Natural antibiotics: a hands-on activity on garlic's antibiotic properties.
Abstract: This work details a science experiment on garlic's antibiotic properties designed to acquaint high school and introductory-level undergraduate students with concepts such as natural antibiotics, bioactive substances, and biosafety. This activity is optimized to be implemented by teachers with limited experience in laboratory activities and/or in poorly equipped schools. A list of materials is provided, along with safety and procedural instructions, discussion topics, and assessment suggestions.

Key Words: Allicin; Allium sativum L.; science experiment; natural antibiotics.
Article Type: Report
Subject: Garlic (Research)
Antibacterial agents (Research)
Bacillus cereus (Research)
Authors: Fonseca, Maria Joao
Tavares, Fernando
Pub Date: 08/01/2011
Publication: Name: The American Biology Teacher Publisher: National Association of Biology Teachers Audience: Academic; Professional Format: Magazine/Journal Subject: Biological sciences; Education Copyright: COPYRIGHT 2011 National Association of Biology Teachers ISSN: 0002-7685
Issue: Date: August, 2011 Source Volume: 73 Source Issue: 6
Topic: Event Code: 310 Science & research
Product: Product Code: 0160072 Garlic; 2834880 Bacteriostats NAICS Code: 111219 Other Vegetable (except Potato) and Melon Farming; 325412 Pharmaceutical Preparation Manufacturing SIC Code: 0139 Field crops, except cash grains, not elsewhere classified; 2834 Pharmaceutical preparations
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 265291905
Full Text: Garlic (Allium sativum L.) is widely used for its culinary and pharmacological properties, which include antimicrobial traits. Garlic extracts are known to inhibit the growth of fungi, protozoa, viruses, and numerous bacteria, namely Salmonella spp., Staphylococcus aureus, and Escherichia colli (Rahman, 2007). Garlic's antimicrobial activity is mainly ascribed to allicin, a bioactive compound present in injured garlic cloves (Harris et al., 2001). Allicin is considered a promising substitute or co-adjuvant for commercial antibiotics (Cutler & Wilson, 2004). In addition to their scientific interest, natural antibiotics like allicin can be used in classroom activities to introduce students to concepts like antibiotics and biosafety as well as basic microbiology techniques (Shimabukuro & Haberman, 2006). Furthermore, assessing bacterial susceptibility to natural antibiotics fosters the discussion of antibiotic resistance, a major public health issue worth addressing in school (Lawson, 2008). This work describes a hands-on activity on the antibiotic effect of garlic shoot juice (GSJ), an allicin-containing aqueous extract. Bacillus cereus, a rod-shaped, spore-forming, Gram-positive food-borne bacterium, is used to encourage students to investigate how aromatic herbs traditionally used in cooking can inhibit microbial flora. Its short generation time provides visual results in approximately one day, and its culturability can be preserved over long periods by freezing spore suspensions. This activity can be conducted in unequipped schools using inexpensive materials available in domestic kitchens.

** Learning Objectives

Students will

* demonstrate the existence of phyto-antimicrobials;

* understand the concepts of antibiotics, antibiotic susceptibility, and biosafety;

* perform basic microbiology procedures;

* interpret and discuss experimental outcomes resulting from qualitative observations; and

* develop creativity skills related with motivation and imagination to devise alternative problem-solving strategies.

** Materials

Bacillus cereus LMG 6923T (strain for teaching purposes, BCCMTM/LMG Bacteria Collection); fresh garlic bulbs; garlic press; 10-mL plastic syringe; gauze; 250-mL glass containers; glass rods; gram scale; microwave oven; petri dishes; Pasteur pipettes and 1-mL pipettes; microbiological loops; agar (bacteriological or available in supermarkets and health food stores); meat (pork or beef); table sugar; kitchen salt; distilled water; 1-L growthmedium flasks (or equivalent microwave glass containers); glass burners; discard container with bleach (20%); ethanol (70%); paper towels; and Falcon and Eppendorf tubes (or equivalent).

** Safety Concerns

This activity requires handling of bacteria. Therefore, students must act responsibly. They must wash their hands before and after the exercise, and they must not eat or drink in the lab. Work surfaces must be disinfected with ethanol (70%), and the materials used must be previously sterilized. Liquids, plastics, and glassware can be sterilized using a microwave oven. Metallic materials can be sterilized using boiling water. All materials in contact with bacteria must be sterilized prior to disposal.

** Methods

This activity is based on antibiotic susceptibility testing using the agar-diffusion method (Figure 1). Students should prepare the growth medium, the bacterial inoculum, and the GSJ extract. Bacterial susceptibility to GSJ on meat agar plates is assessed by observing inhibitory halos surrounding GSJ drops.

