Vpachec3 Week 11
From LMU BioDB 2015
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10 Biological Terms and Definitions
- Aminoglycoside antibiotic
- Fluoroquinolone
- Sessile cells
- cDNA
- Flow cytometry
- Isocitrate Lyase
- Persister
- Exconjugants
- Mutagenesis
- Glyoxylate shunt
Article Outline
- Introduction
- 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
- Strains and Culture Conditions
