Lab 13 Procedure

Measure and Calculate Cell Numbers

• Working in group of 2, obtain one 6-mL 10G bacterial tube,  one 6-mL BW25113 bacterial tube and one 6-mL Nissle 1917 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” 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 WT or Mutant, and either 10G, BW25113 or Nissle 1917.
• Gently mix the bacteria tubes by flickering the tubes with your finger, and then add 2mL of bacteria into each tube.
• Calculate the number of phages to add so the multiplicity of infection (MOI) = 0.1. Be mindful that you have 2mL 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 °C shaker.

Collect Time-Point Samples

• Prepare 6 microfuge tubes and label them with 30 min sand either WT or Mutant, and either 10G, BW25113 or Nissle 1917.
• 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”.
• After all samples are collected, spin all the tubes down in a micro centrifuge for 2 minutes at 4000rpm.
• Remove as much liquid as possible and add 1mL 0.85% saline.
• Gently resuspend the pellet using the 1 mL pipette.
• Spin the tubes down again for 2 minutes at 4000rpm.
• Remove as much liquid as possible and add 1 mL 0.85% saline.
• Dilute the solution by 500 times by adding 2 μL of the bacteria/saline solution to another tube containing 1 mL of 0.85% saline.
• Obtain 3 LB plates and label them with “0 min”,  “30 mins-WT” and “30 mins-Mutant”.
• Add 100 μL of the solution from each microfuge tube onto the corresponding plate. For example, add 100 μL of 10G, BL21 and BW25113 (in total 300 μL) of the 0 min samples on the “0 min” plate.
• Use the bacteria spreader to spread the solution evenly on the plates. Be sure to sterilize the spreader in between use.
• Leave the plates at room temperature for 5 minutes.
• Invert the plates and incubate the plates at a 37 °C incubator overnight.