[FIGURE 1 OMITTED]

Growth Media Preparation

A meat agar medium replaces commonly used growth media such as nutrient agar. To prepare 250 mL of meat agar (enough for approximately 10 plates), boil 150 g of meat and filter the solution through gauze into a 1-L medium flask. Dissolve 6 g of sugar and 1.25 g of kitchen salt, and add 3.75 g of agar. Sterilize the medium using a microwave oven (900 W) for 5 minutes (or an autoclave for 15 minutes). To avoid spillage due to overheating, heat the medium in 1-minute increments interspersed with 15-second cooling periods. Because agar solidifies at ~42[degrees]C, pour it into the sterile Petri dishes (~25 mL per 9-cm-diameter dish) as soon as the medium temperature allows handling. Plating must be performed quickly in a recently disinfected bench. Allow the medium to solidify before using the plates.

Bacterial Inoculum Preparation

To prepare B. cereus spore suspensions, pour 5 mL of sterile distilled water onto a four- to five-day-old culture plate, suspend the spores with a sterile glass rod, and transfer the suspension to a sterile Falcon tube containing 20 mL of sterile distilled water. Dilute the suspension 10-fold (1:9 mL of sterile distilled water). Place the used pipettes in a container with bleach (20%). The suspensions can be stored in a freezer (-20[degrees]C) for at least 2 months.

GSJ Extraction

Peel and wash three or four medium-sized garlic cloves with running water. Using a sterile garlic press, obtain ~5 g of smashed garlic, and then add sterile distilled water in the same proportion (1:1). Mix and filter the homogenate through a 10-mL sterile syringe containing sterile gauze into sterile Eppendorf tubes. The extracts can be stored at 4[degrees]C for at least 3 months.

Bioassay

Transfer 1 mL of spore suspension into each meat agar plate, and distribute it evenly by carefully tilting the closed plates. Position the plates at a slant for 1 minute, and then remove and discard the extra amount of suspension using a sterile pipette. Leave the plates to rest for 10 minutes to allow the bacterial cells to adhere to the medium. Using a Pasteur pipette, apply 1 drop of sterile distilled water (~20 uL) onto one side of each plate (control) and 1 drop of GSJ on the other. Leave the plates to rest for a few minutes, allowing the drops to diffuse into the medium, and then incubate the plates in an inverted position at room temperature for 24 hours (or at 37[degrees]C for ~12 hours).

Sterilize all biological waste in the microwave oven prior to disposal.

O Results & Discussion

In this activity students witness the antibacterial effect of GSJ on B. cereus (Figure 2). Given garlic's pervasive culinary use, students are led to reflect on the health benefits of using it in food processing and preservation. This can lead them to engage in further investigations regarding, for example, the effects of different cooking techniques, times, and temperatures on garlic's antimicrobial activity. Because garlic is most commonly used in cooking, it was decided to prepare an aqueous extract. Students may be interested in testing other solvents used with garlic to cook or season foods, such as wine or vinegar.

Extensions & Discussion Topics

Test the susceptibility of different bacteria to GSJ

Students can compare the inhibitory activity of GSJ on different bacteria by obtaining foodborne isolates from retail meat, for instance. They can distinguish different isolates on the basis of colony shape, size, texture, and coloring. So far, only bacteria naturally thriving on garlic are reported to resist allicin (Shim & Kyung, 1999). Searching for other resistant bacteria is a way to enhance students' motivation and creativity.

[FIGURE 2 OMITTED]

Explore how allicin is produced

Allicin is produced when raw garlic is injured and the vacuolar enzyme alliinase makes contact with allicin's precursor in the cytoplasm, alliin (Harris et al., 2001; Rahman, 2007). A simple experiment assessing the effects of intact and sectioned garlic on bacterial growth demonstrates that allicin production requires cell damage (Figure 3). This experiment can lead to discussion of concepts related to enzymatic reactions, cell structure, and host defense mechanisms.

Screen different organisms for natural antibiotics

Most culinary herbs and spices are rich in antimicrobials, such as phenolic compounds (parsley, laurel), aldehydes (cinnamon, cumin), and acids (vanilla, rosemary) (Vigil et al., 2005). By studying garlic's antibacterial properties, students reflect on the concept of antibiotics and become aware of naturally occurring bioactive substances of pharmaceutical interest. This naturally sparks their curiosity, motivating them to propose experiments to screen different antibiotic-producing organisms.

[FIGURE 3 OMITTED]

Address plant defense mechanisms

Anti-phytopathogenic bioactive compounds, like allicin, participate in chemical plant-defense mechanisms and have promising applications in phytoprotection programs (Slusarenko et al., 2008). Exploring garlic's antibiotic properties introduces students to ecology concepts, such as predation and parasitism, and to physiology topics, including phytoanticipin, phytoalexin, and other secondary metabolites.

