Vpachec3 Week 11

10 Biological Terms and Definitions

1. Aminoglycoside antibiotic
2. Fluoroquinolone
3. Sessile cells
4. cDNA
5. Flow cytometry
6. Isocitrate Lyase
7. Persister
8. Exconjugants
9. Mutagenesis
10. Glyoxylate shunt

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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