Lab 9 Procedure

Phage procurement

• Working in a group of 2, obtain your bacteria culture tubes from the last lab session.
• Briefly vertex the tubes for a few seconds.
• Visually compare the tubes and examine the cloudiness of the cultures; write down your observation.
• Take a picture of the tubes for the mini lab report.
• Take out 3 microfuge tubes. Label them with the mutant identity (e.g. D540H).
• Add 1.5 mL of the mutant bacteria culture tube mixture from the last lab into each microfuge tube.
• There is no need to do the same for the control culture.
• Spin down the tubes at 3000rpm for 2 minutes.
• While the tubes are spinning, take out a syringe with a 0.22 μM filter and obtain an autoclaved small beaker and a 15 mL conical tube.
• After centrifugation, transfer the supernatant from all three tubes to the autoclaved beaker.
• Fill the syringe with as much liquid in the beaker as you can.
• Clear any extra air from the syringe and add the filter to the tip of the syringe.
• Carefully push the supernatant through the filter and into the conical tube (you may lose some samples to the filter and that’s okay).
• Here is a video showing the filtration process:

• Mix the tube by flickering it with your fingers.
• Prepare and label 3 new microfuge tubes and aliquot the filtered solution (contains phages) relatively equally into each tube; label the tubes well.
• You will use the phages for the remainder of the semester.

Plaque Titer Assay

• To calculate the concentration of the phages, you will do five dilutions (serial dilutions) and plate each dilution onto a LB plate.
• Obtain a tube of 0.85% sterile saline solution.
• Take out eight microfuge tubes and label them with the dilutions (10^-1, 10^-2, 10^-3 … 10^-7).
• Add 900 μL of the saline solution into each tube.
• Add 100 μL of the filtered phage mutant into the tube labeled with 10^-1.
• Mix well with a pipette tip, and transfer 100 μL solution from tube 10^-1 to tube 10^-2.
• Repeat the same procedure until the tube 10^-7.
• Obtain 5 pre-warmed LB plates. Label them with your names, group number and phage mutation.
  • For 4 of the 5 plates, phage dilutions 10^-4, 10^-5, 10^-6, 10^-7 will be plated. Label the plates to indicate the dilutions.
  • For 1 of the 5 plates, no phage will be plated. Label the plate as a control plate.
• Here is a quick video showing how to add the soft/top agar onto the LB plate:

• Obtain a tube of 10G bacteria.
• Remove one soft agar tube at a time from the 42 °C water bath.
• Quickly add 100 μL of the 10G bacterial culture and 100 μL of the phage solution.
  • For the control plate, add 100 μL of the 10G bacterial culture and 100 μL of saline.
• Mix the tube by flickering it with your fingers.
• Quickly pour the content of the tube onto the corresponding LB plate, and gently swirl the plate to disperse the soft agar. Try your best to spread the soft agar evenly on top of the LB plate.
• Repeat the same procedures for the rest of the five plates; each with one dilution of phages.
• Keep the unused phage stock in your freezer box.
• Allow the soft agar to harden for a few minutes at room temperature.
• Incubate the plates at 37 °C overnight (do NOT invert plates).