1. Isolation of Bacteriophages in Tropical Soils of Puerto Rico using Mycobaterium smegmatis and
Bacillus cereus as host bacteria, a bacilophage was isolated and it was named Firgaro
Anthony Maldonado Castro, Hernán G. Méndez Colón
RISE Program
Department Biology University of Puerto Rico-Cayey.
Bacteriophages are viruses that infect bacteria, and can be found in various environments,
including tropical soils. The question was if bacteriophages could be isolated from tropical
soils of Puerto Rico; for which we hypothesized that indeed they can be isolated. Therefore
the purpose of this experiment is to effectively isolate a novel bacteriophage from the
tropical soils of Puerto Rico, in order for it to be characterized and annotated its genome.
By isolating and characterizing phage that infect bacteria such as Bacillus cereus and
Mycobacterium smegmatis information is acquired about other related bacteria like
Mycobacterium tuberculosis that are pathogenic. For the methodology, the first part was the
“phage hunt” in which various soil samples were collected from the environment and taken
to the lab for analysis and see if a phage could be isolated from the sample. Once taken to
the lab the samples were enriched and incubated with the two host bacteria and nutrient
medium to allow the phages, if any, to replicate. The supernatant from the enrichment was
used to do streak protocols using agar plates; if a phage was isolated from the sample, clear
spots would develop on the plates. These clear spots, called plaques, are areas of phage
growth, then the phage was purified three times to ensure that it is truly a bacteriophage
and that it is only one type. After various samples a bacilophage, which was called Figaro,
was isolated. This supports our hypothesis that bacteriophages can be isolated from
tropical soils of Puerto Rico. Future work for this experiment includes characterization
and annotation of the novel phage that was effectively isolated.
Bacteriophages are viruses that infect
bacteria in order to use the bacterial
replication mechanism for its own
reproduction. They can be found in
practically every place in the environment,
representing the majority of all biological
entities in the biosphere (Suttle 2005). The
typical structure of phages consists of a
capsid and the tail. The capsid stores the
genetic code, and the tail assists in the
injection of the genetic material into the
bacteria.
Bacteriophages have two different life
cycles namely, the lytic and the lysogenic
cycles. A lytic phage infects the bacteria,
replicates and immediately kills it through
cell lysis. Within 2 minutes after the phage
DNA is injected into the host bacterium,
directs the synthesis of proteins that shut off
the transcription, translation, and replication
of bacterial genes, allowing the virus to take
control of the metabolic machinery of the
host (Snustad 2012). Lysogenic phages
infect the bacteria and integrate into the
bacterial chromosome, where they continue
to divide as the bacteria replicates. If
conditions are favorable, the phage enters
the lytic phase and proceeds to behave like a
lytic bacteriophage (Snustad 2012).
Bacteriophages have specific relationships
with their host bacteria, meaning that they
are dependent on that bacterium for
replication. Mycobateriophages are specific
phages that infect bacteria from the genus

2. Mycobacterium spp., and Bacilophages are
bacteriophages that infect bacteria from the
genus Bacillus spp. This specific
relationship can be useful to learn about the
characteristics of both, the host bacteria and
the phage that infects it. There are various
uses for bacteriophages, which include
therapeutics applications like vaccines and
drug delivery and control of multi drug
resistant bacteria (Pendleton 2013).
The study of bacteriophages and their host
bacteria provides information about related
bacteria. For example, while using
Mycobacterium smegmatis, which is
relatively harmless, other related bacteria
like Mycobacterium tuberculosis or
Mycobacterium leprae are indirectly being
studied (Rubin & Vázquez 2012). The same
mechanism works for the bacteria Bacillus
cereus. Bacteria are abundant in all
ecosystems, and where there are bacteria
there should be bacteriophages, due to their
dependence on their host for replication.
Therefore, we hypothesize that
bacteriophages should be present in tropical
soils of Puerto Rico and can be effectively
isolated. The purpose of this research is to
first isolate and then characterize a newly
discovered bacteriophage with the goal of
contributing to the bacteriophage gene bank.
Materials and Methods
A soil sample was collected using a plastic
spoon wrapped in plastic and a plastic bag to
contain the sample. The sample was
collected at a superficial depth. The
following data about the sample was
recorded and saved: date of collection, time,
depth, moisture content, temperature site
description and GPS location. The sample
was then taken to the laboratory for its
enrichment. All of the following procedures
where done according to the aseptic
techniques described in the SEA-Phages
Resource Guide. For the sample enrichment
two ~0.5g amounts of soil were measured
using a weighing scale. Both amounts were
transferred to two 15mL centrifuge tubes.
Next to one of the tubes 10mL of TSB
Bacillus and 1mL of Bacillus cereus culture
where added using a pipette and a
micropipette. Similarly, to the other tube
10mL Master Mix smegmatis with 1mL of
Mycobacterium smegmatis culture was
added. The two enrichments were incubated
in a shaker at 37°C for at least 24 hours. The
two enrichments were then centrifuged for
15 minutes at 3000 RPM. The supernatants
were filtered using a 5mL syringe and a
.2μm filter, and 5mL was then added to the
syringe. This mix was then filtrated into
another plastic 15mL tube. The filtrate was
then used to strike two agar petri dish. The
two petri dishes were labeled with date,
name of bacteria (Bacillus and Smeg),
initials and numbers from 1-3. For streak
protocol a wooden sterile swab was dipped
into one of each enrichment filtrates; and
was then used to streak the plate starting
from number “1”, then, using a new swab,
continuing to number “2”, and finally to
number “3”. Next 4.5mL of Triptic Soy
Agar with .5mL of B. cereus were added to
plate #1 and 10mL of Top Agar with .5ml of
M. smegmatis culture were added to plate
#2. The plates were incubated at 37°C for 24
hours. After the incubation period, the plates
were examined for plaque formation
indicating bacterial death due to phage
infection and the presence of bacteriophage
growth. This same procedure was followed
in order analyze all the 9 soil samples
collected.
Once a bacilophage was detected in soil
sample number 5, three phage purifications

