Start time: 2pm
Re-prepare M9 medium and quantification of biofilm formed.
- Cultured bacteria (for biofilm optimization trial II next day)
- Single loop taken from streaked petri dishes on 13/12/08 and suspended into 10ml broth in a test tube (incubate at 37 C for 24hrs).
- A new M9 medium was prepared again using a different protocol because the medium prepared earlier appeared cloudy.
New M9 Medium Prep
- Na2HPO4 (10g), Na2HPO4 (10g), NH4Cl (5g), NaCl (2.5g) were dissolved in 1L of distilled water using a volumetric flask ( to give a 5X concentration of M9 salt).
- The flask is autoclaved for 15minutes at 121 degrees.
- 200ml of this sterile 5X M9 Minimal Salt solution is added to 750ml sterile distilled water.
Next, MgSO4 (0.2408g; or 2ml of 1M MgSO4),CaCl2 (0.0147g; or 0.1ml of 1M CaCl2) and glucose (4g of filter-sterilized D-Glucose- use a syringe with filter for to ensure sterility) were added.)
- M9 medium is made and autoclaved the next day (16/12).
Biofilm Preparation (CV staining and Sonication) - Trial I
- The CBD, incubated from 12/13 at 37 degrees for 48 hours was removed for quantification.
- The pegged lid containing biofilm was removed and rinsed in a new microplate containing phosphate buffered saline (PBS) to remove planktonic cells.
- To prepare PBS: 1 PBS tablet was dissolved in 20ml distilled water.
- This solution was then distributed into a separate 96 well microplate, with each well containing approximately 0.2ml of PBS.
- The lid containing the biofilm was then rinsed (dipping it up and down) in this new microplate for about 1 minute.
- Bacteria in the biofilm was then fixed with absolute methanol
- Approximately 0.2ml of methanol was distributed in each well of a new microplate.
- The lid containing the biofilm was transferred from the microplate containing PBS to the microplate containing methanol and soaked for 10 minutes.
- 2 methods used: CV staining and sonication, therefore half the microplate will be stained with CV while the other half will undergo sonication.
Protocol for CV staining (half of 96 wells = 48 wells)
- The biofilm was then stained with 0.005% (w/v) crystal violet
- Preparation of crystal violet
- 5mg crystal violet solid weighed and dissolved in distilled water in a bottle.
- 25ml methanol was measured using a pipette and added to the crystal violet solution.
- Solution topped up to 100ml with distilled water.
- This 0.005% crystal violet solution was distributed into a new microplate, each well containing 0.2ml crystal violet solution.
- The lid containing the biofilm is transferred from the microplate (containing methanol) to another containing crystal violet solution.
- Soaked in crystal violet solution for 20 minutes.
- The biofilm was then rinsed with distilled water to remove excess crystal violet:
- 0.2ml distilled water was added to each well of a new microplate.
- The lid containing the biofilm was transferred from the microplate (containing crystal violet solution) to the microplate (containing distilled water).
- Rinsed (up and down motion) in distilled water for about 1 minute.
- The CV stain on biofilm was then solubilized in glacial acetic acid:
- 0.2ml acetic acid was distributed in each well of a new microplate.
- The lid containing the biofilm is transferred from distilled water to the acetic acid containing microplate.
- It was left to soak until the pegs were destained of blue color.
- The microplate containing the blue stain was separated from the lid.
- The amount of stain was measured using a microplate reader at 600nm. Data is in B2 computer, under Chang Lab folder.
- The absorbance correlating to the amount of stain can be calculated by taking the difference between the readings obtained from plate reader and an absorbance reading of pure acetic acid.
Protocol for sonication (other half of 96 wells = 48 wells): Done after CV staining.
- Each of the 48 wells filled with LB broth (while the other 48 wells contained the destained crystal violet solution from CV staining step (as shown above).
- It was sonicated for about 10minutes.
- 2 of the wells (1 for LB broth and 1 for M9) were plated on an agar plate at 3 dilutions (10^-8, 10^-10, 10^-12).
- Water from the sonicator entered the microplate, affecting the result.
- Styrofoam floats were used to prevent water entry.