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The bactericidal effect of sunlight.

Sunlight is essential for vital biological processes, such as photosynthesis and vitamin D synthesis. However, solar ultraviolet (UV) radiation can be detrimental to living beings: it is associated with coral bleaching, sunburn, and melanoma. By interfering with DNA, proteins, and lipids, UV radiation can induce mutation, cell inactivation, growth reduction, and death (Pourzand & Tyrrell, 1999). Although cells possess UV-protection mechanisms, these do not fully prevent the genotoxicity of natural and artificial UV radiation. This raises obvious human-health concerns that are emphasized by the limited public awareness about the long-term consequences of UV radiation overexposure. The problem is particularly relevant among teenagers, who, in spite of numerous educational interventions, frequently reveal misinformed behaviors (Jones & Saraiya, 2006; Poorsattar & Hornung, 2007). An explanation for the narrow success of programs aimed at promoting safe sunbathing habits among high school and college students may rely on the abstract notions required to understand the mutagenic effects of radiation, and on the need to shift from macro-to micro-levels of conceptualization (Tibell & Rundgren, 2010). In this context, we present a simple and engaging practical experiment to investigate the lethal cellular effect of sunlight using bacteria as a model organism. Bacterial susceptibility to sunlight has long been demonstrated (Downes & Blunt, 1877), and UV-induced damage and repair in these organisms have been thoroughly studied (Goosen & Moolenaar, 2008). As fast-growing unicellular microorganisms, bacteria allow for clear visual evidence within a short period, which is likely to prompt students' curiosity and motivate them to deepen their understanding of the processes involved.

Learning Objectives

In this activity, students expose bacteria to sunlight and witness its deleterious effect on cell survival. The students themselves prepare the growth medium and the plates and conduct the bioassay (Figure 1). In doing so, they

* demonstrate the lethal effect of sunlight on bacteria;

* discuss the impact of UV radiation on living cells;

* perform basic microbiology procedures; and

* interpret and discuss experimental outcomes resulting from qualitative observations.

Materials

* Bacillus cereus LMG 6923T (strain for teaching purposes, BCCMTM/LMG Bacteria Collection)

* Scale

* Autoclave

* Nutrient agar growth medium (available from Fisher Scientific, http://www.fishersci. com)

* Distilled water

* Ethanol (70%)

* Paper towels

* Discard container with bleach (20%)

* Glass burners

* Matches

* Thermometer

* 1-L growth-medium flasks

* Petri plates

* Falcon tubes

* Microbiological loops

* 1-mL pipettes

* Cellophane paper

* Cardboard paper

* Sunglass lenses (glass or plastic, with specified UV-protection features)

[FIGURE 1 OMITTED]

Safety Concerns

This activity requires handling bacteria, and students must act responsibly throughout the experiment. Eating and drinking must not be authorized in the lab. Students must wash their hands before and after the experiment. Work benches must be disinfected with ethanol (70%), and the materials used must be previously sterilized. All biological waste must be sterilized prior to disposal using an autoclave (121[degrees]C, 15 minutes).

Growth Medium Preparation

Prepare 250 mL of nutrient agar growth medium (enough for approximately ten 90 mm x 14 mm Petri plates), by dissolving 5.75 g of the powder in distilled water. Autoclave the medium at 121[degrees]C for 15 minutes. On a recently disinfected bench, as soon as the medium temperature allows handling (~50[degrees]C) pour it quickly into the plates (~25 mL per plate). Allow the medium to solidify and invert the plates. The plates can be used immediately or stored in the refrigerator for 2 to 3 weeks inside plastic sleeves to minimize dehydration. Alternative growth-medium-preparation and sterilization instructions for use in schools with limited equipment and materials can be found in Fonseca & Tavares (2011).

Inoculation

Transfer a loopful of cells from a 24-hour Bacillus cereus culture into a sterile Falcon tube with 5 mL of sterile distilled water. Invert the tube vigorously to obtain a homogeneous suspension. Inoculate the plates with 1 mL of bacterial suspension. Spread the suspension evenly by carefully tilting the closed plates. Slant the plates for 2 or 3 minutes and remove the excess suspension using a sterile pipette. Place the pipettes in a discard container with bleach (20%). Leave the plates without inversion for ~5 minutes, allowing the cells to adhere to the medium.

