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1. Isolating bacteria from tropical contaminated soils in Puerto Rico
Cotto Reyes J, Ruiz Videla CL
Abstract
Bacteria are very popular in mixed populations throughout the environment. However, their
analysis and characterization can only be studied within isolated strains and pure cultures. This is
essential to identifying the properties in which bacteria can produce or resist antibiotics. In order
to study this, two samples were collected, from different soils in Puerto Rico, isolated, and
purified to obtain bacteria colonies. The mentioned bacteria were tested to verify if they
produced antibiotics. Results were negative. Additionally, they were tested to verify their
resistance against various antibiotics. CLRV bacteria showed resistance to four antibiotics;
however, JCR bacteria did not display any resistance against the antibiotics. Such results indicate
that the isolation of bacteria within the first sample (CLVR) effectively resisted against
antibiotics more than the second sample (JCR). It may be due to the diameter it reflected when
the treatments were placed in the mediums. However, their bacterial morphology differs such as
their origin in terms of the location they were administered from. Tell us what the morphology
and origin is. Present those results as well as conclusions in the abstract.
Introduction
Antonie van Leeuwenhoek, the Dutch
microscopist, was the first to observe
bacteria back in 1676 with a single-lens
microscope of his own design. Years later,
in 1859, Louis Pasteur demonstrated the
fermentation process caused by the growth
of microorganism, and he, along with Robert
Koch, invented the germ theory of disease.
Even though the fact that bacteria are the
cause of many diseases was already
common knowledge, it was not until 1910
that the first antibiotic was developed by
Paul Ehrlich. After that, another major
discovery came in 1977 when Carl Woese
recognized that archaea have a separate line
of evolutionary descent from bacteria,
depending on the sequencing of 16s
ribosomal RNA. This also divided
prokaryotes into two evolutionary domains.
Bacteria represent a large realm of
prokaryotic microorganisms which have the
potential to be both beneficial and harmful.
Even though they are often the causes of
human and animal diseases, certain bacteria
produce antibiotics, and others live
symbiotically in different parts of the bodies
of humans and animals, or on the roots of
certain plants. These microorganisms help
break down dead organic matter, and they
arrange the base of the food web in many
environments. Bacteria are extremely
flexible, and they have the capacity for rapid
growth and reproduction. These qualities
attribute to their immense importance in our
world.
The isolation and characterization of
bacteria are common techniques used to
separate and identify the properties in which
a bacteria can be useful to produce effective
antibiotics, cellulose synthesis, and oil
degradation (Unknown author 2015).
Bacteria can be found widely in mixed
populations with other types of
microorganisms in the environment,
denominated as mixed cultures when
collected. However, the analysis and
characterization of microorganisms can be
studied within isolated strains and pure
cultures only. For instance, there are various
methods to separate individual
microorganisms from others. The most
common one is to grow a culture using a

2. spline as an instrument to apply the selected
bacteria into a petri dish. This process is
repeated constantly to observe the success of
differentiation between colonies. Their
properties are also analyzed to distinguish
each colony by size, color, etc.
This procedure has been utilized in other
experiments to determine the specificity of a
bacterium’s resistance to the environment,
according to its characteristics in terms of
growth and reproduction in a certain area.
For example, the effect of ultraviolet
radiation on airborne bacteria implicates the
urgency of utilizing the method of isolation,
identification, measurement and sampling to
discover tolerant bacteria against this
condition (Kobayashi et al. 2015). Also, the
characterization of the bacterial consortium
associated to Euplotes focardii, a
psychrophilic marine ciliate isolated from
Terra Nova Bay, in Antartica at 4° Celsius,
was reported by Sandra Pucciarelli and her
team of investigators in 2015. They propose
to study how its genes can be used to encode
for enzymes involved in the catabolism of
complex substances for energy sources.
Moreover, it is evident that the experiment
of isolation and characterization of bacteria
is performed worldwide by scientists
because it is a gateway for the discovery of
knowledge.
Nowadays, bacteria are creating resistance
against antibiotics, and the necessity to find
more effective treatments is increasing.
Therefore, this investigation focuses on the
isolation and characterization of bacteria
from various soils of Puerto Rico, in order to
discover other antibiotics that bacteria
cannot resist. As hypothesis, the bacteria
collected and analyzed in this experiment
will result in positive outcomes regarding
the antibiotic production, and resistance tests
since they are collected from contaminated
areas.
Materials and Methods
Soil collection and cultivation
Each tropical soil sample was collected
using a sterile spoon and an unopened zip
lock plastic bag. Approximately half of the
bag was taken back to the laboratory. One
gram of the soil sample was measured and
diluted with deionized water. The
supernatant of the soil solution was
transferred to the first micro tube. Then, a
small aliquot was transferred from 100 to 10-
1, so on and so forth until an aliquot from 10-
4 was transferred to the micro tube 10-5
creating a series of dilutions. After that, the
dilutions 100 and 10-5 were each transferred
and spread into a rhizobium defined medium
(RDM) and tryptic soy agar (TSA) plates for
cultivation. The plates were placed in a
30°C incubator and left there for 24 hours,
so that the bacteria could grow.
Isolation and Purification of soil
microorganism
The isolation procedure was started with the
grown bacteria from the dilutions 100
because the bacteria from the 10-5 dilutions
did not grow. Utilizing an inoculator loop, a
small colony of choice was transferred into a
new medium plate, depending on which
plate it originally grew on. Those plates
where incubated at 30°C for 24 hours. These
purifications were done four times with each
bacterium.
Gram stain and bacterial morphology
Once the bacteria were purified, a gram stain
technique was done on each to determine
their bacterial morphology. The gram stain
technique consists of adding deionized water
to a slide mixed with a colony of the
bacteria. Then a few drops of crystal violet
were applied to the smear and left there for a
minute. After cleansing with water and
drying with a hot plate or fire, the same step
was repeated with iodine, alcohol, and
safranin. The slide with the final results was