3. were done for that plate. For this a single
plaque was selected from the plate using a
micropipette, and was added to a microtube
containing 50μl of phage buffer. A petri dish
labelled as “PP1” was streaked following the
same procedure described above to do the
filtrate streaking, but this time the sterile
swab was dipped into the plaque and buffer
solution. The plate was then incubated at
37°C for 24 hours. After the incubation
period, if there was a sign of phage growth,
a second and then a third purification was
done, following the same procedure as for
“PP1”. After the third purification, a second
enrichment was made, adding a plaque from
“PP3” to 10mL of TSB Bacilllus and .5mL
of Bacillus cereus culture, and incubating it
at 37°C for ~24 hours. After the incubation
period, the filtrate was centrifuged for 10
minutes at 3000 RPM. Again, the
supernatant was filtered as described before.
From the filtrate, 10μl were extracted and
added to a microtube containing 90-μmL.
Dilutions were made by extracting 10μL
from the first tube, adding them to the
second tube, vortexing and centrifuging.
Then 10μL from the second tube were added
to the third one, until eight dilutions were
made.
The dilutions were then used to do a spot
test, using a labeled plate in which nine
squared numbered zones were drawn. To the
plate 4.5ml of Top Agar with 0.5mL of
Bacillus cereus were added, without any
streaking. After the agar solidified, 10μL of
each dilution were placed in its
corresponding zone: dilution 1 in zone “1”,
dilution 2 in zone “2”, until reaching zone 8.
The plates were incubated at 37°C. for ~24
hours. After the incubation period, the
plates were examined to look for the best
dilution to use for further characterization.
Using the filtrate from the second
enrichment, an analysis of bacteriophage
capsids proteins was done. First, 1mL of
bacteriophage High titer
Phage Lysate (HTPL) (filtrate) was
transferred to a microtube and centrifuged at
10, 000 Xg for 1 hour at 4ºC. Next, 950µL
of the supernatant was extracted leaving the
pellet in the tube. Into the pellet, 50µL of
LSB and BME were added, and the solution
was boiled for 2 minutes, cooled for 2
minutes, and centrifuged. The samples in the
tube were stained using Coomassie Blue G-
250. A photograph was taken using a white
light box.

4. Results
Sample Coordinates Description Location Date
AMC -
#1a
18.2° 12’ 40’’
N;
66.1° W
Rural, next to houses and close to farm yard with
chickens, surrounded by trees and other plants;
usually a site of organic material composting;
slightly moist.
Temperature: 26°C
Depth: ~2cm
Time: 17:43
Bo. Tomás de Castro, carr.
788, km. 2.9, Caguas,
Puero Rico, 00725
February 2,
2014
AMC -
#2a
18.2°12’ 67’’ N;
66.0° 0’ 41.46’’
W
Low moisture, Rural, fairly away from houses,
used to be a farmyard, area within a tropical
forest.
Temperature: 26°C
Depth: ~1cm
Time: 13:23
Bo. Tomás de Castro, carr.
788, km. 2.9, Caguas,
Puero Rico, 00725
February
17, 2014
AMC -
#3a
18.2° 12’ 9’’ N;
66° 1’ 11.9’’ W
Moderate moisture, Rural, close houses, next to
an asphalt road, surrounded by vegetation.
Temperature: 26°C
Depth: ~3cm
Time: 18:20
Bo. Tomás de Castro, carr.
788, km. 2.9, Caguas,
Puero Rico, 00725
February
23, 2014
AMC -
#4a
18.2° 12’ 1.35’’
N; 66° 2’ 13.7’’
W
Wet, bank of a water stream, houses close by, and
also a road, surrounded by vegetation.
Temperature: 26°C
Depth: ~3cm
Time: 12:03
Bo. Tomás de Castro, carr.
788, km. 2.9, Caguas,
Puero Rico, 00725
March 2,
2014
AMC -
#5a
18.2° 12’ 58’’
N; 66° 1’ 9.8’’
W
Very moist, rural, close to houses, next to an
asphalt road, an area of garbage disposal.
Temperature: 26°C
Depth: ~3 cm
Time: 17:06
Bo. Tomás de Castro, carr.
788, km. 2.9, Caguas,
Puero Rico, 00725
March 9,
2014
HMC –
1b
18° 7' 2.553" N;
66° 6' 58.72" W
Moist, granulated soil, dark in color, under a
mango tree
Temperature: 24°C
Depth: ~1cm
Time: 8:50
205 Avenida Antonio R.
Barceló
Cayey, PR 00736
February 4,
2014
HMC –
2b
18° 7' 7.172" N;
66° 6' 58.33" W
Dry, brown color, near a sidewalk and fountain.
Temperature: 26°C
Depth: ~2cm
Time: 15:49
205 Avenida Antonio R.
Barceló
Cayey, PR 00736
February
18, 2014
HMC –
3b
18° 7' 6.686" N;
66° 9' 46.61" W
Moist, dark color, under moss, covered by
vegetation and next to a building
Temperature:26°C
Depth: ~1cm
Time: 12:14
205 Avenida Antonio R.
Barceló
Cayey, PR 00736
February
24, 2014
HMC –
4b
18° 7’ 8” N;
66° 9’ 45” W
Moist, dark brown soil.
Temperature: 27°C
Depth: ~3cm
Time: 16:51
205 Avenida Antonio R.
Barceló
Cayey, PR 00736
March 6,
2014
Table 1. Soil samples collected in Cayey and Caguas, Puerto Rico. The data was recorded for each soil sample
collected. The only sample in which a bacteriophage was found and isolated was in sample 5a.

