Bacteria Characterization

1. BIOL 3955: Research Seminar
2015
1
Characterization of Bacteria Isolated from Tropical Soils of Puerto Rico
Ann M. González1
, Ramón B. Colón2
1University of Puerto Rico, Cayey, Puerto Rico, Department of Chemistry
2University of Puerto Rico, Cayey, Puerto Rico, Department of Natural Sciences
ABSTRACT
This research was designed to characterize isolated bacteria from soils of Puerto Rico. The
objectives were to collect a soil sample, isolate colonies of bacteria, make some tests, and characterize
the bacteria with the use of microbiological and molecular biology techniques. Techniques such as Gram
stain, PCR and Electrophoresis were used to achieve the purpose of the investigation. As a hypothesis,
positive results in the functional capacities, mainly antibiotic production, were expected. A complete
characterization and isolation were the main objectives of the experiment. Both bacteria (S15UPRC-
RISEAMGP30SP01A1
and S15UPRC-RISERBCR30P01A2
) resulted negative in antibiotic production.
In conclusion, the hypothesis was rejected and the main purpose was not completed. Full
characterization and DNA sequence is planned for future work.
INTRODUCTION
The world has around 7.3 billion habitants.
Every day hundreds or maybe thousands die. We
commonly believe that the majority of the deaths are
caused by illness produced by pathogens, specifically
bacteria. The reality is that bacteria are more than just the
cause of a disease. They are the raw material in the
production of antibiotics; others live symbiotically inside
humans or on the roots of certain plants for the
conversion nitrogen into a usable form. Bacteria put the
flavor in yogurt and the sour in sourdough bread; bacteria
make up the base of the food web in many environments.
Bacteria are of such importance because of their extreme
flexibility, capacity for rapid growth and reproduction.
Each bacterium is different; morphology and
functional capacities as antibiotic production, cellulase
activity, and resistance to antibiotics, and oil, could
characterize them. There are three possible morphologies
that bacteria could have: bacillus (little rod), coccus
(grain or berry) or spirillum (coiled or helical). Also, the
composition of the cell wall is used to distinguish them.
Gram positive bacteria are characterized by a thick layer
of peptidoglycan, while Gram negative have a thin layer
of peptidoglycan and lipids. Membrane and cell wall
characteristics help us to understand the reason why
some bacteria are susceptible to some antibiotics and
others aren’t. Usually, gram negative bacteria are
resistant to antibiotic while gram positive are more
susceptible.
It’s important to comprehend the four possible
capacities that a bacteria could have. Antibiotic
production and resistance are the most important. We can
know if a bacterium is resistant to antibiotics when it has
a mutation. Mutation is a permanent change in the
nucleotide sequence of the genome of organisms. It
results when the DNA has a damage that can’t be
repaired. Researchers right now are looking for bacteria
that could be used as treatments for some diseases.
Another capacity is the cellulase activity. Cellulases are
used in the textile industry for cotton softening and
denim finishing; in laundry detergents for color care,
cleaning; in the food industry for mashing; in the pulp
and paper industries for drainage improvement and fiber

