A Laboratory Activity Demonstrating the Antibacterial Effects of Extracts from Two Plant Species, Moringa oleifera and Allium sativum (Garlic) †
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Authors:
Grace J. Miller1,
Anna M. G. Cunningham1,
Yui Iwase1,
Nicole L. Lautensack1,
W. Matthew Sattley1,*
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Received 07 February 2017 Accepted 18 August 2017 Published 30 October 2017
- ©2017 Author(s). Published by the American Society for Microbiology
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[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
- *Corresponding author. Mailing address: Indiana Wesleyan University, Division of Natural Sciences, 4201 S. Washington St., Marion, IN 46953-4974. Phone: 765-677-2128. Fax: 765-677-2455. E-mail: [email protected].
Abstract:
A variety of plants synthesize natural products that either kill or inhibit the growth of various microorganisms. These plant products may serve as useful natural alternatives to synthetic antimicrobial pharmaceuticals and can be especially important in regions where commercial drugs are often not available. Despite this, the role of plants as producers of natural antimicrobial agents is often understated or even ignored in undergraduate biology curricula. In this laboratory exercise, students extract water-soluble constituents from two plants, Moringa oleifera (moringa) and Allium sativum (garlic), and determine their activity against both a gram-positive (Bacillus cereus strain 971) and a gram-negative (Escherichia coli strain K12) bacterium using a disk diffusion assay on Mueller-Hinton agar. Disks infused with commercially available antibiotics (e.g., penicillin and tetracycline) serve as controls. Following an incubation period of 24 hours, students obtain quantitative data by measuring zones of growth inhibition that develop as a result of strain sensitivity. To determine the effectiveness of the learning objectives, an unannounced quiz was administered both before and after the activity, and the students showed significant gains in their understanding of key concepts. Because this activity combines aspects of two major branches of biology—plant biology and microbiology—it is suitable for use as a laboratory exercise in courses related to either discipline, or it may be used as a laboratory component of a general biology course.
References & Citations
Supplemental Material
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Appendix 1: Pre- and post-lab activity quiz, Appendix 2: Pre- and post-lab activity quiz; instructor key, Appendix 3: Student laboratory report with post-lab assessment questions: antibacterial effects of plant extracts, Appendix 4: Student laboratory report with post-lab assessment questions: antibacterial effects of plant extracts; instructor key, Appendix 5: Laboratory activity student evaluation survey
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Abstract:
A variety of plants synthesize natural products that either kill or inhibit the growth of various microorganisms. These plant products may serve as useful natural alternatives to synthetic antimicrobial pharmaceuticals and can be especially important in regions where commercial drugs are often not available. Despite this, the role of plants as producers of natural antimicrobial agents is often understated or even ignored in undergraduate biology curricula. In this laboratory exercise, students extract water-soluble constituents from two plants, Moringa oleifera (moringa) and Allium sativum (garlic), and determine their activity against both a gram-positive (Bacillus cereus strain 971) and a gram-negative (Escherichia coli strain K12) bacterium using a disk diffusion assay on Mueller-Hinton agar. Disks infused with commercially available antibiotics (e.g., penicillin and tetracycline) serve as controls. Following an incubation period of 24 hours, students obtain quantitative data by measuring zones of growth inhibition that develop as a result of strain sensitivity. To determine the effectiveness of the learning objectives, an unannounced quiz was administered both before and after the activity, and the students showed significant gains in their understanding of key concepts. Because this activity combines aspects of two major branches of biology—plant biology and microbiology—it is suitable for use as a laboratory exercise in courses related to either discipline, or it may be used as a laboratory component of a general biology course.

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Author and Article Information
-
Received 07 February 2017 Accepted 18 August 2017 Published 30 October 2017
- ©2017 Author(s). Published by the American Society for Microbiology
-
[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
- *Corresponding author. Mailing address: Indiana Wesleyan University, Division of Natural Sciences, 4201 S. Washington St., Marion, IN 46953-4974. Phone: 765-677-2128. Fax: 765-677-2455. E-mail: [email protected].
Figures
Images of laboratory procedures. (A) Materials needed for the activity; (B) Moringa seed with hull removed; (C) Mortar and pestle grinding of moringa seed; (D) Ground moringa seed slurry being poured into a 15-mL centrifuge tube; (E) Slurry supernatant sterilized through a membrane filter (0.2–0.45 μm); (F) Sterile disks soaked in sterile extracts; (G) Plating extract-soaked disks onto inoculated plates; (H) Disk placement on plates of either E. coli (left) or B. cereus (right); (I) Zones of inhibition apparent on plates following 24-hour incubation (37°C).