Curricular Framing & Assessment

This activity is framed within the National Science Education Standards for grades 9-12 (National Research Council, 1996), as summarized in Table 1, and it can be adapted to instructional levels from high school through introductory undergraduate microbiology courses. The exercises presented promote students' abilities to conceptualize topics such as natural antibiotics and antibiotic resistance, to plan and execute experiments, and to develop inquiry-based scientific reasoning. The National Science Education Assessment Standards (National Research Council, 1996) recommend that student assessments focus on microbiology and cell biology concepts, performance of laboratory techniques, and awareness about scientific inquiry. Accordingly, students may be asked to produce a report covering basic background information, the hypothesis tested, experimental design, results, discussion, and conclusions. In 10-minute sessions, they can present and discuss their reports and propose alternatives to overcome eventual drawbacks.

** Conclusions

This activity addresses the concept of natural antibiotics by engaging students in a microbiological procedure used to assess antibiotic susceptibility. The exercises proposed promote students' critical, reflexive, and reasoning competencies. Because it uses affordable and easily available materials, the experiment is accessible regardless of a school's laboratory facilities.

DOI: 10.1525/abt.2011.73.6.7

** 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

Cutler, R.R. & Wilson, P. (2004). Antibacterial activity of a new, stable, aqueous extract of allicin against methicillin-resistant Staphylococcus aureus. British Journal of Biomedical Science, 61, 71-74.

Harris, J.C., Cottrel, S.L., Plummer, S. & Lloyd, D. (2001). Antimicrobial properties of Allium sativum (garlic). Applied Microbiology and Biotechnology, 57, 282-286.

Lawson, M.A. (2008). The antibiotic resistance problem revisited. American Biology Teacher, 70, 405-410.

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

Rahman, M.S. (2007). Allicin and other functional active components in garlic: health benefits and bioavailability. International Journal of Food Properties, 10, 245-268.

Shim, S.T. & Kyung, K.H. (1999). Natural microflora of pre-peeled garlic and their resistance to garlic antimicrobial activity. Food Microbiology, 16, 165-172.

Shimabukuro, M.A. & Haberman, V. (2006). An aromatic adventure with allelopathy: using garlic to study allelopathy in the classroom. American Biology Teacher, 68, 242-247.

Slusarenko, A.J., Patel, A. & Portz, D. (2008). Control of plant diseases by natural products: allicin from garlic as a case study. European Journal of Plant Pathology, 121, 313-322.

Vigil, A.L.M., Palou, E. & Alzamora, S.M. (2005). Naturally occurring compounds - plant sources. In P.M. Davidson et al. (Eds.), Antimicrobials in Foods, 3rd Ed. Boca Raton, FL: Taylor & Francis.

MARIA JOAO FONSECA (mjfonseca@ibmc.up.pt) 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. FERNANDO TAVARES (ftavares@fc.up.pt) is Assistant Professor at Faculdade de Ciencias, Departamento de Biologia, Universidade do Porto, Portugal and a researcher at CIBIO - Centro de Investiga$ao em Biodiversidade e Recursos Geneticos, Universidade do Porto, Portugal.
Table 1. Framing of the proposed experiments and discussion
topics within the National Science Education Standards for
grades 9-12 (National Research Council, 1996).

Content Standards                     Activity/Discussion Topic
Life Science
Content Standard C
  The cell                            Allicin synthesis
                                      Enzymatic reaction (concept of
                                      precursor,substrate, and enzyme)
                                      Cell structure (intracellular
                                      location of allicin's precursor
                                      and allinase enzyme)

  The interdependence of              Plant defense mechanisms
    organisms                         (ecological concepts of
                                      parasitism and competition)

The behavior of organisms             Allicin synthesis
                                      Plant response to injury
Science & Technology
Content Standard E
  Identify a problem or design        Screen different organisms for
    opportunity                       natural antibiotics
                                      Test the susceptibility of
                                      different bacteria to garlic
                                      shoot juice

Science in Personal & Social
  Perspectives
Content Standard F
  Personal and community health       Discussion of antibiotic
                                      resistance--natural antibiotics
                                      as substitutes or co-adjuvants
                                      commercial antibiotics
                                      test the susceptibility of
                                      different bacteria to garlic
                                      shoot juice

  Population growth                   Plant defense mechanisms
                                      (concepts of parasitism and
                                      competition)

Natural resources                     Discussion of antibiotic
                                      resistance--antibiotics as natural
                                      resources
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