3. observed under a microscope and the
bacteria were categorized into Gram stain
positive or Gram stain negative. Their
morphology was determined as well.
Storage sample -80°C
A broth was made for each bacterium by
transferring 4mL of the appropriate media
and a colony of the bacteria into a tube, and
incubating it at 30°C for 24 hours. This
broth was then utilized to prepare the
cryogenization of the bacteria. For the
cryogenization, two cryogenic tubes were
labeled as “storage stock” and “working
stock”. Then 1mL of glycerol was added to
the broth, and 1.5mL of that mixture was
added to each cryogenic tube. Those tubes
were frozen at -80°C for 24 hours. The next
day an inoculation of the stocks was
cultivated in a media plate to verify the
viability of the stocks. When the viability of
the stock was tested and they worked, the
stocks were frozen for further use.
Genomic DNA isolation
Primarily, the bacteria need to be
introduced in a boil-proof micro centrifuge
tube with an amount of 200 micro liters of
sterile water containing 200 micro liters of
the liquid culture to start the process of the
polymerase chain reaction (PCR). The
sample is then placed in three stations where
it undergoes _________10 minutes. (The
first one consists of a bucket of ice where
the micro centrifuge tube lays around the
cold cubes for the time required This step
lacks clarity.). T he process of boiling the
tube continues, and finally, it is centrifuged.
Subsequently, 150 microliters from the
supernatant of the culture are transferred to
another tube where it is kept on ice before
running the PCR. The components that are
mixed inside the tube consist of an upward
and downward primer, H20, and a master
mix. The PCR is followed by an
electrophoresis procedure, which is carried
out with the sample of the culture that
resulted from the PCR.
Purification of PCR products
This process remarks? the use of 5
volumes of Buffer PB per 1 volume of the
PCR reaction, which are placed together in a
sterile micro tube for their mixture. This mix
is transferred to a QIA column and
centrifuged for 1 minute at 13,000rpm. Once
this process is finished, the residue is
discarded (flow-through) and the QIA
column is placed again in the same tube.
Furthermore, 750 microliters of Buffer PE
are added to the QIA column and
centrifuged for 1 minute, the flow-through is
discarded, and the QIA column placed in the
same tube. The QIA column is once more
centrifuged in a collection tube of 2
milliliters and the residual wash buffer
discarded. The QIA columns are mixed in
one clean micro centrifuge tube of 1.5mL.
To measure the DNA, 50 microliters of
water are added to the center of the QIA
membrane. The next step consists in
centrifuging the column for 1 minute to
store the purified PCR product in the freezer
(-20 degrees Celsius) or continue to the
electrophoresis protocol.
Antibiotic production and resistant tests
For the antibiotic production test, the
bacteria E. coli and M. luteus were spread in
two separate media. Afterwards, a circular
sterile paper was introduced into a broth of
each of the isolated bacteria. These papers
were inserted in the media plates and were
incubated at 30°C for 24 hours. If the
isolated bacteria produced antibiotic, a circle
without E.coli or M. luteus was supposed to
appear along the papers. The antibiotic
resistance test consisted of inoculating the
isolated bacteria in a media plate and

