Fluent Essays

Ive attached the instructions and two articles for the assignment. Please follow the instructions and NO plagiarism. you should design a concept map for each section on the article. primary_literature_analysis_guidelines.docx _028x_66.9.1543.pdf a_liquid_based_method.pdf Unformatted Attachment Preview Primary Literature Analysis Guidelines PURPOSE: The purpose of this literature analysis is to help the students understand the structure of a scientific paper and therefore help them to properly structure their own written report. GUIDELINES: Each student will analyze two primary literature articles of their choosing. For each: 1. Print the abstract of the article and attach it to the analysis. 2. Create a concept map/flow chart of each section of the article that outlines the ideas presented and in the order in which they are presented. This is meant to be a summary of the structure to show how each section is organized to provide a clear line of thought and “story” throughout the article. 3. A brief statement explaining the structure of each section of the article, immediately following each concept map/flow chart. For example, how did they progress through the material in the Introduction so that you were provided with enough background information but also lead to the specific objective of the study? 4. An overall summary paragraph providing reflection on how the article is structured and how different aspects of that structure help or hinder in your ability to follow the story or understand the study. For example, if the study incorporated multiple experimental components, did the authors always present the information about these components in the same order throughout each section of the article? Example Introduction Section Flow Chart from the article “The fungal fastlane: Common mycorrhizal networks extend bioactive zones of allelochemicals in soils” (Barto et al. 2011, PLoS One) BIG IDEA: Allelopathy defined, conditions that affect it explained, problem with using synthetic soils in lab to study complex interactions in natural soils. Knowledge gap identified- mycorrhizal fungi are sometimes targeted, but unknown how they might transport allelochemicals Identify their specific focus and proposed mechanism of chemical transport via mycorrhizal network What is known: Summary of what is known about mycorrhizal transport of water, nutrients, etc. Known to transport water, nutrients. Might transport chemical signals How this interaction could transport allelochemicals and prevent microbial degradation of those chemicals Likely increase flow rates of chemicals between source and target Problem with previous experiments- lack ability to evaluate fully formed mycorrhizal networks in short term greenhouse experiments Objective of current study- in longer greenhouse experiment determine if networks are important to allelochemical transport between plants Fully develop networks in experimental plots, or disrupted networks Apply herbicidal chemical as model, then use actual plant-released allelopathic compounds. Brief summary of findings & conclusions- network provided direct path for chemical transport, inhibited neighboring plants. Important overlooked component in allelopathy. 1543 Journal of Food Protection, Vol. 66, No. 9, 2003, Pages 1543–1549 Copyright q, International Association for Food Protection Interaction of a Free-Living Soil Nematode, Caenorhabditis elegans, with Surrogates of Foodborne Pathogenic Bacteria GARY L. ANDERSON, 1 KRISHAUN N. CALDWELL, 2 LARRY R. BEUCHAT, 2 1Department AND PHILLIP L. WILLIAMS1* of Environmental Health Science, University of Georgia, Athens, Georgia 30602-2102; and 2Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, GrifŽ n, Georgia 30223-1797, USA MS 02-455: Received 13 December 2002/Accepted 28 March 2003 ABSTRACT Free-living nematodes may harbor, protect, and disperse bacteria, including those ingested and passed in viable form in feces. These nematodes are potential vectors for human pathogens and may play a role in foodborne diseases associated with fruits and vegetables eaten raw. In this study, we evaluated the associations between a free-living soil nematode, Caenorhabditis elegans, and Escherichia coli, an avirulent strain of Salmonella Typhimurium, Listeria welshimeri, and Bacillus cereus. On an agar medium, young adult worms quickly moved toward colonies of all four bacteria; over 90\% of 3-day-old adult worms entered colonies within 16 min after inoculation. After 48 h, worms moved in and out of colonies of L. welshimeri and B. cereus but remained associated with E. coli and Salmonella Typhimurium colonies for at least 96 h. Young adult worms fed on cells of the four bacteria suspended in K medium. Worms survived and reproduced with the use of nutrients derived from all test bacteria, as determined for eggs laid by second-generation worms after culturing for 96 h. Development was slightly slower for worms fed gram-positive bacteria than for worms fed gram-negative bacteria. Worms that fed for 24 h