Difference between revisions of "Vpachec3 Week 11"
From LMU BioDB 2015
(→Article Outline: Started outline) |
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#* | #* | ||
#Materials and Methods | #Materials and Methods | ||
| − | #*Strains and Culture Conditions | + | #*'''Strains and Culture Conditions''' |
#** 18 strains in the ''Burkholderia cepacia'' group | #** 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 | ||
==Links== | ==Links== | ||
Revision as of 09:04, 16 November 2015
10 Biological Terms and Definitions
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
