- Learn how to culture viruses in a host cell
- Quantitate viruses in a specimen
- Identify viral plaques in a bacterial lawn
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.
MATERIALS NEEDED: per table
- 10-3 dilution of the bacteriophage ( this 1/1000 is already made for you ) –either T4 or phi 174
- micropipette and tips
- Pi-pump and 10 ml pipets
- 5 test tubes for dilutions
- 1 bottle of saline solution (0.9% NaCl) to make dilutions
- 50º C 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 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.
- 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.
Detailed procedure with diagram
Set up 5 saline (0.85% NaCl) dilution tubes labeled 10-4, 10-5,10-6, 10-7, and 10-8. Into each tube, place 9ml pf into which you will dilute the viral solution. You will be making 1/10 dilutions.
- Starting with the 10-3 dilution of the virus that you picked up (or were given by your instructor), transfer 1 ml to the dilution tube marked 10-4 and mix.
- Make 4 more dilutions out to 10-8.
- Into 6 microtubes, add 100 microliters of each viral dilution, plus 300 microliters of E. coli. Let sit at room temperature for 10 minutes while the virus infects the bacteria. Mix these well.
- Take the tubes over to the water bath and transfer the entire contents of E. coli - phage tubes into 6 soft agar tubes, using a sterile plastic transfer pipet. Mix well. KEEP SOFT AGARS INSIDE OF WATER BATH SO THEY DO NOT SOLIDIFY.
- 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.
- Allow the plates to harden and incubate at 37º C right side up.
- Lay the 6 plates right side up, from lowest dilution towards highest dilution.
- 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!)
- Get an accurate count of that plate. Fill in the formula for viral counts.
- Calculate the number of viruses per ml. of original specimen.
- Why are viral counts expressed as plaque forming units (PFUs)?
- Did the two phages grow on both E. coli strains? Why or why not?
- Give the plaque count/ml for your viral specimen.
Jackie Reynolds, Professor of Biology (Richland College)