on bacterial lawns formed on tryptic soy agar dispersed bacteria over a 3-h period when they were transferred to a bacteria-free agar surface. The results of this study suggest that C. elegans and perhaps other free-living nematodes are potential vectors for both gram-positive and gram-negative bacteria, including foodborne pathogens in soil. The agricultural impacts of plant and animal parasitic nematodes have long been recognized and, by virtue of their direct effects on fruit and vegetable production, have been extensively studied. Comparatively little is known regarding the impact of free-living microbivorous nematodes on produce production and safety, although these nematodes are the most abundant and widespread soil mesofauna. They play an important role in microbial degradation processes and nutrient  ow in soil (20), which includes the inoculation of new substrates by phoretic transport or through the excretion of viable microorganisms. The colonization of new substrates by microorganisms is favored by the presence of free-living bacterivorous nematodes (10, 18). Free-living nematodes are reportedly attracted to soils receiving agricultural inputs, including organic amendments and fertilizer (16, 29), and thus have the potential to play a role in colonizing agricultural soils with foodborne pathogens originating from animals. Bacterivorous nematodes have been shown to ingest and transmit a broad range of bacterial species, including plant and human pathogenic species. Chantanao and Jensen (12) compiled a list of bacteria ingested and transmitted in viable form by Pristionchus iheritieri (Nematoda: Diplogasterinae), a free-living saprozoic nematode. The list included several species of plant pathogenic bacteria. Jensen (24) demonstrated that Rhabditis spp. are able to ingest and void viable spores of various bacteria that are pathogenic * Author for correspondence. Tel: 770-542-0606; Fax: 770-543-7472; E-mail: pwilliam@arches.uga.edu. to plants. In addition, rhabditid nematodes fed a diet of Escherichia coli defecated viable cells (2). Nematodes are also able to protect bacteria present in their digestive tracts from chlorination (28). Salmonella ingested by P. iheritieri is reported to have survived the chlorination of wastewater at a treatment facility (33). Thus, nematodes may harbor, protect, and disperse bacteria, including those ingested and passed in viable form in feces. The association of free-living nematodes and various genera of bacteria has been studied. While it is recognized that free-living nematodes avoid certain bacteria (4, 31), it is clear that they do not uniformly avoid foodborne pathogens. Wasilewska and Webster (35) reported that a few species of bacterivorous nematodes can disseminate plant or human pathogenic bacteria. Two human enteric pathogens, Salmonella and Shigella, are reportedly ingested and defecated by free-living saprozoic nematodes (11). Salmonella Typhimurium is known to infect the free-living soil nematode Caenorhabditis elegans. Infected nematodes survive for up to 11 days, thus potentially serving as reservoirs for this foodborne pathogen. Darby and Falkow (13) reported that C. elegans was unaffected by 9 of 10 pathogens and fed on them as they do on nonpathogenic E. coli. From these reports, it appears that free-living nematodes may be important vectors of pathogenic bacteria, including some bacteria capable of causing human disease. Soil is a source of microbial contamination for fruits and vegetables, as evidenced by the isolation of soil-residing pathogenic bacteria from produce (6, 17). Jay (23) stated that the microbiota of soil-grown fruits and vegetables 1544 ANDERSON ET AL. may be expected to re ect the microbiota of soils in which they grow. Microbivorous nematodes are among the primary grazers of bacteria in soil (34). The possibility that nematodes may transmit foodborne pathogens to raw produce and thus play a role in the epidemiology of foodborne disease has been given only meager research attention. In a survey undertaken to determine the presence of amoebae and Salmonella in vegetables, Rude et al. (32) recovered nematode eggs and larvae by a nacconol-ether method. The recovery of nematodes from uncooked vegetables indicates that agronomic conditions and marketing practices may be conducive to the survival of nematodes on fresh produce (32). This result also indicates that if free-living nematodes are present on raw produce, they may serve as vehicles for contamination with pathogenic bacteria, either by surface contact or via eggs or voided material from their gastrointestinal tracts. We undertook a study to evaluate the interaction of C. elegans with bacterial surrogates for foodborne pathogens occasionally occurring or persisting in soil. Nematode-bacterium interactions were characterized to determine the propensity of young adult worms to be attracted to bacterial colonies, to compare the feeding and development of young adult worms cultured on this diverse group of bacteria, and to examine the