The document summarizes research conducted by students Christopher Harness and Victoria Torres to discover and identify bacteriophages from a soil sample. Through a process of enrichment, purification, and isolation, they identified a phage they named "Phernando" that is similar to members of the Siphoviridae family. Electron microscopy analysis revealed its tail and head structure. The students gained experience in scientific research techniques like restriction enzyme analysis, gel electrophoresis, and calculating phage concentrations. Their work provided hands-on learning about identifying unknown bacteriophages through established lab procedures.

1. Harness 1
Christopher Harness
Dr. S. Conant and J. Thompson
ReBUILD SEA Phages
1 December 2015
Discovering and Identifying Bacteriophages
Medical practices over the past century have led to very beneficial discoveries on curing
diseases that run rampant throughout mankind and other living organisms. One of these modern
practices involves combating bacterial infections through the use of bacteriophage, or viruses
aimed to infect and replicate towards specific bacteria via their receptive genes. An example in
which phage-based therapy is effective against other treatments such as antibiotics is when the
infection is inaccessible through the use of these treatments. According to research performed by
Dr. Stephen T. Abedon and his team, situations where antibiotics perform poorly “include
osteomyelitis, diabetic infections of the feet, burns and infections of the central nervous system,
which can be protected from antibiotics due to the presence of the blood-brain barrier (but which
does not necessarily prevent adequate phage penetration to combat infections)”. In a research lab
performed by student of the University of Detroit Mercy, they were tasked in collecting a soil
sample of any location, in which they would purify, isolate, and identify the sample for possible
bacteriophage held from within. The purpose of this was to learn of research lab etiquette and
how to analyze unknown bacteriophage.
In the SEA Phages lab, the research team of students Christopher Harness and Victoria
Torres collected their soil sample on September 6th, 2015 at 10:36 AM in Christopher’s local
park, Moravian Park of Sterling Heights, Michigan. The approximate location, according to the
GPS reading, was 42.545778 degrees North and 82.975505 degrees West. The sample was

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located in a shaded, forest area when the air temperature was 25℃, while the sample itself was
dark, damp, and dug up from a 13.1763 cm depth below ground level. The sample was then
taken back and underwent an enrichment process in order for it to properly adsorb with the
bacteria, Mycobacterium smegmatis (M. smeg for short). This process had the soil sample mix
with materials such as H2O, 10X 7H9/glycerol broth, and even a small sample of M. smeg in
order to become a proper enrichment sample. From there, this sample underwent serial dilutions
via phage buffer (down to the 10-6 dilution factor) and put on a petri dish plate once it had
properly adsorbed with 0.5 mL of M. smeg and has been solidified by top agar. The results were
successful, each plate having plaque morphologies.
The next process was to create a spot plate choosing plaque morphologies on any
enrichment plate. Four plaques (labeled A – D) where chosen on the 10-1 Enrichment Plate,
having spots B and C contain the most plaques to sample off of for further tests. The chosen spot,
C, underwent a purification process known as “streak plating,” where the chosen sample would
be streaked across a petri dish in order to further isolate given plaques. This process was repeated
up to three times, each new streak was taken from an isolated plaque from the previous plate, but
an isolated plaque was chosen from the second streak plate for further testing due to the third
streak plate having its top agar not solidifying properly.
Having the plaque undergo titer assays furthered the process of isolating and purifying
the sample phage, but was proven to be the most difficult due to reoccurring issues. Although the
first titer assay resulted in consistent plaque morphologies, the second assay (and multiple
disregarded ones) all contained two types of plaques: lysogenic (A) and lytic (B). The
researchers decided to create proceeding titer assays on both morphologies which resulted in the
lysogenic “Plaque A” containing a hybrid lytic-lysogenic plaque population in its next titer

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(whereas B had the same two populations). Another titer assay was performed on this population,
having the 10-3 result in a “webplate” (a plate where it almost fully consists of plaques, but has
enough bacteria present) and become flooded by phage buffer to help create a Medium Titer
Lysate.
Medium Titer Lysate (MTL) contains a purified phage sample, but its concertation (in
plaque count over milliliter or pfu/mL) is determined via spot testing, this time recording the
actual plaque count contained within the range of 5 – 50 plaques within any dilution factor
placement (up to 10-10). The concentration was calculated by the equation:
# 𝑝𝑓𝑢
5 𝜇𝐿
×
1000 𝜇𝐿
1 𝑚𝐿
×
𝐷. 𝐹𝑎𝑐𝑡𝑜𝑟; in this case, the plaque count was 6 located in the 10-5 region, resulting in the
calculation 1.2 × 108
𝑝𝑓𝑢/𝑚𝐿. To create identical and factored webplates, the amount of MTL
needed (in mL) is calculated by the area of the plate over the area of the plaque, over the MTL
concentration. This calculation resulted in 7.0 × 10−3
𝜇𝐿 of MTL needed to create a 1X Factor
webplate. Out of the plates made, a 5X plate became the only successful web out of the group.
From there, four 5X plates were created for flooding purposes to create a High Titer Lysate
(HTL).
The process to determine the HTL concentration is similar to the MTL’s; however, 10-6 to
10-12 dilution plates are created instead of a single spot plate. Calculations for this concentration
are similar, but instead of
#𝑝𝑓𝑢
5𝜇𝐿
being in the equation, it is:
#𝑝𝑓𝑢
50𝜇𝐿
. For this calculation, 17 spots
were found on the 10-6 plate, resulting in 3.4 × 108 𝑝𝑓𝑢
𝑚𝐿
being the concentration for this HTL.
From there, the HTL sample undergoes both DNA isolation and purification, and Transmission
Electron Microscopy (TEM Imaging).

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Under the DNA purification process, the DNA concentration totaled out to be 51.5
𝑛𝑔
𝜇𝐿
.
From there, 9.7𝜇𝐿 of this DNA concertation was exposed to the restriction enzymes: BamHI,
ClaI, EcoRI, and HaeIII (HindIII being left out), to help identify a possible DNA sequence based
on how many fragments were “cut” by the enzymes. These measurements were determined by
the placements of the enzyme cuts under the process of gel electrophoresis. The gels would
display the enzymes distance from the starting “wells” after a period of time has elapsed. This
resulted in the DNA exposed to the HaeIII restriction enzyme having the highest distance from
the well; although, the DNA in this experiment may have been denatured in the purification
process.
As the HTL sample underwent TEM Imaging, curtesy of Wayne State University,
clusters of bacteriophage were identified, all of which shared similar characteristics to the
Siphoviridae type of bacteriophage (only consisting of a present “tail” and head). Upon
completion of this experiment, the researchers nicknamed their phage “Phernando” for the sake
of alliteration (and pun) to the word “phage” and to give it a human-like characteristic after
months of hard work to identify it. This work experience in the lab has allowed them to become
more familiar with the rigorous tasks present in any scientific research lab, and how to
collaborate as a team to properly complete the task at hand. The knowledge obtained could one
day be used in their careers in the medical field whenever they are tasked to cure a possible
illness or to even diagnose/treat a patient who is in great illness from a disease.

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Works Cited:
Abedon, Stephen T et al. “Phage Treatment of Human Infections.” Bacteriophage 1.2 (2011):
66–85. PMC. Web. 30 Nov. 2015.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/>.
Conant, Stephanie, and Jack Thompson. “SEA Phages.” University of Detroit Mercy. 2015.
PowerPoint. 29 November 2015.