Log Phase Growth Curve and Cell Count in Liquid Media

Updated 02/23/2020 JB
This protocol differs from the Streptococcus Growth Curve and Cell Count in Liquid Media protocol because this protocol focuses on discovering how the cell count changes during the lag phase. It is a very similar protocol, but includes more sampling during the log phase. As in the more general protocol, note that this protocol skips measuring the lag phase.

1. The night before: From the -80* freezer, streak out what bacterial strains you want onto a blood plate. If you have any questions about what tubes correlate to strains, consult the strain spreadsheet on the lab wiki. Incubate in the 37*.
   1. Note: You can also streak these out up to three days in advance, but incubate the plate in the 34* incubator.
2. Set up the biosafety hood. Ensure you have a vortex, necessary pipettes, and a tube rack in your hood.
3. Add 3mL BHI, 30uL catalase, and a line of cells from the plate. Incubate for approximately four hours in the 37* at 5% CO2.
4. During incubation:
   1. Turn on the spectrophotometer (must be turned on at least 15minutes before its first reading. I recommend turning it on once you go into the lab so you don’t have to think about it later).
   2. Set out 12 tubes, one for each hour of the growth curve. In the hood, add 2670 uL BHI and 30 uL catalase to each tube.
   3. In a deep 96 well plate, add 900uL PBS to all wells. Cover with tin foil so the PBS does not evaporate.
   4. Obtain 3-4 blood plates.
5. After four hours, vortex the tube and check its O.D.
   1. If the O.D. is at 0.1, proceed to step 6.
   2. If the O.D. is lower than 0.1, continue to incubate (note: the more you vortex the tube, the slower S. pneumo will grow)
   3. If the O.D. is between 0.1 and 0.3, dilute the tube to 0.1 and continue to step 6.
   4. If the O.D. is higher than 0.3, dilute to approximately 0.05 and wait until the tube’s O.D. reaches 0.1 again.
6. Add 300 uL from the tube at 0.1 to each of the tubes you set up at step 4b. Incubate at 37*.
7. After 1 hour, check the first tube. Vortex and record its O.D.
8. Use one row of the 96 well plate (prepared in step 4c) to do 8 10-fold serial dilutions.
   1. In the first well of the row, add 100uL of the tube to the 900uL PBS. Mix, then move 100uL to the next row, and so on.
9. On one blood plate, pipette 10uL of each well (and of the undiluted tube) into dots on the blood plate. Let dry then incubate.
   1. Typically, you can fit 3 hours’ worth of 10 uL dots onto one plate.
10. Repeat step 7-9 every 15 minutes until the O.D. is 0.7 (which correlates to stationary phase).
11. After the O.D. is 0.7., wait two hours and then repeat steps 7-9 for one more tube.
12. Leave one tube in the incubator overnight and proceed to clean up.
13. Next day:
    1. Repeat steps 7-9 for the overnight tube in the incubator. Remember to check the next day as well.
    2. Record at which dilution you are able to see single colonies for each time point.