dispersal of bacteria by C. elegans after feeding on monoxenic cultures. J. Food Prot., Vol. 66, No. 9 in diameter (area, ca. 0.31 cm2), and the total area covered by colonies was ca. 3.74 cm2. Approximately 22.1 cm2 of the agar surface was free of bacterial colonies. Nematodes were placed on a colony-free area in the center of the plate. Their movement to bacterial colonies was monitored and documented with video capture and image analysis (NIH Image v1.59, www.cc.nih.gov/cip) for 16 min. Following ‘‘tracking,’’ nematodes were monitored for 24 h to determine whether they remained associated with bacteria or tended to disperse into the colony-free area of the agar surface. Selection and culture of bacteria. The bacteria chosen for the investigation were E. coli OP50, an avirulent strain of Salmonella Typhimurium, Listeria welshimeri, and Bacillus cereus. E. coli OP50 is a uracil-deŽ cient strain commonly used as a food source in the culturing of C. elegans. The virulence of Salmonella Typhimurium is attenuated owing to an alteration of the rpoS gene caused by natural mutation, rendering it incapable of infecting mice according to standard pathogenicity assays (38). A laboratory stock strain of L. welshimeri and an atoxigenic strain (F3812/ 84) of B. cereus isolated from pasteurized milk were used. These four test bacteria served as surrogates for pathogenic E. coli, Salmonella, L. monocytogenes, and B. cereus. Ingestion of bacteria by C. elegans. The feeding of C. elegans on bacteria can be quantiŽ ed by monitoring the change in optical density at 570 nm (OD570) of bacterial suspensions inoculated with known numbers of worms (25, 26). A modiŽ cation of a method described by Anderson et al. (3) was used to measure the ingestion of four test bacteria by C. elegans. C. elegans was placed in suspensions of bacteria (ca. 109 CFU/ml of K medium, 1 ml per well) in 12-well tissue culture plates. Four wells containing bacterial suspension were inoculated with young adult worms. Two wells containing bacterial suspension not inoculated with worms served as controls. Duplicate determinations of OD570 values for bacterial suspensions were made with the use of a spectrophotometric plate reader after feeding intervals of 4 and 20 h. The consumption of bacteria by C. elegans was expressed as DOD570 per 100 worms over deŽ ned periods (net DOD570 5 DOD570 for suspension with worms 2 DOD570 for suspension without worms). The number of worms in each well was determined from digital images by image analysis (3). The quantitative relationship between OD570 and the number of bacteria in a suspension varied among test bacteria. For each test bacterium, a calibration curve establishing the relationship between the number of cells in a suspension and OD570 was constructed. Populations of bacteria were determined by serially diluting suspensions in tryptic soy broth (TSB), surface plating the dilutions on TSA, incubating the plates at 378C for 24 h, and counting colonies. Populations (CFU/ml) were plotted against the OD570 values for suspensions to construct calibration curves. The dependence of feeding by C. elegans upon the density of bacterial cell suspensions was determined. Young adult worms were fed bacteria in suspensions of various cell densities as determined by the initial OD570 values. For each trial, four suspensions of bacterial cells were prepared by diluting 24-h TSB cultures in K medium (at culture/K medium ratios of 4:1 to 0.5:1). Two or three trials were carried out for each of the four test bacteria; thus, feeding was determined at 8 to 12 cell densities for each of the test bacteria. For each trial, the feeding of C. elegans (DOD570 per 100 worms) in suspensions containing each population of bacteria was calculated relative to the average across the test populations (relative feeding 5 feeding at initial OD570 [c-n]/ average feeding for initial OD570 values [c-1, c-2, c-3, and c-4]). The initial OD570 values (c-1, c-2, c-3, and c-4) were determined for four dilutions ranging between 4:1 to 0.5:1 stock solution to K medium; thus, a 2.4-fold range in cell density was tested for each feeding trial. These procedures make it possible to explore the relationship between cell density and feeding even when changes in OD570 vary between trials. Attraction of C. elegans to bacterial colonies. C. elegans may be either attracted to or repulsed by bacteria, and a number of methods are available to assess its behavior in this regard. We developed a simple method to rapidly screen for the attraction of C. elegans to bacterial colonies. Brie y, 12 replicate colonies of a single bacterial species were established on the periphery of the surface of tryptic soy agar (TSA, pH 7.3; BBL/Difco, Sparks, Md.) in 6-cm-diameter petri plates. Each colony was ca. 0.63 cm Dispersal of bacteria by C. elegans. Three-day-old adult C. elegans fed for 24 h on 24-h colonies of E. coli OP50, Salmonella Typhimurium, L. welshimeri, or B. cereus on TSA. Worms 1 to 2 cm away from colonies were picked with a sterile