Wet Lab (Hands-On):

Be sure to use proper aseptic techniques throughout. This includes disinfecting lab benches before and after use, washing hands before and after the experiment, flaming test tubes after opening and before closing, using sterile pipets and tubes, and cleaning up spills with disinfectant solution. Bacterial waste (including contaminated pipets and tubes) should be discarded in a designated biohazard container for later sterilization.

1. Start by serially diluting the concentrated Phi X 174 bacteriophage stock solution (provided in tube A):

• Label 2 sterile snap-cap tubes B and C.
• Add 4.5 ml of sterile, distilled water to tubes B and C.
• Add 0.5 ml of Phi X 174 bacteriophage stock solution (in tube A) to tube B. Mix gently.
• Add 0.5 ml of tube B solution to tube C. Mix gently.

(Eppendorf Pipettors, Image from Bio-Link.Org, NSF 2002)

2. For each of the bacterial strains you will test, using the sharpie pen, label the bottom (not the interchangeable lid!) of four base agar plates with the name of the strain and the name of your group. Mark the first plate as the (negative) control plate. Label the other three plates as A, B, and C.

3. Take one tube of soft agar out of the 47’C water bath and wipe the outside with a Kimwipe. Quickly add 0.2 ml of the appropriate bacterial culture to the soft agar tube and mix gently. Pour the mixture over the base agar on the control plate. Replace the plate lid. You may have to tilt the plate around to evenly spread the soft agar.

(Stovall Belly Dancer Hybridization Bath, Image from Bio-Link.Org, NSF 2002)

4. Take another soft agar tube and wipe the outside. Quickly add 0.2 ml of the same bacterial strain, plus 0.2 ml of the tube A bacteriophage solution. Mix gently as before, and pour the mixture over the base agar in plate A. Spread evenly.

5. Repeat step 4 for plates B and C.

6. Repeat steps 2, 3, 4 and 5 for each bacterial strain to be tested.

7. Once the soft agar on all the plates has solidified, incubate the plates upside down in a 37’C incubator overnight.

(Cellstar Radial Shaker Incubator, Image from Bio-Link.Org, NSF 2002)

8. The next day, count the number of plaques on each plate. Numbers in excess of 300 plaques per plate should be recorded as TNTC (Too Numerous To Count). Also record plaque morphology (shape, size, clear vs. opaque, and presence vs. absence of a halo).