The samples were immediately frozen at -80°C. Wound topology Eight VLU chosen because they were particularly large and recalcitrant to healing had a MediRule II template (Briggs Corporation, Des Moines, IA) placed over the wound and the wound was outlined on the template grid. Multiple areas of the wound were chosen on the templates grid system and a variety of sample points chosen arbitrarily, which represented edge and center portions of the wound. Once these areas were marked on the template Selleckchem Gemcitabine and the wound, the wound was then prepared. This was
done by using normal saline irrigation along with a cotton gauze to gently remove surface debris. None of the wounds required local anesthesia and the areas that had been identified on the wound (as marked on the template) were then sampled. Individual sterile stainless steel curettes were used to debride an approximately 1.0 cm diameter sample of the biofilm
down to the host tissue. Any bleeding at the sample sites was controlled with pressure. The patients reported no additional discomfort from the procedure. The samples were individually placed in separate this website sterile 2 cc Eppendorf tube (Fisher Scientific, Pittsburgh, PA), labeled with the patient’s study accession number and grid location. The samples were then frozen at -80°C until subsequent molecular analysis. DNA extraction After thawing, the debridement samples were centrifuged at 14,000 rpm for 30 seconds and resuspended in 500 μl RLT buffer (Qiagen, Valencia, CA) (with β-mercaptoethanol).
A sterile 5 mm steel bead (Qiagen, Valencia, Cisplatin clinical trial CA) and 500 μl sterile 0.1 mm glass beads (Scientific Industries, Inc., NY, USA) were added for complete bacterial lyses in a Qiagen TissueLyser (Qiagen, Valencia, CA), run at 30 Hz for 5 min. Samples were centrifuged briefly and 100 μl of 100% ethanol added to a 100 μl aliquot of the sample supernatant. This mixture was added to a DNA spin column, and DNA recovery protocols were followed as instructed in the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) starting at step 5 of the Tissue Protocol. DNA was eluted from the column with 30 μl water and samples were diluted accordingly to a final concentration of 20 ng/μl. DNA samples were quantified using a Nanodrop spectrophotometer (Nyxor Biotech, Paris, France). Massively parallel bTEFAP and bTEFAP titanium Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed as described previously [9] at the Research and Testing Laboratory (Lubbock, TX.). The new bacterial tag-encoded FLX-Titanium amplicon pyrosequencing (bTETAP) approach is based upon similar principles to bTEFAP but utilizes Titanium reagents and titanium procedures and a one-step PCR, mixture of Hot Start and HotStar high fidelity taq polymerases, and amplicons originating from the 27F region numbered in relation to E. coli rRNA. The bTEFAP procedures were performed at the Research and Testing Laboratory (Lubbock, TX) based upon RTL protocols http://www.researchandtesting.com.