- Took 5 ml from 10-ml overnight E coli culture into 150 ml broth for 5hr incubation at 37 C (inoculum for biofilm optimization trial II)
- Autoclave new M9 medium (1 liter)
Biofilm Optimization (Trial II)
- 3 CBDs were prepared to be incubated at 3 temperatures: 30, 37 and 42 C at 30 rpm for 48 hours. The procedures are same as Trial I.
- 5-hr incubated E coli broth adjusted to get 1.0 McFarland Std (Abs = 0.257 at 600nm). 0.45ml broth culture : 2.55ml broth to get 0.267 reading.
- For minimal medium M9, E coli broth centrifuged at 3000g for 10 minutes, supernatant LB Broth discarded (without disturbing sedimented E coli cells). Previously prepared M9 medium added to the E coli cells. Absorbance at 600nm adjusted until it matched 1.0 McFarland Std. 0.55ml broth culture : 2.45ml broth to get 0.25 reading.
- 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 18/12/08 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 (in used by others)
- Inoculum volume in wells reduced to 200 ul to prevent spillage of liquid upon replacement of pegged lid.