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FIGURE 1
Images of laboratory procedures. (A) Materials needed for the activity; (B) Moringa seed with hull removed; (C) Mortar and pestle grinding of moringa seed; (D) Ground moringa seed slurry being poured into a 15-mL centrifuge tube; (E) Slurry supernatant sterilized through a membrane filter (0.2–0.45 μm); (F) Sterile disks soaked in sterile extracts; (G) Plating extract-soaked disks onto inoculated plates; (H) Disk placement on plates of either E. coli (left) or B. cereus (right); (I) Zones of inhibition apparent on plates following 24-hour incubation (37°C).
Measuring zones of inhibition after 24-hour incubation. Inhibition of bacterial growth was evaluated by measuring the diameter (mm) of each zone of inhibition. Comparisons of individual antibacterial agents can be made between the gram-negative E. coli (A) and the gram-positive B. cereus (B). Disks clockwise from top left: P, penicillin; T, tetracycline; G, garlic; M, moringa.

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FIGURE 2
Measuring zones of inhibition after 24-hour incubation. Inhibition of bacterial growth was evaluated by measuring the diameter (mm) of each zone of inhibition. Comparisons of individual antibacterial agents can be made between the gram-negative E. coli (A) and the gram-positive B. cereus (B). Disks clockwise from top left: P, penicillin; T, tetracycline; G, garlic; M, moringa.
Average improvement of student performance on the pre- and post-lab activity quiz. Significant gains in student understanding were evident for questions 3, 4, and 5. The questions presented to the students are given in Table 1 and in Appendices 1 and 2 . Data were compiled from the responses of 29 students. Statistical differences were determined by converting the pre- and post-lab quiz scores to percentages, which were then normalized for the statistical analysis. In this form, the chi-squared test could be applied using the CHITEST function of Excel. The resulting p value calculated for these 29 sets of pre- and post-lab scores was 4 × 10−38 (<0.05).

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FIGURE 3
Average improvement of student performance on the pre- and post-lab activity quiz. Significant gains in student understanding were evident for questions 3, 4, and 5. The questions presented to the students are given in Table 1 and in Appendices 1 and 2 . Data were compiled from the responses of 29 students. Statistical differences were determined by converting the pre- and post-lab quiz scores to percentages, which were then normalized for the statistical analysis. In this form, the chi-squared test could be applied using the CHITEST function of Excel. The resulting p value calculated for these 29 sets of pre- and post-lab scores was 4 × 10−38 (<0.05).
Student evaluation of the laboratory activity. The survey results showed that almost 90% of the students strongly agreed or agreed that they enjoyed the activity (question 1), the instructions were easy to follow (question 2), and they were interested in the results (question 3). Nearly 94% of the students strongly agreed or agreed that the techniques were relevant and useful (question 4). Perhaps most importantly, about 85% of the students agreed (nearly one-half strongly agreeing) that the exercise highlights the value of medicinal plants as potential producers of antibacterial agents (question 5), which was identified as a specific goal of the activity (Objective 6). The questions as presented to the students are given in Appendix 5 .

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FIGURE 4
Student evaluation of the laboratory activity. The survey results showed that almost 90% of the students strongly agreed or agreed that they enjoyed the activity (question 1), the instructions were easy to follow (question 2), and they were interested in the results (question 3). Nearly 94% of the students strongly agreed or agreed that the techniques were relevant and useful (question 4). Perhaps most importantly, about 85% of the students agreed (nearly one-half strongly agreeing) that the exercise highlights the value of medicinal plants as potential producers of antibacterial agents (question 5), which was identified as a specific goal of the activity (Objective 6). The questions as presented to the students are given in Appendix 5 .