4. inserting them into different antibiotic disks.
Some of the antibiotic disks that were used
were: Kanamycin, Penicillin, Tetracycline,
Gentamicin, Chloramphenicol, Bacitracin,
Vancomycin, and Streptomycin. After
incubating the media plates for 24 hours, the
results were analyzed. If a circle appeared
along the antibiotic, it meant that the
bacterium was not resistant, but if a circle
did not appear, it meant that the bacterium
was resistant to the subsequent antibiotic.
Results Tables need to be integrated into
the results and mentioned in the text with
references such as See Table ____ or See
Figure ____. There are no figures of the
plates.
After collecting and cultivating the
samples on TSA and RDM media, two
different bacteria grew from the Ruiz C #1
sample, and one from the Cotto J #1 sample.
Based on the results, the isolated bacteria
demonstrated positive feedback in regards
to the gram stain procedure within two of
the three samples collected.
CLRV30TSA01A represented a bacterial
morphology of a coccus and the other
samples were observed as bacillus.
Transitionally, in the PCR and
electrophoresis conduction the bacteria
showed a negative effectiveness towards the
characterization of these microorganisms.
However, the coccus sample was
implemented over these procedures once
again (This sentence is not clear.). It
produced a positive outcome which could be
due to an error made in the initial PCR
experimentation.
None of the bacteria produced antibiotics.
However, the bacteria did show resistance to
various antibiotics. The smaller the diameter
the more resistance the bacteria would be to
a certain antibiotic. According to the
observed diameters, CLRV30TSA01A and
CLRV30M01B were resistant to Penicillin,
Chloramphenicol, Bacitracin, and
Vancomycin. On the other hand,
JCR30TSA01A did not resist any of the
antibiotics that it was exposed to.
.
Table #1: Sampling locations and their descriptions
Sample Coordinates Environmental description Location Date/Time
Ruiz C
- #1
18°6’55.1”N;
66°9’36.2”W
Moist (underneath grass), 5 cm;
72°F
Cayey,
PR
January/28/15
7:30 p.m.
Cotto J
- #2
18.15º N
66.18º W
Moist environment (above
grass), 76º
Cidra, PR January/29/15
8:00 a.m.

5. Table #2: Results from different procedures
Table #3: Antibiotic Resistance Test Results:
Discussion This discussion for me is very
confusing. It seems to me that you are
reviewing literature when you should be
discussing the results of your study. I see
this whole section as a review of literature
and not a discussion of findings.
(The objective of this investigation
was to characterize and identify isolated
bacteria. Characterization included
determining antibiotic efficiency and
bacterial morphology. Genomic sequencing
.was left for future work. This sentence
contains too much information and does not
have a clear message. I tried to revise it for
clarity). For instance, in a study of blood cell
that were incubated as “bacteria free”
cultures were analyzed using the
BACTEC™ FX system. Gram staining
helped determine whether or not bacteria
were actually growing (Peretz et. al. 2015).
They discovered that 15 cultures defined as
negative by the BACTEC FX system at the
end of the incubation were found to contain
microorganisms when Gram-stained. This
finding raises a problematic issue
concerning the need to
perform Gram staining of all blood cultures,
which could overload the routine laboratory
work, especially laboratories serving large
medical centers and receiving a great
number of blood cultures. In addition, the
development of new instrumentation for
PCR procedures can be a key to identifying
and quantifying the target pathogen in
infected samples that do not require reliable
Process CLRV30TSA01A CLRV30M01B JCR30TSA01A
Gram Stain Negative Positive Positive
Bacterial Morphology Coccus Bacillus Bacillus
PCR/Electrophoresis #1 Negative Negative Negative
PCR/Electrophoresis #2 Positive Negative -
Antibiotic Production Negative Negative Negative
Antibiotic CLRV30TSA01A CLRV30M01B JCR30TSA01A
Kanamycin 2.0 cm 1.0 cm 2.0 cm
Penicillin 0.0 cm 0.0 cm -
Tetracycline 1.6 cm 2.4 cm 2.2 cm
Gentamicin 1.9 cm 0.9 cm -
Chloramphenicol 0.0 cm 0.0 cm 2.5 cm
Bacitracin 0.0 cm 0.0 cm 1.5 cm
Vancomycin 0.0 cm 0.0 cm -
Streptomycin 1.6 cm 1.0 cm 1.9 cm

6. methods, like DNA extraction . In a related
study, this fact was proven to be effective,
using a SYBR Green-based real-
time PCR assay for the rapid, specific, and
sensitive detection of Acidovorax avenae
subsp. Citrulli is a serious disease of
cucurbit plants (Cho et al. 2015). This
assay targets specifically the YD-repeat
protein gene of A. avenae subsp. Citrulli is
seen as a single band in the product of this
experiment. In synthesis, the resistance of
antibiotics was an important issue to cover
because of the diversity in microorganisms
this technique can be useful on. As a matter
of fact, the staphylococcus aureus was
grown in a medium where antibiotics like
Tetracycline, Penicillin, co-trimoxazole,
erythromycin and vancomycin were present
(Shahmohammadi R et. al. 2015). The most
effective treatments were vancomycin,
having all of its isolates as susceptible
organisms and subsequently, penicillin with
high antibiotic resistance. In future works,
the cellulose activity and oil degradation can
be a situation to study under the analysis of
the properties that the bacteria in this
experiment carry.
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