wire and transferred to the surface of an uninoculated TSA plate. Nematodes were allowed to move about freely on plates. After 3 h, worms were killed with a hot wire, and plates were incubated for 24 or 48 h at 378C to allow the growth of bacteria. Digital image MATERIALS AND METHODS Nematode culture. Cultures of N2 (wild-type) strain C. elegans were used. A stock suspension of dauer-larval-stage worms in M9 buffer was kept at 208C and renewed monthly according to procedures described by Donkin and Williams (15). The dauers were used to generate age-synchronous adult worms. Dauers were placed on the surface of K agar (36) in petri plates with an established lawn of E. coli OP50 (7) and incubated at 208C. After 3 days, eggs and worms were collected from the plates and treated for 15 min in Clorox solution (1\% NaClO and 0.013 M NaOH) to isolate eggs (22). Eggs were then placed on K agar with an E. coli OP50 lawn and incubated for 3 days to obtain age-synchronous adults. J. Food Prot., Vol. 66, No. 9 INTERACTIONS OF C. ELEGANS WITH SOIL MICROFAUNA 1545 analysis was used to quantify the extent of bacterial dispersal on the basis of the number of foci of bacterial growth. A second series of bacterium dispersal experiments was carried out with worms that had fed on bacteria and were then surface sanitized by washing with sodium hypochlorite before being placed on uninoculated TSA. Worms were collected in 10 ml of K medium (37) in a 15-ml test tube and allowed to settle to the bottom. The K medium was removed, and worms were suspended in 0.4 mM sodium hypochlorite at 208C for 5 to 6 min. The hypochlorite solution was removed, and the worms were rapidly washed in K medium (twice, 10 ml per wash) before they were deposited on the surface of TSA. The dispersal of ingested cells, as well as the presumably small number of cells that may have survived hypochlorite treatment on the external surfaces of worms, was determined as described for worms not treated with sodium hypochlorite. RESULTS AND DISCUSSION E. coli OP50 served as a standard against which the responses of C. elegans to other test bacteria were compared. The relationship between C. elegans and Salmonella Typhimurium is unique in that Salmonella is among the few pathogens known to infect nematodes (11). Salmonella Typhimurium has occasionally been isolated from fruits and vegetables and can persist in soil for several months (6). B. cereus, a gram-positive sporeformer, is commonly found in soil. L. welshimeri, another gram-positive bacterium, is not pathogenic to humans, but Listeria species, including the pathogenic L. monocytogenes, are known to reside in soil as saprophytes (5). Attraction of C. elegans to surrogates. Young adult worms placed at the center of a TSA plate on which 24-h colonies of test bacteria had formed migrated into the colonies. Digital images documenting the movement of nematodes into colonies were captured at 1-min intervals for 6 min and at 16 min (Fig. 1). Worms were rapidly and strongly attracted to colonies of all four test bacteria. Over 80\% of the worms migrated to colonies of Salmonella Typhimurium, L. welshimeri, and B. cereus within the Ž rst 4 min. After 6 min, .90\% of the worms were located in colonies of these bacteria. Eighty-nine percent of the worms were associated with colonies of E. coli OP50 within 6 min. At the end of the 16-min tracking period, 95, 95, 95, and 99\% of the nematodes were located in colonies of E. coli, L. welshimeri, Salmonella Typhimurium, and B. cereus, respectively. Andrew and Nicholas (4) reported that C. elegans is strongly attracted to E. coli, Pseudomonas  uorescens, and Pseudomonas aeruginosa. These gram-negative bacteria were found to support the growth and reproduction of the nematode. Bacillus megaterium, a gram-positive sporeformer, was found to repel C. elegans. We observed that C. elegans was strongly attracted to 24-h colonies of gram-negative as well as gram-positive bacteria. Behavior and development of C. elegans on surrogates. The association between nematodes and 24-h colonies of test bacteria growing on TSA plates was monitored over a 96-h period following inoculation with 3-day-old C. elegans adults. As observed in the attraction assays, worms became strongly associated with bacterial colonies within FIGURE 1. Attraction of C. elegans to 24-h colonies of bacteria growing on TSA. Twenty to 30 young adult worms were placed on the surface of TSA ca. 0.5 mm away from colonies. The percentage of worms found within colonies was calculated from digital images captured at deŽ ned intervals up to 16 min after inoculation. 16 min after they were deposited on the surface of TSA. The percentage of worms migrating to bacterial colonies was determined at 1-h intervals for 4 h. More than 90\% of the worms were found within colonies of E. coli throughout this 4-h period. The association was also strong for the three other test bacteria, with $95\% of worms migrating to bacterial colonies. After 20 h, the a ... Purchase answer to see full attachment