2. BIOL 3955: Research Seminar
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modification, and they are even used for pharmaceutical
applications (Sethi et al. 2012). Bacteria with this
capacity usually live in aquatic environments. Last but
not least, bacteria can degrade oil. Commonly, they are
used to biodegrade oil pills. This can be seen in the oil
spill accident of June 2010 when bacteria ate up the
contamination leaked in the Gulf of Mexico (Kemsley
2013).
The scientific study of bacteria is really
important; new bacteria would help us to keep
developing treatments for diseases or innovating uses for
the microorganisms. For example, lactic acid bacteria are
a good source of novel bacteriocinogenic that could be
exploited as an alternative for use as biopreservatives in
foods (Martins and Augusto 2014). There are hundreds
of examples; simply, bacteria are worth to research.
The purpose of this research is to study bacteria
collected from the soils of Puerto Rico and investigate
them. The objectives are to isolate, purify, characterize,
and identify the functional capacities of the collected
bacteria. We would identify the bacteria using
bioinformatics. We expect an affirmative response in the
antibiotic production capacity from the bacteria in study.
MATERIALS AND METHODS
Aseptic techniques were used throughout the whole
experiment.
Sample Collection
The main objective in this research is to isolate
and characterize bacteria collected from tropical soils of
Puerto Rico. To collect the sample two places where
chosen from Bo. Toita in Cayey and Bo. Asomante in
Aibonito. Aseptic technique was used to handle the
samples with the purpose of avoiding the contamination
of the sample. The soil was collected with a plastic spoon
and stored in a Ziploc bag. A thermometer was used to
measure the air temperature. The location and physical
descriptions were recorded.
Cultivation of Soil Microorganisms and pH
Determination
Once the soil samples were collected, the
cultivation of the microorganisms was the next step.
First, 1.0g was weighed and mixed in the vortex with 5-
10mL of sodium chloride (NaCl). The pH of the solution
was determined after it was homogenized using pH
strips. Also after the agitation, the soil settled to the
bottom of the conical tube and 1mL of the solution were
transferred to centrifuge tubes to eventually make
dilutions of the bacteria with different concentrations.
The dilutions were from [0] to [-5]. To cultivate in agar
plates only [0] and [-5] dilutions were used. R2A and
ISP4 mediums were assigned to cultivate the bacteria.
From each sample two plates were used. The samples
were streaked, using the “L” method and placed in the
incubator at 30°C for 24 hours until the bacterial colonies
grew.
Purifications
Twenty-four hours after the cultivation, bacterial
colonies grew. To separate the colonies, another ISP4 or
R2A plate was used (depending in which medium the
bacteria grew). With an inoculating loop, the selected
bacterium was streaked onto the new plate. Finally, the
bacterium incubated at 30°C for 24 hours for new
bacterial proliferation. This process was made in
triplicate to ensure complete isolation.
After the isolation, cryogenics, that is freezing
the samples, was performed to allow the retention of the
bacteria for future experiments. In this procedure broth
was added to provide food to the isolated bacteria, and
glycerol to protect cell membranes.
Gram Stain
Usually, the first step in the identification of a
bacterial organism is the Gram stain. This method helps
in the differentiation of bacteria into two large groups
namely, gram-positive and gram-negative. The first step
in this method is to place the bacterium on a glass slide
and add a drop of deionized water. With heat from a
Bunsen burner the bacterium is adhered to the glass and
is ready to be differentiated. The dyes crystal violet and
safranin are used. First, one drop of the crystal violet dye
is applied. After one minute, the glass slide is washed
with deionized water and a drop of iodide is added. The
iodide binds to the crystal violet and traps it in the cell.
One minute later, the slide is rinsed with water and one
drop of alcohol is added. The alcohol should be removed
almost immediately. The alcohol decolorizes the cells by

3. BIOL 3955: Research Seminar
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forming a thin peptidoglycan layer, while the dye
remains in the cells by forming a thick peptidoglycan
layer (gram-positive). Finally, one drop of safranin is
added and immediately rinsed for one minute. The
second dye gives a pink color to the decolorized cells
(gram-negative). When the Gram stain is completed, the
glass slide is studied under the microscope to determine
cell morphology.
Purification and amplification of Genomic DNA
The DNA of each bacterium is needed for future
and more complex experimentation. Purification of
genomic DNA involves disruption and lysis of the
bacteria followed by the removal of proteins and other
contaminants and finally recovery of the DNA. To isolate
the DNA, 500ųL of bacterial broth were transferred to a
labeled micro tube and heated at 100°C for ten minutes.
After that, the bacteria broth was placed in an ice bath for
ten minute. Next, the sample was centrifugation for ten
minutes. Finally, 300ųL of the supernatant were
transferred to a new tube, labeled, and ready for future
procedures.
The isolated DNA was amplified using two
primers: dNTP and Taq. This procedure consists of
adding 6ųL of the previously isolated DNA to a PCR
tube and placing it in a thermal cycler to start the heating
and cooling cycle.
To confirm successful DNA isolation and
amplification, it is necessary to run a gel electrophoresis.
Further analysis of the results could be done using
bioinformatics
Antibiotic Production
To characterize the bacteria in the study, it is
necessary to study their capacities. Antibiotic production
is one such capacity. Each bacterium was placed on a
paper disc and placed in two agarose plates streaked with
E.coli and M. luteus. The plates should be placed in the
incubator at 30°C for 24 hours for bacterial proliferation.
If there is any antibiotic production, bacterial inhibition
should be observed.
RESULTS AND DISCUSSION
The main objective in this experiment was to
isolate bacteria from tropical soils of Puerto Rico and
characterize them. The expectation was to experiment
and determine whether or not the samples were antibiotic
and oil resistant and if they produce antibiotic or
cellulase. Unfortunately, because of the lack of time, the
experiment could not be concluded. Still, the results
obtained from the procedures already done are a
significant part of the research.
Two bacteria were studied and partially
characterized. Each bacterium had a bacterial designator
to distinguish them and both were collected from
different places. Location and the most important details
of the soil collection are shown in Table 1. Dilutions
were made to purify the bacteria. Once the bacteria were
isolated it was possible to start with the characterization
tests.
Gram stain was the first test made. Bacteria
S15UPRC-RISEAMGP30SP01A1
was a gram positive
Cocci (Figure 1). When a gram positive cocci bacteria
colony is clustered, usually it
is characteristic of
Staphylococcus. Most are
harmless and reside on the
skin and mucous membranes
of humans and other
organisms. Nevertheless, a
small component of soil
microbial flora can also
contain it. It is characterized
as an antibiotic resistant
bacteria and it does not produce antibiotic. The sample
S15UPRC-RISEAMGP30SP01A1,
like a Staphylococcus,
demonstrated negative results in antibiotic production for
Table 1: Soil Collection Details
Bacterial Designator Location
Approximate
Temperature
pH
S15UPRC-
RISEAMGP30SP01A1 Cayey 77°F 5.5
S15UPRC-
RISERBCR30P01A2 Aibonito 68°F 5.0
Figure 1: Positive Cocci

