Isolation of E.

coli-Specific Bacteriophage (Coliphage) from Sewage Water

Al Jay Mejos, John Warner Carag, Jojiemar De Pano, Allison Vincent Labador, Erika Mari Macapagal
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City

Abstract
Bacteriophages are host-specific viruses targeting only certain types of bacteria. E. coli-infecting bacteriophages are specifically called coliphage. The
process of isolation of viruses by infecting host exploiting the specificity of the viruses is also employed in the isolation of bacteriophage. This
experiment determined to isolate coliphage from 15 mL sewage water procured from the College of Education by introducing them to 18-24 hour old
pre-grown E. coli after suspension in 5 mL chloroform for 1 hour. After infection of the E. coli samples for 5 minutes, 5, 10 and 0 drops of the purulents
were transferred to 3 mL tubes of soft (0.7%) Nutrient Agar labeled A, B and C respectively, shaken, transferred to NA plates and incubated*. Plaques
from the NA plates were transferred to E. coli broths and reincubated* and then observed. Erroneous data have been observed which are attributable
to misexecution, while only the third set of data showed agreement to the expected results.

Introduction
Bacteriophages or phages are viruses that infect prokaryotic
cells. Bacteriophage that infect E. coli are referred to as coliphage.
Phages can replicate only within host cells. Phage must attach to a
receptor on the surface of a bacterial cell in order to initiate an
infection. This interaction between the phage and receptor is very
specific - a given phage type only will bind to a specific receptor
molecule. Thus, no two phages are alike. A coliphage can replicate
only within E. coli.
When a virion initiates an infection on a flat surface, a zone of
lysis may occur that results in a clear area in the lawn of growing
host cells. This clearing is called a plaque, and it is assumed that each
plaque has originated from replication events that began with one
virion. By counting the number of plaque-forming units, one can
calculate the number of virus infectious units present in the original
sample.
This procedure also permits the isolation of pure virus
strains. If a plaque has risen from a single virion, all the virions in this
plaque should be genetically identical. Some of the virions from this
plaque can be picked and inoculated into a fresh bacterial culture to
establish a pure virus line. (Madigan, et al, 2007).
The objective of this experiment is to determine and isolate
the presence of coliphage or E.coli bacteriophage from the sewage
sample.
Methodology
Treatment of Sewage Culture
Sewage water was collected from the sewage canal alongside the
UP College of Education. To 15mL of raw sewage water 5mL of
chloroform is added to treat it. The water-chloroform mixture was
mixed thoroughly and allowed to stand for 60 minutes at room
temperature.
Isolation of Bacteriophage
5mL of chloroform-treated sewage water is introduced into a 5mL
18-24 hour old culture of E. coli and was allowed to stand for 5 minutes
at room temperature. To 3 tubes of 3mL soft NA (0.7%) maintained at
50oC, sewage-culture filtrate was to added test tubes A, B, and C
respectively, with the following amounts: A – 5 drops; B - 15 drops, C –
0 drop. After shaking to mix the contents, each was poured separately
into newly prepared NA plates. After allowing the soft agar to solidify,
plates were incubated for 24 hours, and were observed.
Purification of the Isolate
After touching a plaque in the NA plate it was inoculated into a 24-
hr. old broth culture of E. coli. And after incubating for 24 hours, zones
of clearings were observed.
Results
Table 1. Microbial growth in varying pH, temperature and UV exposure.
Group Control 1 mL 2 mL
1 2 7 n/a
2 1 n/a n/a
3 9 67 132
Discussion
This experiment focuses on the isolation of bacteriophage from
sewage water obtained near the vicinity of the Institute of Biology
along the College of Eductaion . The obtained sewage water was
first treated with chloroform in order to eliminate other
microorganisms contained in the sewage water. After a period of
time, the chloroform-treated sewage water was then introduced
into a culture of E. coli. This is an essential step since bacteriophages
are known to infiltrate other microorganisms (bacteria).
Bacteriophages need these bacteria because they lack certain
enzyme systems that would permit them to synthesize materials
needed for their life cycle in order to proliferate. These materials
are present inside the host bacteria (E. coli) and are used by the
“intruder” bacteriophage in order to proliferate. The combination of
the sewage water with the E. coli culture is now called a sewage-
culture filtrate. This sewage-culture filtrate is then inoculated on 3
soft NA tubes. The experiment utilized soft NA in order to take
advantage of its jelly like consistency, helping the researchers
visualize the plaques produced after inoculation of the samples to
the NA plates. The 3 soft NA tubes were used for the three set-ups
with different sewage-culture filtrate concentrations in the
experiment. The 3 soft NA tubes with the sewage-culture filtrate
were shaked and then inoculated to 3 NA plates.
After incubation for 24 hours, clearings or plaque formations
were seen in some of the NA plates. These plaque formations
indicate bacterial lysis or the presence of bacteriophages in the NA
plates. By looking at results, one can see that there was plaque
formation in the all three control set-ups. This is not consistent with
the theoretical result since this set-up did not receive any sewage-
culture filtrate and thus did not contain any bacteriophages that can
lyse the bacterial E.coli. Human errors in performing the experiment
and bacteriophage contamination are one of the few reasons that
could explain the control and group 2 experimental results. The 1
mL set-up of group 2 and both 2 mL set-ups of groups 1 and 2 were
labeled as n/a since no plaque formation can be observed since the
soft NA did not properly settle in the NA plate. This error shows
how important it is to give time for the soft NA to settle properly in
order for the researchers to be able to observe and count the
plaques produced by the bacteriophages. Only group 3 had shown
similar results with the theoretical since a significant increase in the
plaque formation or bacterial lysis can be seen between the lower
concentrations of sewage-culture filtrate (1 mL) and the higher
concentrations of sewage-culture filtrate (2 mL).
References
http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html. by
Dr. Mark Sutherland. Date accessed: Sept. 25, 2009
http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Media.htm by Dr.
David D. Fankhauser. Date accessed: Sept. 24, 2009.
Madigan MT & Martinko JM. 2006. Brock Biology Of Microorganisms, 11th ed.
USA: Pearson Prentice Hall, Pearson Education, Inc.
http://www.mansfield.ohio-state.edu/~sabedon/biol4022.htm Date
accessed: Sept. 24, 2009.

*incubation for 24 hours at 26°C