Lab 11 Procedure
Measure and Calculate Cell Numbers
- Working as a group of 2, obtain one 12-mL 10G bacterial tube.
- Gently mix the bacteria tube by flickering the tubes with your finger.
- Take out a microfuge tube and label it with “0min”.
- Transfer 1 mL of bacterial cell culture to the microfuge tube and obtain the OD600 readings from the UV spectrometer.
- Record the OD600 reading in your lab notebook. This reading will be the baseline reading for the experiment.
- Return the 1 mL bacterial cells to the microfuge tube and keep the tube on ice.
- Calculate the number of bacterial cells using the OD600 reading (see in-lab instruction).
- Obtain and label three bacterial culture tubes with “control”, “WT” and “Mutant”.
- 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) = 0.2. Be mindful that you have 3 mL of bacteria cells in each condition.
- MOI means the number of phage particles present relative to bacterial cells. For example, if two thousand phages are added to one thousand bacterial cells, the MOI is 2.0.
- Here is a quick video showing how to calculate the volume of phages to add:
- 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 3 microfuge tubes and label them with “30 mins” and “control”, “WT” or “Mutant”.
- Prepare another set of 3 microfuge tubes for the 60-min time point.
- 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 time point samples.