- 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 trial III)
Biofilm Optimization (Trial III)
- 3 CBDs are prepared to be incubated at 3 different temperatures: 21, 30 and 37 C at 30 rpm for 24 hours. The procedures below follow from previous trials, except minor adjustments to procedures in: M9 inoculum resuspension sequence => broth matched with 1.0 McFarland first before centrifuging.
- Following 1.0 McFarland Std (A600 = 0.257), 5-hr incubated E coli broth adjusted to match A600 value. Approximately 9X (0.3ml broth culture to 2.7ml broth) dilution to match standard.
- For minimal medium M9, absorbance of E coli broth at 600nm adjusted until it matched 1.0 McFarland Std. E coli 1.0 McFarland inoculum 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, same volume at 1ml.
- Using multipipette (8-pronged), 200 uL of adjusted inoculum pipetted into each well of CBD.
- Columns 1-6: rich M9 Medium
- Columns 7-12: minimal LB Medium
- CBD covered with pegged lid. Incubated at desired temperatures at 30 rpm. Will check on 12/19 to score for biofilm growth.
- Plated LB-based 1.0 McFarland E coli inoculum at 10^-10 to 10^-20 dilutions to verify cell number.
- Plated M9-based 1.0 McFarland E coli inoculum at 10^-6 to 10^-10 dilutions to verify cell number.
- Plated planktonic cells from CBD wells at 10^-2, 10^-4, 10^-6 dilutions for both LB and M9 wells of 21, 30 and 37 C CBDs.
- To be checked after ~24 hrs for colony counting.