- Start 9:15 AM
- Took 5 ml from 10-ml overnight E coli culture into 150 ml broth for 5-6 hr incubation at 37 C (inoculum for biofilm optimization )
- Prepared LB Broth (400 mL)
Biofilm Optimization (Trial I)
- Following 1.0 McFarland Std (A600 = 0.257), 5-hr incubated E coli broth adjusted to match A600 value. A 13.6X dilution was required to match standard.
- For minimal medium M9, E coli broth centrifuged at 3000g for 10 minutes, supernatant LB Broth discarded through careful pipetting, without disturbing sedimented E coli cells. Previously prepared M9 medium used to resuspend E coli cells. Absorbance at 600nm adjusted until it matched 1.0 McFarland Std. Approximately 6X dilution to match standard.
- Further dilution was again carried out (i.e. 1 inoculum : 29 medium)
- Using multipipette (8-pronged), 230 uL of adjusted inoculum pipetted into each well of CBD.
- Columns 1-6: rich LB Medium
- Columns 7-12: minimal M9 Medium
- CBD covered with pegged lid. Incubated at 4:00 PM at 37C, 150 rpm. Will check on Monday to score for biofilm growth.
- Plated 10^-8, 10^-10, and 10^-12 dilutions to verify cell number after 5-hr incubation. To be checked after ~24 hrs for colony counting.
- M9 medium not homogeneous, may be due to saturation or skipping of autoclaving step. Must recalculate and redo later.
- Unsure of second step above, regarding minimal medium. Do we culture a separate inoculum using M9 medium?? If so, starting cell number is not consistent....
- Need to check with RPM value from literature.... 30 RPM from Tre-hardy et al. 150 RPM used was too fast, may have caused the spillage during biofilm growth