5. The samples were divided in two groups,
four of them were collected in Cayey, and
five of them were collected in Caguas, and
labeled as “a” and “b”, as shown in Table 1.
A total of nine soil samples were collected.
The only sample in which a bacteriophage
was found and isolated was sample number
5a. This was a plate containing Bacillus
cereus, so it is a bacillophage.
The results from the protein polyacrilamide
electrophoresis can be observed in figure 2
and figure 3.
Figure 3. Gel 2 form the protein electrophoresis. After
running for 30 minutes the samples travelled all the way
through the gel. The band patter for Figaro can be observed
in row eight.
Discussion
Figure 1. Third purification plate showing clear spots:
plaque formation due to phage growth.
All three phage purifications were done with
positive results. After the third purification,
shown in figure 1, three spot test were done,
but with negative results in the three of them
due to no phage growth. After this, a fourth
purification was made, but using the filtrate
instead of a plaque from “PP3” and
incubated at 37°C for ~24 hours. The plate
showed no plaque formation; there was no
phage growth.
Figure 2. Gel 1 from the protein electrophoresis. After running for
30 minutes the samples only travelled halfway through the gel. The
band patter from Figaro can be observed in row number eight.
The soil samples from Cayey did not
showed phage growt; no bacteriophages
could be isolated from those samples. The
first soil samples collected from Caguas, as
the ones from Cayey, did not showed sign of
phage growth after the analysis was
performed. At the time of the first two
sample collections, problems with the
bacterial culture at the labs arose, probably
affecting our results. In this samples there
could have been bacteriophages, but since
the bacterial cultures were defective, there
was no phage growth. From the third sample
onwards, however, the bacterial cultures
were working properly, but there was no
phage growth until sample #5. Possible
reasons for this lack of phage formation
could be contamination, experimental errors
during the procedure, or simply that the
samples did not contain phages. One thing
that has to be mentioned is that the samples
were always collected a day before the
analysis. The soil had to remain in a sealed
plastic bag for approximately 12-24 hours,
during which organisms living in the

6. samples could be affected by the isolated
environment. All of these could be causes
for the negative results obtained in the first
samples. Sample #5 was collected from
under a trash can, were the soil was in
contact with decomposing organic material,
and bacilophage Figaro was isolated. This
unlike the previous soil samples, which were
isolated from natural, pristine sites yet no
phages were isolated from those. With this
we can conclude that bacilophages can be
more easily isolated from sites containing
decomposing, or even rotting, organic
material. It appears to be that that is the type
of environment in which the host bacteria
will develop and, since bacteriophages
depend on their host for replication, it is
more likely for them to also thrive in such
environments. We can also conclude that
indeed novel bacteriophages can be isolated
from tropical soils of Puerto Rico.
The isolated bacteriophage is a bacilophage,
since it grew in the Bacillus cereus plate.
We decided to name it Figaro in honor of
the famous character of the same name from
the opera “Il Barbiere di Siviglia”, by
Gioachino Rossini. In figure 1 the third
purification done after the phage isolation
can be observed. The white round spots are
the plaques formed due to cell lysis, which
means that the phage has effectively
developed.
Now that we finally isolated a bacilophage,
future work includes characterization and
annotation of the bacteriophage genome.
Acknowledgements
Special thanks to:
RISE Program
Howard Hughes Medical Institute
PHAGES Program
Dr. Michael Rubin
Giovanni Cruz
Gustavo Martínez
Nicolle Rosa
Cristopher Qintanal
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