BACTERIOPHAGES

Bacteriophages are viruses which infect bacteria. PHAGE (as in phagocytosis) means "toeat", and generally refers to a virus. Most bacteria have phages that are able to parasitizethem. In fact, the ability to be infected with a known phage type is used to identify somestrains of bacteria (like Staph), known as phage typing . As the virus infects bacterial cellsthat it has been mixed with, the lytic infection destroys the bacteria. The bacteria have beenpoured into what is called a bacterial lawn on the agar plate. As the surrounding cells areinfected and killed by the released viruses, a clear spot on the agar---in the bacterial lawn---develops, called a plaque. The plaques can be counted and the number of virus particles orvirions in the original specimen, can be quantitated as viruses/ml of plaque-forming units/ml(PFUs).

In this lab, 2 kinds of bacteriophages will be used---T4 and phi 174 viruses. Their hostbacteria are 2 different strains of E. coli, so these bacteriophages are called coliphages. Thepurpose of using 2 different viruses is to show the specificity of a virus for its host, even forthese little bacterial viruses. The liquefied tryptone soft agar, into which the bacteria andviruses are placed, has less agar concentration than normal liquefied agar. It allows betterdiffusion of the viruses and better contact with the bacteria.

The procedure is really very easy.

The phage specimen you will use is already diluted to 1/1000, and you will dilutefurther.

Bacteria and phage are mixed together in tubes of soft agar. The mix is incubated inthe water bath.

After incubation the mix is added to the soft agar and poured over the tryptone agarplates.

OBJECTIVES:

Learn how to culture viruses in a host cell.Quantitate viruses in a specimen.Identify viral plaques in a bacterial lawn.

MATERIALS NEEDED:

per table

10-3 dilution of the bacteriophage (this 1/1000 is already made for you) –either T4 or phi 1741ml pipettes and pi-pump5 - 9ml saline for dilutions of bacteriophages50oC water bath

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2420

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1 strain of E. coli (B or C) in TSB6 TSA plates6 - 3 ml liquefied soft agar tubes (kept in water bath)

THE PROCEDURE:

Be SURE to mix the dilutions.Change pipettes between dilutions.Each table will use a different combination of a phage and an E. coli host

T4 phage and E. coli B T4 phage and E. coli C Phi 174 phage and E. coli C Phi 174 phage and E. coli B

Set up 5 saline (0.85% NaCl) dilution tubes labeled 10-4, 10-5, 10-6, 10-7, and 10 -8. Youwill be making 1/10 dilutions.

into 6 soft agar tubes, using a sterile plastic transfer pipet. Mix well. 4. Take the tubes over to the water bath and transfer the entire contents of E. coli-phage tubes

KEEP SOFT AGARS INSIDE OF WATER BATH SO THEY DO NOT SOLIDIFY. .

3. Into 6 microtubes, add 100 microliters of each viral dilution, plus 300 microliters of E. coli.

Mix these well.Let sit at room temperature for 10 minutes while the virus infects the bacteria.

-82. Make 4 more dilutions out to 10 .-4instructor), transfer 1ml to the dilution tube marked 10 and mix.

-31. Starting with the 10 dilution of the virus that you picked up (or were given by your

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5. Remove 1 soft agar tube at a time and pour it onto the TSA agar plates, gently rotatingthe plate WELL so as to distribute the phage-bacteria all over the agar.

6. Allow the plates to harden and incubate at 37oC right side up.

INTERPRETATION

1. Lay the 6 plates right side up, from lowest dilution towards highest dilution.2. Pick each plate up, hold it up to the light, and determine which one has between 30-

300 plaques (you can also use the Quebec colony counters---good backlighting!)3. Get an accurate count of that plate. Fill in the formula for viral counts.

# viruses/ml = PFUsdilution of tube X amount plated

4. Calculate the number of viruses per ml. of original specimen.

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LABORATORY REPORT SHEET

QUESTIONS:

1. Why are viral counts expressed as plaque forming units (pfu)?

2. Why was E. coli added to the soft agar overlays?

3. Why did the two phages not grow on both E. coli strains?

4. Give the plaque count/ml for your viral specimen.