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Created different concentrations of cells:
1) Put 10mL culture (pvj4) into 6, 15mL centrifuge tubes
original culture (20 cultures)
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Used Beckman Centrifuge
To balance tubes:
- Weigh the tubes that will be in one rotor (standing them up in a styrofoam block is easiest)
- Weigh tubes that will be placed in other rotor
- If unbalanced, put empty tube in the lighter styrofoam block and add water to tube until both block are the same mass.
- For bacterial cells, centrifuge for about 10 minutes at a speed a little higher than 1000rpm.
To create bacteria in mid-logarithmic phase
- Bacteria double every 20 minutes
- An overnight culture (15hrs) should reach stationary phase (od about 10)
- If cell culture density is known (after an overnight culture)-- dilute down to .05 .1 cell density (to make it simple-- can calculate amount of cells after a certain amount of time
- Cells in stationary phase don't grow much -- culture od would probably be out of spec's od range (0.2-0.7) so dilute it down to the linear range and then to 0.05 or 0.1 and let incubate for about an hour
Mid-logarathmic phase: optical density less than 1-- dilute culture in stationary phase (greater than 2 or 3) to 0.05od or 0.1od and then incubate for 45 minutes to an hour to reach mid-logarithminc phase (remember doubling time for E.Coli is once every 20 minutes)
To grow cultures, use a culture solution that is 1/4 the volume of the flask
- Insert any setbacks we had today or lessons to learn from!