23 December 2008

Date: 23 December 2008; Time: 0930; Agenda: Quantification of 72hr biofilm at 3 temperatures and biofilm formation at 21deg and 37deg for 72hr.

Biofilm formation:

• Similar procedures carried out to grow biofilm, only that now they are left to grow at 72hr at 21deg and 37deg. 30rpm.
• Now we try to inoculate the bacteria in M9 and LB media and then use those to grow the biofilm (ie. instead of centrifuging)
• Columns 1-4: M9 culture(centrifuged); Columns 5-8: M9 culture (inoculated); Columns 9-12: LB culture
• Similar plating done to verify McFarland standards.

Quantification of biofilm:

• This time we quantified the biofilm formed based on both CV and sonication with subsequent plating.
  • We first stained (half) the pegs with CV and then the absorbance read to quantify the amount of biofilm formed.
  • Those not stained with CV were used to carry out the sonication.
    • Result of sonication: SONICATOR TOO AGGRESSIVE --> Try the sonicator in B4 Lab (Prof Kunn) next time.

Results for plating of serial dilutions done on 20 December.

• For LB McFarland Standard:
  • 10^-12: 105
  • 10^-14: 50

• For M9 sample (with the same starting number of cells as LB McFarland Standard)
  • 10^-16: 359

• Results of plating seems inaccurate as the starting number of cells in M9 and LB medium should be theoretically similar assuming the centrifuging was done properly. If the starting number of cells are the same, the LB agar plates (at a certain dilution) should contain similar number of colonies for both M9 and LB media. However, the number of colonies vary by a lot (compare at 10^-16: for LB: Less than 50; for M9: 359). Hence, the centrifuging may have been inaccurate!