Lab 12 Procedure
Measure and Calculate Cell Numbers
- Obtain one 12-mL 10G bacterial tube and one 12-mL BW25113 bacterial tube.
- Gently mix the bacteria tubes by flickering the tubes with your finger, then remove 1 mL of each bacterial cell culture to a microfuge tube labeled “0 mins” (one with 0 mins 10G and one with 0 min BW25113) and obtain the OD600 readings from the UV spectrometer.
- Record the OD600 readings in your lab notebook.
- Return the 1 mL bacterial cells to the microfuge tube and put it on ice.
- Calculate the approximate number of bacterial cells using the OD600 reading (see in-lab instruction).
- Obtain and label six bacterial culture tubes with either control, WT or Mutant, and either 10G or BW25113.
- Gently mix the bacteria tubes by flickering the tubes with your finger, and then add 3mL of bacteria into each tube.
- Calculate the number of phages to add so the multiplicity of infection (MOI) = 0.5. Be mindful that you have 3mL of bacteria cells in each condition.
- Add the amount of phages needed to the corresponding “WT” and “Mutant” tubes.
- Shake the bacterial tubes in a 37-degree shaker.
Collect Time-Point Samples
- Prepare 4 microfuge tubes and label them with 30 min sand either WT or Mutant, and either 10G or BW25113.
- Prepare another 4 microfuge tubes and label them with 60 mins and either WT or Mutant, and either 10G or BW25113.
- 30 minutes after shaking starts, take out the tubes and remove 1 mL solution of each culture tube to the corresponding microfuge tubes labeled “30 mins”.
- Put the microfuge tubes on ice.
- Return the bacterial tubes to the shaker.
- Obtain the OD600 readings from the UV spectrometer for the “30 mins” samples.
- Record the OD600 reading in your lab notebook.
- Return the 1 mL bacterial cells to the microfuge tubes and keep the tubes on ice.
- Repeat this procedure for the 60-min time point.
- After 60 minutes, stop shaking and identify trend in the OD600 readings for the control, WT and mutant samples. Re-measuring OD600 if necessary.
- Discuss with the teaching team if anything is not clear or if anything is unusual.
- Discard the collected samples.