Chang Lab:Notebook/CBE/08/148/2008/12/15

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  • The M9 medium that prepare could not dissolve fully, therefore we went online to search for the new protocol.

New M9 Protocol: M9 salts : 5X concentration

  • The following components were weighed using a weighing machine and added into the a 1L flask:
    • Na2HPO4 (10g)
    • KH2PO4 (5g)
    • NH4Cl (5g)
    • NaCl (2.5g)
  • M9 minimal salts is dissolved in 1L of distilled water.
  • The flask is autoclaved for 15minutes at 121 degrees.

M9 Medium:

  • 200ml of this sterile 5X M9 Minimal Salt solution is added to 750ml sterile distilled water.

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) Syringe with filter was used to ensure sterility!

Biofilm Quantification(Sonification and CV Staining) Trial 1

CV Staining

  • CBD incubated on the 13th Dec at 37 degrees for 48 hours was used for quantification.

Steps involved in quantifying the biofilm:

  • The peg lid containing biofilm is removed and rinsed with phosphate buffered saline (PBS)

(PBS was prepared by dissolving 1 PBS tablet with 200ml of distilled water)

    • After about 1 minute, the peg lid was then put into a new microplate containing ~95% methanol. This is to fixed the bacteria.
    • The peg was then soaked for 10 minutes
    • A new 96 wells microplate was then used to prepare for CV staining. As we are using two methods to quantify the biofilm:CV staining and sonification. Only half of the wells were filled with 0.2ml of CV solution.
    • 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.
    • The peg was left to soaked for 20 minutes.
    • The peg was then rinsed with water to remove extra CV solution.
    • The peg was then soaked into a new microplated which contain Acetic Acid Solution. It is left till all the CV stain was removed from the peg.
    • The amount of stain was measured spectrophotometrically by reading the microplate at 600nm using a microplate reader.
  • 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.


  • 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.
  • 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.