Determination of phages in soil

Bacteriophages are abundant just about anywhere. Phages are abundant in fresh and sea water
as well as soil. We will be analyzing soil from Mt. Clef or Kingsmen creek to determine the
concentration of phages in various locations.
The overall process is to collect soil and grow the phages on E. coli in order to amplify the
phage. We will also do a second amplification step to increase the probability of success of the
experiment. Since the phages we will isolate infect E. coli, they are sometimes called
coliphages.

Materials
E.coli strain B, overnight culture (days 1, 3, 4)
Lambda agar plates (9 per group) and 50 ml lambda top agar [10 g tryptone, 2.5 g NaCl per liter;
12 g agar/liter for plates and 7 g agar/liter for top agar] (day 4)
Tryptone broth media (lambda broth): 10 g tryptone, 2.5 g NaCl per liter
37C incubator
Syringe (10 ml)
Sterile filter (0.45 m)
Sterile tubes (16)

Methods
NOTE: Be sure to use sterile techniques!

Day 1

1) Put on your safari hat.

2) Obtain a small collection vial and hike to Mt. Clef/Kingsmen creek.
3) Locate the desired soil site. If you are collecting under a plant, be sure to collect the sample
from near the roots of the plant. Put about 30 ml of soil into the vial. The exact amount isn’t
important.
4) Hike back to the laboratory and then take off your safari hat.
5) Mix 1.0 ml of E. coli and 60 ml of media to produce an “E. coli broth.”
6) Pour the 60 ml of broth into the culture vessel.
7) Incubate the sample overnight at 37ºC. The culture works best if it is frequently and gently
mixed during the first three hours, so wait a few minutes, and then mix the sample. Before
leaving, remind your instructor to continue mixing the sample.

Day 2

8) The soil will have settled to the bottom of the vial. The samples will probably smell bad. Do
not mix the sample! Without shaking, pour the solution through a cheese cloth or filter.
Collect 5 ml or more into a sterile tube. You do not need the entire solution, so don’t worry
about getting every last drop.
9) We will now filter the solution to remove bacteria. Obtain a sterile 0.45 m filter, a syringe,
and a sterile tube. Do not open the filter package yet.
Last printed on: January 31, 2013 1
 Soil phages
10) Draw 5 ml of solution into the syringe.
11) Open the filter package and attach the syringe to the filter by twisting it on.
12) Slowly depress the syringe plunger and collect the filtrate into a new sterile tube. You need
to collect at least 2.5 ml into your tube.
13) Label the sample and place it in the refrigerator until the next lab. This is tube A. Do not
put it in the freezer!

Day 3

14) We will now amplify the phage. Add 9 ml of sterile broth to each of two tubes.
15) Add one drop of E. coli B to each tube.
16) Add 1.0 ml of your filtrate to one tube. The other is a control.
17) Incubate the sample overnight at 37ºC. The next day you may see a difference in cloudiness
between the two tubes. Put the tube with viruses (tube B) in the refrigerator and discard the
control.

Day 4

18) Filter the tube B overnight culture through a syringe filter as before.
19) We will now do plaque assays on the tube. See the T4 plaque assay handout for the
protocols, but add only one drop of E. coli B per plate. Do the following dilutions of phage:

Plate Phage tube Dilution

1 A None
2 B None
3 B 10-1
4 B 10-2
5 B 10-3
6 B 10-4
7 B 10-5
8 B 10-6
9 Control (no phage) None

20) Incubate the plates overnight at 37C. Save the A and B tubes for future analysis.
21) Observe plaques! There will probably be different types of plaques. Count the plaques on
each plate. If there are too many plaques to count, state this fact in your lab results.
22) Save the plates with plaques for later analysis.

Analysis
Determine the concentration of phage in both tubes A and B.

References
Carolina Biological Supply Company (1981). Soil Bacteriophage Kit.