Bioassay

Because glass and plastic filter some UV radiation, remove the lids from the inoculated plates. To avoid contamination, wrap each plate in cellophane paper and store the lids in the clean area. Keep one plate (control) and expose the inoculated surface of the other plates to sunlight, organized into three sets: (1) test-plates, wrapped in cellophane paper; (2) control-plates, with a sunglass lens on top of the cellophane; and (3) control plates, covered by cardboard. Remove and label one plate of each set every hour. Monitor the temperature every 30 minutes and, if there is a photometer available, the sunlight irradiance as well. Remove the cellophane paper and place the lids on the plates. Invert and incubate the plates at room-temperature for 48 hours (or at 37[degrees]C for 20 hours).

Results & Discussion

The results obtained demonstrate sunlight's lethal effect on B. cereus (Figure 2). There is an obvious decrease on the number of bacterial colonies, following the increase of sunlight exposure (Figure 2, [A.sub.1]-[A.sub.3]). The bacterial growth in the control plate that was not exposed to sunlight assures the viability of the bacterial inoculum (Figure 2, B). Mesophilic B. cereus strains grow optimally at 20-40[degrees]C. The growth in the control plates covered by cardboard demonstrates that the inhibition observed in the test plates was not temperature-induced (Figure 2, C). Finally, the growth pattern in the plates protected by sunglass lenses, acting as UV-excluding filters, indicates that the deleterious effect of sunlight is mainly due to solar UV radiation (Figure 2, D). These results were obtained using a UV-A/ UV-B protecting glass lens from a renowned brand. Lenses with different protection features are likely to produce variable results. This aspect is worth exploring, namely by discussing with the students the quality of the lens and its expected efficiency in protecting the human eye from solar UV radiation.

[FIGURE 2 OMITTED]

Extensions & Discussion Topics

Address DNA damage and repair mechanisms. UV-induced mutagenic lesions are mainly ascribed to the dimerization of adjacent pyrimidine bases, leading to structural changes in the DNA molecule that interfere with replication and transcription processes. However, living organisms have developed protection and repair mechanisms to counteract the effects of solar UV radiation. These include the production of UV-absorbing pigments, like melanin, that act as physical barriers, and DNA repair systems, such as photoreactivation (photolyase enzyme) and excision repair (base and nucleotide excision repair). A detailed description of DNA repair mechanisms is available in Sinha and Hader (2002). This activity can be used to introduce students in introductory undergraduate courses to concepts like mutation, mutagenic agents, and DNA repair mechanisms.

Test different organisms for the effect of UV radiation. In this activity, B. cereus, a resistant spore-forming, Gram-positive bacterium, was used to illustrate the severity of sunlight's bactericidal effect. However, bacterial resistance to UV radiation varies. For instance, bacteria such as B. subtilis that produce photoprotective pigment-covered spores (Riesenman & Nicholson, 2000) are particularly resistant to solar UV radiation. Alternatively, yeast cells (Saccharomyces cerevisiae) can also be used. Testing the deleterious effect of sunlight with different organisms introduces students to ecological and evolutionary concepts such as biodiversity, adaptation, and natural selection.

Test different UV radiation wavelengths and UV screens. Ultraviolet-induced DNA damage is wavelength-dependent. Ultraviolet-A radiation (320-400 nm) causes only indirect damage through the production of reactive oxygen species, whereas UV-B (280-320 nm) and UV-C (100-280 nm) radiation also induce direct damage, because DNA strongly absorbs radiation at wavelengths below 320 nm (Fernandez Zenoff et al., 2006). The most effective bactericidal effects occur from 200 to 280 nm (McDonnell, 2007), although DNA's absorption spectrum covers the UV-B wavelength. Students can test the effect of specific UV wavelengths on bacterial survival using different lamps. Since UV radiation is used in numerous applications, including forensics, phototherapy, and tanning beds, students may be motivated to discuss the human health effects of UV exposure. By exploring the protective efficacy of screens such as glass, plastics, photographic UV filters, or sunscreen-impregnated membranes, students are made aware of how personal decisions affect health.

