Vpachec3 Week 11

Article Outline

1. Introduction
2. Materials and Methods
   • Strains and Culture Conditions
     • 18 strains in the Burkholderia cepacia group
     • Plated on agar plates at 37 degrees Celsius over night
     • Cultures diluted in broth and incubated at 37 degrees Celsius
   • Minimal Inhibitory Concentration (MIC)
     • Used EUCAST broth microdiluation protocol to find MICs
   • Quantification of Persister Cells in Biofilms and Planktonic Cultures
     • Aim: to see how many surviving persister cells there are
     • Day old biofilms and planktonic cultures were treated with tobramycin and ciprofloxacin
     • Used 96 well micro titer plate for growth
     • After 24 hrs, the supernatant was removed and put into antibiotic solution mixed in physiological saline
     • Cells were harvested and plated onto agar plates
     • Planktonic cultures needed extra preparation to reach optical density of 1
   • RNA Extraction and Microarray Analysis
     • Biofilms exposed to either antibiotic or 0.9% salt solution
     • After 24hr, the sessile cells were prepped to take out the RNA
     • Right before microarray analysis, the samples were concentrated and amplified
     • Gene expression analysis was executed
     • Data used 2 color Agilent microarrays
     • T-test analysis was used
   • Quantitative RT-PCR
     • Chose 11 selected genes to use RT-qPCR on
     • RNA was extracted from biofilms
   • Flow Cytometry
     • Aim: to see induction of reactive oxygen species by tobramycin
     • Stained biofilms with ROS-specific dyes
     • Dye sits for 30 mins in the dark
     • Rinsed off and physiological saline was added in well
     • Cells harvested and resuspended in diluted physiological saline
     • Cells were measured using flow cytometer
     • 50,000 cells per sample

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