BIOL 3955: Research Seminar 2015

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 Chemistry2University 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

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BIOL 3955: Research Seminar 2015

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 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 CollectionThe 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.

PurificationsTwenty-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.

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BIOL 3955: Research Seminar 2015

Gram StainUsually, 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 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 ProductionTo 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.

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BIOL 3955: Research Seminar 2015

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 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

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Figure 1: Positive Cocci

Figure 2: Antibiotic Production E. coli

Figure 3: Antibiotic Production M. luteus

Figure 4: Morphology1

Figure 5: Positive Bacillus

Figure 6: Morphology2

Figure 7: Antibiotic Production E. coli

Table 1: Soil Collection Details

Bacterial Designator LocationApproximateTemperature

pH

S15UPRC-RISEAMGP30SP01

A1

Cayey 77°F 5.5

S15UPRC-RISERBCR30P01A2 Aibonito 68°F 5.0

BIOL 3955: Research Seminar 2015

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.

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/2013/985685/

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.

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