Discuss the impact of atmospheric pollution. The anthropogenic release of atmospheric pollutants during recent decades has resulted in a noticeable depletion of the stratospheric ozone layer that typically shields Earth from UV-C and the majority of UV-B radiation (Rastogi et al., 2010). This has resulted in an increase of solar UV-B surface irradiance, which raises serious health concerns. The visualization of sunlight's lethal effects on bacteria is an appealing way to engage students in the discussion of the environmental and personal health consequences of human interference in nature. Relevant information on atmospheric emissions is available from the Tropospheric Emission Monitoring Internet Service (http://www.temis.nl/index.php).

Curricular Framing & Assessment

This activity addresses content standards in the National Science Education Standards for grades 9 through 12 (National Research Council, 1996), as summarized in Table 1. While engaging in the design and execution of experiments, students are introduced to microbiology, genetics, and ecology concepts; perform laboratory techniques; and develop scientific-reasoning skills. In small groups, students can manipulate one or more of the variables mentioned in the core and extension activities--for instance, exposure period, filter material, and model organism. They may be asked to convey their findings in an activity report, comprising the research question(s), methods, results, and main conclusions. A scoring rubric covering general procedural, methodological, and conceptual elements is provided in Table 2. Students can also be assigned research exercises focusing on the discussion topics suggested. Ten-minute presentations can be organized, allowing students to share with their colleagues and reflect upon the outcomes of their investigations.

Conclusions

This simple and visually appealing hands-on activity fosters opportunities to familiarize middle school and high school students with abstract biology notions, such as mutation and mutagenic agent. Furthermore, it can be a persuasive strategy to promote increased awareness about the importance of adopting healthy sun-protection practices.

DOI: 10.1525/abt.2011.73.9.8

Acknowledgments

The authors are grateful to Catarina L. Santos for helpful comments and suggestions on the manuscript. M.J.F. is supported by the Fundacao para a Ciencia e a Tecnologia fellowship SFRH/ BD/37389/2007.

References

Downes, A. & Blunt, T.P. (1877). Researches on the effect of light upon Bacteria and other organisms. Proceedings of the Royal Society of London, 26, 488-500.

Fernandez Zenoff, V., Sineriz, F. & Farias, M.E. (2006). Diverse responses to UV-B radiation and repair mechanisms of bacteria isolated from high-altitude aquatic environments. Applied and Environmental Microbiology, 72, 7857-7863.

Fonseca, M. & Tavares, F. (2011). Natural antibiotics: a hands-on activity on garlic's antibiotic properties. American Biology Teacher, 73, 342-346.

Goosen, N. & Moolenaar, G.F. (2008). Repair of UV damage in bacteria. DNA Repair, 7, 353-379.

Jones, S.E. & Saraiya, M. (2006). Sunscreen use among US high school students, 1999-2003. Journal of School Health, 76, 150-153.

McDonnell, G.E. (2007). Antisepsis, Disinfection, and Sterilization: Types, Action, and Resistance. Washington, D.C.: ASM Press.

National Research Council. (1996). National Science Education Standards. Washington, D.C.: National Academy Press.

Poorsattar, S.P. & Hornung, R.L. (2007). UV light abuse and high-risk tanning behavior among undergraduate college students. Journal of the American Academy of Dermatology, 56, 375-379.

Pourzand, C. & Tyrrell, R.M. (1999). Apoptosis, the role of oxidative stress and the example of solar UV radiation. Photochemistry and Photobiology, 70, 380-390.

Rastogi, R.P., Richa, Sinha, R.P., Singh, S.P. & Hader, D.-P. (2010). Photo-protective compounds from marine organisms. Journal of Industrial Microbiology & Biotechnology, 37, 537-558.

Riesenman, P.J. & Nicholson, W.L. (2000). Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Applied and Environmental Microbiology, 66, 620-626.

Sinha, R.P. & Hader, D.P. (2002). UV-induced DNA damage and repair: a review. Photochemical and Photobiological Sciences, 1, 225-236.

