect Bacteria mini kit (Qiagen HIIlden, Germany).the mixture was incubated on a shaker-incubator at room temperature for 10 minutes. Seven hundred μl RNeasy lysis buffer; RLT (with 10 μl β-mercaptoethanol per ml) was added and vortexed vigorously. Then, 96-100%