4. BIOL 3955: Research Seminar
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E. coli and M. luteus (See Figures 2 and 3). These
confirmed that the bacteria do not kill or inhibit bacterial
growth.
The morphology turned out to be circular with
convex elevation and an entire margin (Figure 4). At last,
the result of the PCR on products electrophoresis gel
resulted to be negative. A
negative result in a PCR
product could be affected
by the primer that was
used. Possibly, it was not
appropriate for the test.
On the other hand, the bacteria colony
S15UPRC-RISERBCR30P01A2
was a gram positive
Bacillus (Figure 5).
Turnbull and Baron
(1996) indicate that
Bacillus species are a
rod-shaped microbe,
endospore-forming,
aerobic or facultative
anaerobic, gram positive
bacteria. In some species
cultures may turn gram
negative with age. The
bacterium has morphology
of a circular form with flat
elevation and curled
margin (Figure 6). In the production of an antibiotic test,
results do not show antibiotic production (See Figures 7
and 8). The bacteria do not inhibit the growth of the
E.coli or M. luteus bacteria. When the electrophoresis
gel was done the product was negative. Like the
previous bacteria, the primer may have caused the
negative result.
The hypothesis was not accepted because the
results of the production of antibiotics were negative.
Each bacterium could not be characterized with
specificity because we only know its family, but not its
genus. It can be done with Bioinformatics, but the results
were not good enough to proceed to DNA sequencing.
Other experimentation that could be done to identify each
bacterium are the test of resistance to antibiotics, oil test,
production of cellulose and another electrophoresis gel
with other primers. If future electrophoresis results are
positive, DNA sequence could be done. Then, the
bacteria may be completely characterized.
Figure 2: Antibiotic
Production E. coli
Figure 3: Antibiotic
Production M. luteus
Figure 8: Antibiotic
Production M. luteus
Figure 4: Morphology1
Figure 5: Positive Bacillus
Figure 6: Morphology2
Figure 7: Antibiotic
Production E. coli

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CITED LITERATURE
1. Kemsley T.2013.Bacteria Naturally 'Eat Up' Oil
Spill Contamination by Supplementing Diet with
Nitrogen [Internet].Nature World News; [cited
2015 Mar 13].URL: http://www.natureworldne
ws.com/articles/3757/20130830/bacteria-
naturally-eat-up-oil-spill-contamination-
supplementing-diet-nitrogen.htm
2. Martins L, Augusto L.2014.Antagonistic lactic
acid bacteria isolated from goat milk and
identification of a novel nisin variant
Lactococcus lactis. BMC Microbiology
[Internet]; [cited 2015 Mar 12].14:9 pages.URL:
http://0-www.ncbi.nlm.nih.gov.elis.tmu.edu.tw/
pmc/articles/PMC3930553/
3. Sethi S, Datta A, Lal B, Gupta S.2012.
Optimization of Cellulase Production from
Bacteria Isolated from Soil. [Internet]; [cited
2015 Mar 12].ISRN Biotechnology Vol 2013:
approximately 7 pages. URL:
http://www.hindawi.com/journals/isrn/2013/985
685/
4. Turnbull P, Baron S. 1996. Medical
Microbiology: Bacillus [Internet]. 4th Ed.
Galveston (TX): Univ.of Texas Med. Branch at
Galveston; [cited 2015 May 6]. URL:
www.ncbi.nlm.nih.gov/books/NBK7699/
5. RISE Program 2015 LabManual.2015. Cayey
(PR): RISE Program. Vol 1-Vol 4.