Tibell, L.A.E. & Rundgren, C.-J. (2010). Educational challenges of molecular life science: characteristics and implications for education and research. CBE Life Sciences Education, 9, 25-33.

MARIA JOAO FONSECA is a high school biology teacher and Ph.D. student at IBMC--Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, and Faculdade de Ciencias, Departamento de Biologia, Universidade do Porto, Portugal;e-mail: mjfonseca@ibmc.up.pt. FERNANDO TAVARES is Assistant Professor at Faculdade de Ciencias, Departamento de Biologia, Universidade do Porto, Portugal and a researcher at CIBIO--Centro de Investigacao em Biodiversidade e Recursos Geneticos, Universidade do Porto, Portugal; e-mail: ftavares@fc.up.pt.
Table 1. Contextualization of the extension experiments
and discussion topics in the National Science Education
Standards for grades 9-12 (National Research Council,
1996).

Life Science

Content standard C

The cell

Address DNA damage and repair mechanisms
Test different bacteria for the effect of UV radiation

Molecular basis of heredity

Address DNA damage and repair mechanisms

Biological evolution

Test different bacteria for the effect of UV radiation

Science and Technology
Content standard E

Implement a proposed solution
Evaluate the solution and its consequences

Test different UV radiation wavelengths and UV screens

Science in Personal and Social Perspectives
Content standard F

Personal and community health

Address DNA damage and repair mechanisms
Test different UV radiation wavelengths and UV screens
Discuss the impact of atmospheric pollution

Environmental quality

Address DNA damage and repair mechanisms
Discuss the impact of atmospheric pollution

Natural and human-induced hazards

Discuss the impact of atmospheric pollution

Table 2. Scoring rubric for the evaluation of general procedural,
methodological, and conceptual elements.
The total score ranges from 0 to 24, according to the key presented.

                 The student/group of students ...                Score

Procedural       ... registers observational data and results       1
Items
                 ... prepares (or gives input about)                1
                 the experimental setup

                 ... respects all biosafety instructions            1

                 ... successfully completes the activity            1

Methodological   ... interprets        ... one set of plates        1
Items            and discusses the
                 results obtained      ... two sets of plates       2
                 for ...
                                       ... three sets of plates     3

                 ... discusses the influence of variables,
                 such as ...

                 ... length and time of the exposure period         1
                 (considers the UV index, the ozone column,
                 cloudiness ...)

                 ... temperature effect                             1

                 ... viability and concentration of the             1
                 bacterial inoculum

                 ... type of filter used, namely:

                 Cellophane paper--What is the purpose of using     1
                 it? Does it filter solar UV radiation?

                 Cardboard--What information do the results         1
                 obtained for the plates covered with cardboard
                 give about the effect of temperature?

                 Cardboard/sunglass lenses--Does cardboard          1
                 filter the same types of radiation as sunglass
                 lenses?

                 Sunglass lenses--How is the protection             1
                 capacity related to the quality of the lens?

                 ... addresses         ... formulating new          1
                 unexpected            explanations and
                 results ...           hypotheses

                                       ... redefining the           1
                                       experimental setup

                                       ... readjusting the          1
                                       variable control

                 ... understands the notion of model organism
                 and is able to discuss the following questions:

                 Would the same results be obtained if different    1
                 bacteria were used?

                 How different would the results be if a            1
                 multicellular organism were used instead
                 of a unicellular one?

Conceptual       ... formulates hypothesis about the effect of      1
Items            solar UV radiation on bacteria

                 ... predicts the bioassay results                  1

                 ... is aware of positive and negative effects      1
                 of sunlight

                 ... extrapolates the results obtained in light
                 of a human health context:

                 understands the importance of healthy sun          1
                 exposure habits, mentioning the need to avoid
                 excessive exposure and to use adequate
                 protection;

                 understands that human activities resulting in     1
                 the emission of atmospheric pollutants that
                 damage the ozone layer can lead to increased
                 solar UV surface irradiance.

                 Total Score                                       24
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Title Annotation:HOW-TO-DO-IT
Author:Fonseca, Maria Joao; Tavares, Fernando
Publication:The American Biology Teacher
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2011
Words:2565
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