- Start 8:20 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 trial II)
- Autoclave new M9 medium (1 liter)
Biofilm Optimization (Trial II)
- 3 CBDs are prepared to be incubated at 3 different temperatures: 30, 37 and 42 C at 30 rpm for 48 hours. The procedures below follow from Trial I.
- Following 1.0 McFarland Std (A600 = 0.257), 5-hr incubated E coli broth adjusted to match A600 value. Approximately 6X (0.45ml broth culture to 2.55ml broth) dilution 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 5.4X dilution (0.55ml to 2.45ml broth) to match standard.
- Using multipipette (8-pronged), 200 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 12/18 to score for biofilm growth.
- Plated 10^-10, 10^-12, and 10^-14 dilutions to verify cell number after 5-hr incubation. To be checked after ~24 hrs for colony counting.
- CBD incubated at 30 C was transferred to static incubator. Cannot do 3 CBDs next time, number of incubators limited.
- Inoculum volume in wells changed to 200 ul due to overflow of liquid upon replacement of pegged lid.