University of Puerto Rico-Cayey

Department of Biology / RISE Program

BIOL 3955 Seminar Investigation: Biomedical Techniques Spring 2015

Bacteria Report X Final Report

Written Report Assessment Criteria May _____ 2015

Students’ Names: 15.Camille Ruiz / 3.Justin Cotto

Research: Isolating bacteria from tropical contaminated soils in Puerto Rico

Evaluator’s Name Dr. Eneida Díaz and Dr. Elena González Total Points ____55/75_____

Content Poor Fair Average Good Excellent

Introduction

1. Adequately presents the theme too basic 1 2 3 x4 5

2. Summarizes related studies well 1 2 x3 4 5

3. Includes adequate CSE citations in the text/Cited Literature 1 x2 3 4 5

4. Hypothesis and/or problem well formulated 1 2 3 4 x5

5. Indicates significance of the study 1 2 3 4 5x

Experimental Design and Methodology

6. Describes effectively the lab strategy and/or methods 1 2 3 x4 5

7. Presents only the necessary steps to understand the strategy 1 2 3 x4 5

Results

8. Presents results adequate for data collected 1 2 3x 4 5

9. Visual aids enhance comprehension of results 1 2x 3 4 5

Discussion of results

10. Includes whether or not the hypothesis(es) was proven and or research question(s) answered

1 2 3x 4 5

11. Refers to implications for the future 1 2 3x 4 5

Language and report organization Poor Fair Average Good Excellent

12. Uses correct grammar, syntax, punctuation, spelling 1 2 3 4 x5

13. Demonstrates clarity 1 2 3 4x 5

14. Demonstrates coherence with appropriate connectors 1 2 3 4 x5

15. Body of report well organized 1 2 3x 4 5

Isolating bacteria from tropical contaminated soils in Puerto RicoCotto 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 identifyingy 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, which it came out 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) were effectively to resisted against antibiotics more than the second sample (JCR). because of theIt 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 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.

MethodologyMaterials and Methods

Soil collection and cultivationEach 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. Six micro tubes were labeled from 100 to 10-5 and where filled 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 rRhizobium dDefined mMedium (RDM) and tTryptic sSoy aAgar (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 microorganismThe 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 morphologyOnce 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 observed iundern 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 _________each of them for 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.). Right after it is done,T the 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 oin 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 ion 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. TAfterwards, the QIA column is once more centrifuged once more in a provided 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 theits DNA, a quantity of 50 microliters of water are added to the center of the QIA membrane. The next step consists ion 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 resistancet test consisted of inoculating the

isolated bacteria in a media plate and 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 circledid 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 were capable to demonstrated a 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 polymerase chain reaction 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.). and Iit promoted produced a positive outcome as a difference, which could be due to an error made ion the initial PCR experimentation.

NMoreover, none of the bacteria produced antibiotics. However, twever, the bacteria did show resistance to various antibiotics. The smaller the diameter the , more resistance the bacteria would present to thatbe 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 to any of the antibiotics that it wasere 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/28/157:30 p.m.

Cotto J- #2

18.15º N66.18º W

Moist environment (above grass), 76º

Cidra, PR January/29/158:00 a.m.

Table #2: Results from different procedures

Process CLRV30TSA01A

CLRV30M01B JCR30TSA01A

Gram Stain Negative Positive PositiveBacterial Morphology Coccus Bacillus Bacillus

PCR/Electrophoresis #1 Negative Negative NegativePCR/Electrophoresis #2 Positive Negative -Antibiotic Production Negative Negative Negative

Table #3: Antibiotic Resistance Test Results:

Antibiotic CLRV30TSA01A

CLRV30M01B

JCR30TSA01A

Kanamycin 2.0 cm 1.0 cm 2.0 cmPenicillin 0.0 cm 0.0 cm -

Tetracycline 1.6 cm 2.4 cm 2.2 cmGentamicin 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 cmVancomycin 0.0 cm 0.0 cm -Streptomycin 1.6 cm 1.0 cm 1.9 cm

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 is investigation was helpful to analyze, characterize and identify isolated bacteria. Characterization included determining within different parameters, where its antibiotic efficiency and , bacterial morphology. and Ggenomic sequencinge .was wereleft proved for future work.s 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 s that were incubated as “bacteria free” culturess were analyzed usingby the BACTEC™ FX system. were observed with Ggram staining helped determineto see whether or not it is real that bacteria wereare actually growing or not (Peretz et. al. 2015). They discovered that 15 cultures which were defined as negative by the BACTEC FX system at the end of the incubation were found to contain microorganisms when Gram-stained., having a slow growth as a quality. 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, t

Also, the development of new instrumentation for PCR procedures can be a key to establish an improvement when identifying and quantifying the target pathogen in infected samples that does not require reliable methods, like DNA extraction , which can lead to errors within the experiment. 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 S 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.

Cited Literature

Cho S, Park H, Ahn Y, Park S. 2015. Rapid and specific detection of Acidovorax avenae subsp.citrulli by SYBR Green real-time PCR using YD repeat protein gene. J Microbiol Biotechnol. Vol and pages[Internet; cited: 2015 March 17]. Recovered at: http://www.ncbi.nlm.nih.gov/pubmed/25951847

Kobayashi F, Maki T, Kakikawa M, Yamada M, Puspitasari F, Iwasaka Y. 2015. Bioprocess of Kosa bioaerosols: Effect of ultraviolet radiation on airborne bacteria within Kosa (Asian dust). J Biosci Bioeng. [Internet; cited: 2015 March 17]. Doi:10.1016/j.jbiosc.2014.10.015. Recovered at: http://www.ncbi.nlm.nih.gov/m/pubmed/25735592/?i=3&from=isolation%20of%20bacteria%20from%20soil

Online Practices of Microbiology for pharmacists [Internet]. [2015]. Granada (ES): CEI Biotic; [cited: 2015 March 17]. Recovered at: http://www.pomif.com/pages/practicas/bacteriologia/aislamiento

Peretz A, Isakovich N, Pastukh N, Koifman A, Glyatman T. 2015. Performance of Gram

Staining on Blood Cultures Flagged Negative by an Automated Blood Culture System. [Internet; cited: 2015 March 17] Recovered at: http://www.ncbi.nlm.nih.gov/pubmed/25877009

Puciarelli S, Devaraj RR, Mancini A, Ballarini P, Castelli M, Schrallhammer M, Petroni G, Miceli C. 2015. Microbial Consortium Associated with the Antarctic Marine Ciliate Euplotes focardii: An Investigation from Genomic Sequences. Microb Ecol. [Internet; cited: 2015 March 17]. Doi: 10.1007/s00248-015-0568-9. Recovered at: http://www.ncbi.nlm.nih.gov/pubmed/25704316

Shahmohammadi R, Nahaei R, Akbarzadeh A, Milani M. 2015. Clinical test to detect mecA and antibiotic resistance in Staphylococcus aureus, based on novel biotechnological methods. Artif Cells Nanomed Biotechnol. [Internet; cited: 2015 March 17] Recovered at: http://www.ncbi.nlm.nih.gov/pubmed/25950954