Bacteriophage

Bacteriophages are viruses that infect bacteria. Bacteriophages (phages or Φ for short) are abundant in natural ecosystems, and may be one of the main things limiting the world's bacterial populations. Phages are also widely used as tools for molecular biology. A phage’s ability to induce a cell to make huge numbers of copies of a specific DNA molecule can be quite useful to anyone who is trying to clone a gene. In this lab, you'll learn to plate with bacteriophage and use PCR to detect phage DNA.

Plating with phage

Working with phages in the lab requires a different plating technique than the one you've used so far in Bio 6B. In this lab, you'll use bacteriophage T2, a pathogen that will kill E. coli cells by causing lysis. The challenge will be that you need to see where the cells were killed on your plates. Up to now, you have used plasmids that give the cells the ability to grow on antibiotic plates. You used the antibiotic to wipe out the cells that don't have plasmids, and the ones that survived were the ones you wanted.

To see cells that have been killed by the phage, you will need to create a bacterial lawn. A lawn is a dense growth of bacteria, spread evenly over a plate. This is very different from the colonies that you've seen so far. To create a smooth lawn for phage plates, you need to have the bacteria growing embedded in the agar, rather than spread on top of the agar. You'll start with a plate containing agar, as usual. We can call this the hard agar. Next, you'll mix together E. coli cells, T2 phage, and melted soft agar, and pour thiis mixture onto a plate. The soft agar will solidify, and the cells will grow embedded in the soft agar. The cells will spread through the soft agar until they create a smooth bacterial lawn, making the entire plate cloudy (but with no colonies on top of the agar). If a single bacteriophage infects a single cell, the bacteriophage will reproduce in the cell. The cell will lyse, releasing more viruses. These viruses can infect neighboring cells, but they can't spread far because they're in the soft agar. Eventually a mini-epidemic is created, leaving a small clear spot on the plate where all the cells have been killed and huge numbers of viruses have been released. This clear spot in the bacterial lawn is called a plaque. Each plaque starts from one phage infecting one cell, so if you count the number of plaques on your plate, you'll know how many individual bacteriophage particles (or virions) you initially put onto the plate. Since the viruses reproduce prolifically in the plate, you can easily recover a million times as many viruses as you started with.

The Polymerase Chain Reaction (PCR)

In the plasmid labs (pARO180 conjugation and pGLO transformation), you collected plasmid DNA from the cells and directly visualized it using electrophoresis. In this lab, you'll use PCR to confirm the presence of bacteriophage DNA in the cells that were infected with phage. See PCR for detecting phage for details on that part of the experiment.

Procedure, day 1: plating

Materials

For each lab group, obtain the following:

  • 4 TPA or similar agar plates
  • 1 tube SM liquid medium for diluting phage
  • T2 Bacteriophage stock. The phage concentration is unknown, so we’ll call it 1x.
  • 4 empty sterile glass culture tubes
  • 3 1.5-ml microcentrifuge tubes
  • E. coli liquid culture
  • 4 melted soft agar tubes (leave these in the warm water bath until you’re ready to use them)
  • Pipetmen, tips, and a beaker for waste tips

Plating method

The procedure for this lab is simple, but timing is important. Make sure you plan out what you’re doing before you start. If you’re a little too slow, your experiment won’t work. The basic procedure is to prepare a phage dilution series, add the E. coli cells to the diluted phage, add melted soft agar to the phage/cell suspension, and pour the whole mess onto an agar plate. The bacteria will grow embedded in the soft agar; this will create a good lawn and let you see your plaques well.

Before you start, the soft agar needs to be fully melted and ready at about 50° C in the water bath. Before doing anything else, check to make sure that this is so.

  1. Label the bottoms of your 4 plates 10-1x, 10-2x, 10-3x, and “no phage.”
  2. Make a 10-fold dilution of your T2 phage stock as follows. Pipet 20 µl of phage stock into a microcentrifuge tube, then pipet 180 µl of sterile SM medium into the same tube. Mix gently. The concentration of the phage stock is called 1x for convenience; your 10-fold dilution is 0.1x, or 10-1x. Write “10-1x” on the tube.
  3. Prepare 200 µl of 10-2x phage by making a 10-fold dilution of our 10-1x phage. Pipet 20 µl of 10-1x phage into a new tube, then pipet 180 µl of sterile SM medium into the same tube. Mix gently, and label this tube “10-2x”.
  4. Prepare a tube of 10-3x phage by diluting some of your 10-2x phage, using the amounts listed above. Mix gently, and label the tube “10-3x”.
  5. Pipet 500 µl of E. coli culture into each of your 4 empty culture tubes. Label the tubes 10-1x, 10-2x, 10-3x, and “no phage.” (Label the tubes with tape, which you can remove at the end of the lab.) Add 100 µl of the 10-1x phage to the cells in test tube labeled 10-1x, and mix gently. (Don’t add phage to the other tubes yet.)
  6. Take one tube of melted soft agar from the water bath and bring it back to your lab table. Pour the entire contents of the soft agar tube into the 10-1x test tube. Pour in one quick motion to ensure that everything gets mixed. You are combining the phage and cells with the soft agar.
  7. Immediately pour the entire contents of the soft agar tube (now containing soft agar, E. coli cells, and diluted phage) onto the plate labeled 10-1x. Quickly spread the agar by swirling the plate once, then let this plate sit until the agar solidifies completely. (Make sure that the soft agar is completely liquid and lump-free when you pour it. If there are lumps in the agar, you should do another plate; you won’t see your plaques well on a lumpy plate. Also, don't move the plates until the agar is solid, or you may end up with lumpy plates.)
  8. Repeat these steps to make the 10-2x and 10-3x phage plates. For the “no phage plate, use 100 µl of plain SM instead of the diluted phage.
  9. When you’re done with all the plates, make sure the agar is all solid, then put the plates in the 37° incubator. Put the plates upside down so condensation from the lid doesn’t drip on the plates. You don't need to tape them together or seal them.

