BIO163-2L Microbiology and Immunology Laboratory

3rd Quarter SY 2014-2015

ABSTRACT

Bacteriophages infecting Bacteroides are potentially a good tool for fecal source tracking, but different Bacteroides host
strains are needed for different geographic areas. A feasible method for isolating Bacteroides host strains for phages present
in human fecal material is described. Useful strains were identified for application in Spain and the United Kingdom. One
strain, GA-17, identified as Bacteroides thetaiotaomicron, was tested in several locations in Europe with excellent performance
in Southern Europe.
Keywords: Escherichia coli, bacteriophage, isolation, phage, DNA, RNA, bacteria, virus, virions

1. INTRODUCTION
A bacteriophage is a virus that infects and replicates within
a bacterium. Bacteriophages are composed of proteins that
encapsulate a DNA or RNA genome, and may have
relatively simple or elaborate structures. Their genomes
may encode as few as four genes, and as many as
hundreds of genes. Phages replicate within the bacterium
following the injection of their genome into its cytoplasm.
Bacteriophages are among the most common and diverse
entities in the biosphere.
Phages are widely distributed in locations populated by
bacterial hosts, such as soil or the intestines of animals.
One of the densest natural sources for phages and other
viruses is sea water, where up to 910-8 virions per milliliter
have been found in microbial mats at the surface, and up to
70% of marine bacteria may be infected by phages. They
have been used for over 90 years as an alternative to
antibiotics in the former Soviet Union and Central Europe,
as well as in France. They are seen as a possible therapy
against multi-drug-resistant strains of many bacteria.
2. MATERIALS AND METHODS
2.1 MATERIALS
Chloroform is an organic compound with formula CHCl3.
Chloroform treatment is generally used to kill any bacteria
present.
Escherichia coli is a Gram-negative, facultatively
anaerobic, rod-shaped bacterium of the genus Escherichia
that is commonly found in the lower intestine of warmblooded organisms (endotherms). Coliphage use E. coli as
a host organism, these phage can be found in places where
E. coli are plentiful.

A. Treatment of Sewage Culture

15 mL of raw sewage was added into 5 mL of chloroform.
Then, it was shook vigorously. After shaking the mixture, it
was left to stand for 60 minutes at room temperature.
B. Isolation of Bacteriophage
The chloroform treated sewage water was introduced into a
5 mL 24 hour old culture of E. coli. The sewage-culture
filtrate was left to stand at room temperature for 5 minutes.
Then, two 3 mL soft NA tubes, which were maintained at 50
degrees Celsius, was inoculated with the sewage-culture
filtrate. The first tube was inoculated with 5 drops of filtrate
and the second tube was added with 15 drops of the filtrate.
The tubes were shook to mix the contents. They were then
poured separately into newly prepared NA plates.
The NA plates were allowed to solidify and were incubated
at 37oC for 24 hours.
After 24 hours, the NA plates were observed for plaque
formation.
C. Purification of Isolate
A clearing area was touched using an inoculating loop and
was inoculated into a 24 hour old broth culture of E. coli.
The broth culture was incubated at 37oC for 24 hours and
was then observed for clearing areas.
3. DISCUSSION OF RESULTS

2.2 METHODS

Experiment 08Date 24 March 2015

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BIO163-2L Microbiology and Immunology Laboratory

3rd Quarter SY 2014-2015

Unfortunately, the results cannot be seen because all of the

plates, broths, and NA were cleaned prior to the incubating
period. However, in order to remedy this, below is a picture
of plaque formation.

4. BBC Horizon (1997): The Virus that Cures

Documentary about the history of phage medicine in Russia
and the West
5. Keen, E. C. (2012). "Phage Therapy: Concept to Cure".
Frontiers in Microbiology 3. doi:10.3389/fmicb.2012.00238.
PMC 3400130. PMID 22833738
6. Singleton P (1999). Bacteria in Biology, Biotechnology
and Medicine (5th ed.). Wiley. pp. 444454. ISBN 0-47198880-4.
7. W.M.A. Mullan (2001). Isolation and purification of
bacteriophages.

4. CONCLUSIONS AND RECOMMENDATIONS

It is highly recommended that during delicate and very time
sensitive experiments like this, the custodians or laboratory
assistants should be informed so that they will not touch or
clean the NA plates and broth. Because of
miscommunication, there is no conclusion that can be
reached and no appropriate recommendation can be given.
5. REFERENCES
5.1 Journals
1. Mc Grath S and van Sinderen D (editors). (2007).
Bacteriophage: Genetics and Molecular Biology (1st ed.).
Caister Academic Press. ISBN 978-1-904455-14-1.
2. Wommack, K. E.; Colwell, R. R. (2000). "Virioplankton:
Viruses in Aquatic Ecosystems". Microbiology and
Molecular Biology Reviews 64 (1): 69114.
doi:10.1128/MMBR.64.1.69-114.2000. PMC 98987. PMID
10704475
3. Prescott, L. (1993). Microbiology, Wm. C. Brown
Publishers, ISBN 0-697-01372-3

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