• 11 Petri dishes
• Marker
• Escherichia coli bacteria culture
• Essential oils: orange oil, thyme oil, lemongrass oil
• Plant extracts: aloe vera extract, ginger extract
• Filtered paper disks
• Sterile forceps
• Alcohol
• Bunsen burner
• Designated tray
• Incubator
• Ruler

Procedure:

1. Labeling Petri Dishes:
   • Label all 11 Petri dishes with the date, group number, Escherichia coli bacteria, and the assigned essential oil or plant extract.
   • Divide every Petri dish into two halves with a marker, except for one. This Petri dish will be used as a control and contain only the inoculated bacteria and one paper disk containing one drop of water. Each oil and plant extract, except for the water control, should have two Petri dishes assigned to them.
2. Inoculation:
   • Inoculate all Petri dishes with the Escherichia coli bacteria.
3. Drying:
   • Let all the Petri dishes dry for one minute.
4. Sterilization:
   • Sterilize forceps by dipping them into alcohol, then into the flame of the Bunsen burner.
5. Preparing Paper Disks:
   • With the sterilized forceps, pick up a filtered paper disk and place it into the designated tray.
6. Re-sterilization:
   • Re-sterilize forceps.
7. Adding Oil or Plant Extract:
   • Add one drop of the assigned oil or plant extract onto the paper disk.
8. Placement of Paper Disc:
   • Using the forceps, place the paper disk onto one of the halves of its designated Petri dish.
9. Re-sterilization:
   • Re-sterilize forceps.
10. Repeating for Each Petri Dish:
    • Repeat steps 5 through 9 until every Petri dish has two paper disks on either side containing their assigned oil or plant extract.
    • After placing two paper disks on either side of a Petri dish, let it sit for at least 30 seconds, then put the lid back on.
11. Incubation:
    • After all Petri dishes are prepared, place them on the designated cart for transfer to the incubator.
    • Incubate Petri dishes for 48 hours.

Part 2: Measurement and Recording:

1. Measurement:
   • Measure and record the diameter of clear zones around each paper disk in millimeters (mm).
2. Interpretation:
   • If the clear zone is larger than the paper disk and there is no visible bacterial growth on the plate, mark it as “complete inhibition”.
   • If there is no clear zone, but bacterial growth is observed, indicate as “no inhibition”.
   • Compare your results with other groups who used different bacteria.

Ensure all steps are conducted under sterile conditions and proper safety measures are followed throughout the experiment.

Lab Abstract

Purpose: We are a part of UNM’s ECURE project to enrich our undergraduate education in STEM. In our Microbiology for Health Sciences course, we engaged in a research to evaluate the antibacterial properties of the essential oils (EOs) and plant extracts used in traditional medicines.

Significance: Antibiotic resistance has been a major public health concern worldwide. New measures to prevent the development of antibiotic resistance are becoming inevitable. One such approach to counter the antibiotic resistance has been the search of biologically active pharmacophores possessing novel modes of action from roots, barks, leaves, flowers, seeds and fruits.

Research Question: Numerous studies have discovered promising novel antimicrobial candidates from plant-derived EOs. EOs are particularly interesting as some oils have been used by native groups for curative purposes in the past. In our research, we have evaluated whether the selected commercial EOs inhibit the growth of Gram-positive and -negative bacterial strains.

Methods: The inhibitory effect of three commercial EOs – orange oil, thyme oil, lemongrass oil against four bacterial strains Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis was tested using a disc diffusion method.

Results: Of the three tested EOs, thyme oil and lemongrass oil showed inhibitory effect against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis.

Conclusion: Natural products have been investigated and utilized to alleviate disease since early human history. Our findings may provide additional knowledge to the antimicrobial research and alternative therapy to tackle the antibiotic resistance.

Photo Results

Report Instructions

1. Materials and Methods (4 points):
   1. Bacterial strains used in your group – describe the bacteria used in the experiment, name and description of the bacteria (Gram + or – , where did we get the bacteria (this is our lab culture), etc.). Follow your Group information. – one paragraph
   2. List of essential oils (EOs) used in your experiment – list the name of EOs and information about the EOs (you have to do literature search to learn about each EO). – one paragraph
   3. Methods – describe how did your group do the experiment (follow your Group experiment method) – one paragraph

2. Results (3 points):
   1. Describe your group’s results – one paragraph
   2. Depends on your experiment, include your group experiment result picture with appropriate captions or/and include your group Table with appropriate title.

3. Conclusions (2 points): Interpret your group’s experiment results and give conclusions about your project – how your experiment results are significant to tackle antibiotic resistance, to find natural antimicrobials, and to the drug discovery. In this section, relate the results with our project objectives, antibiotic resistance issue, natural antimicrobials that you described in your previous “Literature Survey” report

– one paragraph

4. References (1 point): references should be done in appropriate apa format

Submit your report in Times New Roman, 12 font, 1″ margins, single line space, left aligned, Word file or PDF file