Before you leave

You should have four plates in the incubator.

Used pipet tips & microfuge tubes, including the tubes with your diluted phage solutions, go in the biohazard trash.

Gloves, paper towels, etc. go in the regular trash (not the biohazard, unless they’re contaminated).

Beakers used for waste tips: dump the tips into the biohazard trash and put the beaker into the dirty-glassware tub on the cart (if there’s not a tub, check with the instructor).

Glass culture tubes with lids: peel off the tape labels and put the tubes in a tube rack in the dirty-glassware tub on the cart. Don’t set the tubes on their sides; the bacterial cultures will leak out. Don’t leave the tubes in a beaker. Leave them in a rack. These tubes will be autoclaved to kill the bacteria and then the tubes will be washed and reused.

Bad plates that you may have messed up should be put into the biohazard waste can.

Procedure, day 2: plate results and lysates

Today you'll observe your plates, extact DNA from the plates, and perform PCR to test for the presence of phage DNA in the plates.

Plate results

Start by looking at your plates. You should have three plates with different numbers of plaques, and one plate with no plaques. Record the number of plaques. Your three plates were prepared with 10-fold dilutions of phage, so the 10-2x plate should have 1/10 as many plaques as the 10-1x plate.

Prepare lysates for PCR

If there are plaques on the plate, it means that some cells have been lysed. You should be able to recover the virus DNA that was released from the lysed cells and use this DNA as  template for PCR. All you need to do is put some buffer on the plate, and the virus particles will diffuse into the liquid. However, most of the virus is trapped in the soft agar, so you will need to give it some time to diffuse into the liquid.

  1. Choose the plate that has the largest number of plaques (should be your 10-1x plate), and also the "no phage" plate.
  2. Pipet or pour about 4 ml SM buffer onto each of these two plates. You want enough medium to completely cover the plate, but not so much that it will spill. (Later, you’ll want to pipet off the SM.)  Swirl the plates gently and let them sit for at least an hour. (You’ll recover more virus by letting it sit longer.) While you're waiting, you can start preparing for PCR.
  3. Label one  microcentrifuge tube “phage” and another “no phage”.
  4. After 1 hour, recover your plate lysates as follows: Tilt the plate slightly and pipet off the SM into a microcentrifuge tube. Recover 1 ml of buffer into each tube. If there’s not enough liquid to pipet, add some more SM to the plates. Add this amount to the total volume of SM added. The two tubes contain your plate lysates. You should have two: phage lysate and no phage lysate. You can now use these lysates for PCR. Be sure to write your initials and the date on the tubes.
  5. Spin the tubes for 5 min on high. These lysates are now ready to use as PCR templates, with no further preparation.

 Go to the bacteriophage PCR page for the next steps. By the time your lysates are ready, you should have your PCR cocktail ready.

Alternate protocol: plug lysates

Plate lysates, as described above, are prepared by pouring buffer over the whole plate. That method works best when there are hundreds or thousands of plaques. If you have a small number of plaques, use this plug lysate protocol to get a higher concentration of phage. In this method, you cut a few plaques out of a plate and put them into a small volume of buffer.

  1. Pipet 500 µl SM buffer into each of two 1.5-ml micro tubes.
  2. Choose a plate that has at least one plaque for your phage plug lysate, and also use your no-phage plate as a negative control.
  3. Phage plate: Cut the tip off a sterile disposable plastic transfer pipet. Use the pipet to cut into the agar of your plate and pick up an agar plug containing a plaque. Put the agar plug into a the tube with the SM. Using the same pipet, put three more plugs into the same tube, so you have four agar plugs in one tube with SM.
  4. No phage plate: Repeat this procedure with another tube and no-phage plate to make a negative control. If there are no plaques on this plate (the expected result), just pick agar plugs. If there are any plaques, pick those.
  5. Vortex the tubes extensively and then incubate at 37°C for at least an hour to allow the phage particles to diffuse out of the agar. Vortex occasionally during the incubation. Spin the tubes for 5 minutes at high speed, and the plug lysates are ready to use for PCR.

Review

Terms and concepts

  • Bacterial lawn
  • Dilution series
  • Lysis
  • Plaque
  • Plate lysate
  • Soft agar and hard agar

Review questions

  1. How is the procedure for plating with phage different from the procedure for plating with cells that have been transformed with a plasmid?
  2. Why do you have hard agar and soft agar in this experiment?
  3. How many molecules of phage DNA did you put onto your 10-3x phage plate?
  4. Why did you do different dilutions of phage?

References

Lambda Bacteriophage, Wikipedia. This is different from the T2 (or T-even) phage that we're using, but the page has some relevant information.

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