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. E. coli in LB dilutions
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. E. coli in M9 dilutions
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.
Serial Plating
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.
Notes
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
