- 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.
- 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
- 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)
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.
- 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.
- 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.