15 December 2008

Date: 15 December 2008; Time: 1400; Agenda: Reprepare M9 medium and quantification of biofilm formed.

• A new M9 medium was prepared again using a different protocol because the medium prepared earlier appeared cloudy, which should not be the case.
• New M9 medium protocol:
  • In 1L of volumetric flask, the following components were weighed using a weighing machine and added into the flask:
    • Na2HPO4 (10g)
    • KH2PO4 (5g)
    • NH4Cl (5g)
    • NaCl (2.5g)
  • This 1L 5X concentrate of M9 minimal salts is dissolved in 1L of distilled water.
  • 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, the following components were added:
    • MgSO4 (0.2408g; or 2ml of 1M MgSO4)
    • CaCl2 (0.0147g; or 0.1ml of 1M CaCl2)
    • Glucose (4g of filter-sterilized D-Glucose)
      • Use a syringe with filter for to ensure sterility!
  • Once the M9 medium is made, it was stored and will be autoclaved the next day (16/12).

Quantification of biofilm formed (CV staining and Sonication):

• The biofilm that has been incubated in the 37 degrees incubator was removed after 48 hours.
• The lid containing the biofilm is removed from the microplate and the pegs 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 (up and down motion) 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 is transferred from the microplate containing PBS to the microplate containing methanol.
  • It was left to soak in methanol for 10 minutes.

Protocol for CV staining:

• As the quantification of biofilm will be based on 2 methods (ie. CV staining and sonication), 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 sufficient distilled water in a bottle.
    • 25ml methanol is 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 about 0.2ml crystal violet solution.
  • The lid containing the biofilm is transferred from the microplate containing methanol to the microplate containing crystal violet solution.
  • It was left to soak 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 is transferred from the microplate containing crystal violet solution to the microplate containing distilled water.
  • It was rinsed (up and down motion) in distilled water for about 1 minute.
• The stained 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 the microplate containing distilled water to the microplate containing acetic acid.
  • It was left to soak until the pegs are destained of blue colour.
• The microplate containing the blue stain was separated from the lid.
• The amount of stain was measured spectrophotometrically by reading the microplate at 600nm using a plate reader.
• The results are as follows:
• 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):

• Each of the 48 wells were soaked in LB broth (while the other 48 wells contained the destained crystal violet solution from CV staining step (as shown above).
• The microplate was then placed in a sonicator.
• However, water from the sonicator entered the microplate, rendering the experiment slightly inaccurate.
  • With regards to this, we decided to make use of a styrofoam to levigate the microplate the next time so that water does not enter.
• 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).
• Counting and subsequent back calculation of the number of colonies may provide an estimate for the amount of biofilm formed for both M9 and LB Broth medium.

This marks the end of quantification of biofilm.