16 December 2008

Date: 16 December 2008; Time: 0820; Agenda: Quantification of 48hr biofilm at 3 temperatures.

• 5ml of overnight culture was mixed with 150ml LB Broth and inoculated at 37degrees for 5 hours.
• Previously prepared M9 broth autoclaved just before usage.

Biofilm formation:

• After M9 is autoclaved, we decided to divide each CBD into 2 parts: One half (ie 48 pegs) filled with M9 broth and the other half (other 48 pegs) filled with LB Broth. This way, we can see how biofilm growth differs in both mediums.
• For this trial:
  • Columns 1-6: LB Broth; Columns 7-12: M9 Broth
• The protocol for the exact biofilm formation can be found on the procedures written for 15 december.
• The only difference for this trial, is that now we introduce M9 medium instead on only LB Broth:
  • For M9, because we only inoculated 1 flask of culture in LB Broth, when we obtain the McFarland standard for LB Broth, we need to also obtain the McFarland standard for M9 Broth. In order to do this:
    • We used a pipette and obtained about 1-1.5ml of LB Broth culture and placed it in a 1.5ml centrifuge tube(after 5hrs of inoculation - see above). This was repeated to prepare 4 such tubes.
    • These 4 centrifuge tubes were then placed in the centrifuge (made sure that they are 'balanced') and made to centrifuge for 10 minutes at 3000g.
    • At the end of centrifuging, the supernatant (LB Broth) is discarded and the residue diluted with the same amount of M9 medium (ie. 1-1.5ml). This is the corresponding M9 culture, similar to the LB Broth culture obtained above (ie. they both should have the same starting cell number since they are obtained from the same cell culture -> EDITED: See 18 December because this is not McFarland Standard!).
    • A McFarland standard for M9 is prepared: Approximately 5.4X dilution (0.55ml to 2.45ml M9 broth) needed to reach McFarland Standard.
• Both (LB and M9) McFarlands are diluted 1:29 with LB Broth and M9 respectively and then 200ul (reduced from 230ul from the previous time as the contents in CBD tend to overflow when the lid is replaced) added to each well in the CBD.
• The lids were replaced and the CBD parafilmed. It is then placed in the incubators. 3 such CBDs were prepared such that they were each placed in different temperatures of 21degrees, 30degrees and 37degrees for 48hrs.
  • The incubator was set to 150rpm.

• The culture after 5 hours of incubation was also plated and the number of colonies counted the next day to gain an estimate of the concentration of cells in the culture.