BjornsMethods/ExperimentLog

Bjorn's Experimental Log

December 14, 2005

• The first step of my adaptation protocol requires me to reproducibly grow bacteria to a concentration of 5e7 - 2e8 cells/mL in 1 hour

• Today I am taking one of my frozen aliquots of bacterial cells (BL21), thawing it, diluting it down to 2 different concentrations, and growing it back up to hopefully find out all the details of what initial concentration of cells will give me the desired concentration at the end of 1 hour.

• Going to try the following initial volumes of my frozen aliquot: 200uL, 500uL

1. X uL of my Klett 80 (4e8 cells/mL) E. coli in ~9.9 mL LB solution in 125 mL flask
2. Grow in orbital water bath at 37C, 200 rpm for 1 hr (or until back to Klett 80)
3. Put flask on ice
4. Serial dilute to get a 1e6 dilution of the Klett 80 solution and create 4 plates: 1x200uL, 2x100uL, 1x50uL
5. Grow overnight in warm room (37C)

Results:

• After 1 hr, the 200uL sample was only at Klett 3 and the 500uL sample was at Klett 16. I let the Klett 16 sample continue to grow for another hour at which it was at Klett 36. At the end of one hour I still proceeded to plate and grow the 500uL sample to practice my technique.

December 15, 2005

1. Remove my plates from the warm room
2. Count colonies and calculate the true concentration of my Klett 16 solution

NOTE: I realized when I came in this morning that I diluted down my Klett 16 solutions as though they were of Klett 80, thus I had very few colonies on my plates this morning. Live and learn ehh???

Today I'm going to repeat yesterday's protocol but with an initial bacteria aliquot volume of 1 mL and 1.5 mL since I was not able to get enough bacteria grown at yesterday's initial concentrations.

RESULTS: After 1 hr the 1 mL sample grew to Klett 18 and the 1.5 mL sample grew to Klett 35. I diluted and plated them to grow overnight in the 37C room so I can count them in the morning.

December 16, 2005

Counted the colonies I grew yesterday and confirmed the following bacterial concentrations:

• 1.5 mL starting aliquot had 1.08e8 cells/mL
• 1.0 mL starting aliquot had 7.5e7 cells/mL

These results now let us know how much of the initial cell aliquot to add to the LB solution so we have btw 5e7 - 2e8 cells/mL after 1 hour of growth.

December 17-19, 2005

Been working on my multi-agent systems simulator all weekend. Its pretty cool ;)