Experiments

It has been determined that several lab tests will be conducted to observe and understand Cutibacterium and biofilm. The importance of these tests is to give proof and make sure that our research, data, and results are viable and can be replicated. Other lab tests are completed to obtain statistics and analytics about our experiment in whole so the data can be analyzed and processed to reach a stable conclusion.

• PCR testing done on the plasmid to ensure its viability
• qpcr is a type of PCR which monitors the real-time amplifications of the DNA-molecule. We got our primers,DNA Oligo, from Integrated DNA technologies.
• primers centrifuged first in order to get all the DNA to the bottom
• The primers are resuspended in distilled water. 245 microliters for one and 275 microliters for the other.
• Master Mix is created for 4 DNA samples and then the primers are added to the Master Mix. PCR mix used is here: https://www.thermofisher.com/order/catalog/product/A25741
• The qpcr is then set up with correct time and temperature as the procedure states. Within 2 hours, our pcr was completed.
• gel electrophoresis is also conducted as a part of PCR to determine the validity of the fragments in the plasmid.
• This is set up by using a 5% Agarose gel powder. 100ml for 1.5 gram of powder. It was then mixed and microwaved for around 1 min until clear.
• mini-prep plasmid purification done to isolate plasmid DNA in order for it to be sequenced and have PCR done.
• Spectrophotometer used to measure the concentration
• useful when trying the grow biofilm as there must be a certain concentration of bacteria to grow.
• This is also being used when determining the biofilm prevalence. The machine accounts for absorbance at certain wavelengths, in our case is around 570nm.
• Crystal Violet Staining Process with dnase
1. Spun bacteria plate down at speed 3500 for 5 minutes
2. Replaced media on 96 well biofilm plate from 8/29
DNase procedure:
1. Keep dnase vials in ice for a few minutes
2. Take them out of the ice and keep them in a tray to defrost them
3. Dilute the DNase into 4 different sections of concentrations: control, 0.5 mg, 1 mg, 5 mg,
4. Dilution using m1v1=m2v2 resulted in initial volumes of 5 microliters for the 0.5 mg sample, 10 microliters for the 1 mg sample, 50 microliters for 5 mg sample
5. divided the 96 well plate into the 4 sections of concentration: (control, 0.5 mg, 1 mg, 5 mg) 24 wells for the 0.5 mg, 24 wells for the 1 mg 22 wells for the 5 mg and 26 for the control
6. Pipetted the DNase into each individual well
7. Plate goes back into the incubator for 24 hours!
Then This procedure was followed from the study--DNase inhibits early biofilm formation in Pseudomonas aeruginosa- or Staphylococcus aureus-induced empyema models: Changes: Bacteria was first centrifuged at 3500 rmp for 5 min. After Acetic Acid was used, multiple washings of pbs followed to get the crystal violet staining out. Centrifuged for 5 min at 3500 each time pbs went in.

Crystal Violet Staining Protocol
Neuroscience Article on Crystal Violet
NCBI Article on Biofilm Research