BACTERIOPHAGES

Bacteriophages are viruses which infect bacteria. PHAGE (as in phagocytosis) means "to
eat", and generally refers to a virus. Most bacteria have phages that are able to parasitize
them. In fact, the ability to be infected with a known phage type is used to identify some
strains of bacteria (like Staph), known as phage typing . As the virus infects bacterial cells
that it has been mixed with, the lytic infection destroys the bacteria. The bacteria have been
poured into what is called a bacterial lawn on the agar plate. As the surrounding cells are
infected 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 or
virions 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 host
bacteria are 2 different strains of E. coli, so these bacteriophages are called coliphages. The
purpose of using 2 different viruses is to show the specificity of a virus for its host, even for
these little bacterial viruses. The liquefied tryptone soft agar, into which the bacteria and
viruses are placed, has less agar concentration than normal liquefied agar. It allows better
diffusion 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 dilute
further.
 Bacteria and phage are mixed together in tubes of soft agar. The mix is incubated in
the water bath.
 After incubation the mix is added to the soft agar and poured over the tryptone agar
plates.

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 174
1ml pipettes and pi-pump
5 - 9ml saline for dilutions of bacteriophages
50oC water bath
1 strain of E. coli (B or C) in TSB
6 TSA plates
6 - 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. You
will be making 1/10 dilutions.

1. Starting with the 10-3 dilution of the virus that you picked up (or were given by your
instructor), transfer 1ml to the dilution tube marked 10-4 and mix.
2. Make 4 more dilutions out to 10-8.
3. Take your E. coli over to the water bath, and inoculate 0.3ml into 6 (LABELLED 10 -3 to
10-8 ) soft agar tubes. Keep these soft agars in the water bath to keep from
solidifying.
4. Now take your 6 viral dilutions (10-3 to 10-8) in a rack over to the water bath, and
transfer 0.1ml of each dilution into 1 soft agar. Mix these well and LET THEM SIT
FOR 10 MINUTES. This allows the phage to attach to the bacterial cells.
5. Remove 1 soft agar tube at a time and pour it onto the TSA agar plates, gently rotating
the 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.

2
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 = PFUs
dilution of tube X amount plated

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

QUESTIONS:

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

2. Did the two phages grow on both E. coli strains? Why or why not?

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

LAB MANUAL: TABLE OF CONTENTS

Fall 2011 – Jackie Reynolds, Richland College, BIOL 2421