Testing Plant Substances as Potential Medicines:
Purpose: To find what plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria.
Materials:
Purpose: To find what plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria.
Materials:
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Procedure:
Part II
4. Grind 2 g of plant tissue (leaves) with 10 mL of deionized water in mortar and pestle, and let sit for 3 minutes. Filter in 11 cm funnel, sterilize extract with syringe filter, and collect 1 mL of extract in labelled 1.7 mL microtube.
5. Repeat Step 4, except replace deionized water with methanol. Place 1.7 mL tube with 1 mL of methanol extract in 65*C heat block (caps open) for 24 hours to evaporate methanol. Reconstitute dry matter in microtube with 1 mL of deionized water.
6. Repeat Step 4 and 5 for six samples and label them.
7. Drop filter paper disks in each filtered extract tube using sterile forceps (sterilized by being flamed in alcohol).
8. Prepare three negative control disks of only methanol and sterile and distilled water.
9. Prepare six positive control disks of ampicillin solution.
10. Allow disks to be saturated with the extract (perhaps overnight).
11. Close tubes. Store all samples at 4*C until ready to use.
Part III
12. Transfer 1 mL of the E. Coli broth to middle of Petri dish with sterile pipet. Sterilize spreading loop with alcohol and flame and spread bacteria culture in Petri dish. Cover and let culture soak in agar for at least 15 minutes.
13. Separating the methanol-extracted samples and the water-extracted samples in different dishes, place one disk on each quadrant at least 2 cm away from edge of dish. Block excess liquid before placing disks in.
14. Repeat step 13 twice. (3 methanol extraction replicates and 3 deionized water extraction replicates)
15. Place one of negative control disks in center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
16. Finish with 6 Petri plates with a negative control in the center, a positive control, and 3 sample disks. Record which solvents and plant extracts are in each quadrant. Let soak for a few minutes.
17. Ensure disks adhere to surface of agar. Invert the plates and incubate at 37*C for 24 to 48 hours.
18. After incubation, look for at the plates with plant extract disks for zones of inhibition, clear area formed by inhibitory (decrease in action) action of a substance in the plant material around the disk. Photograph the plates, labeling any inhibition of bacterial growth.
19. Create a data table for the replicates and averages. Include descriptions of the bacterial lawn around each disk. Record the diameter and clarity of any cleared areas around the disks in quantitative measurements.
Part II
4. Grind 2 g of plant tissue (leaves) with 10 mL of deionized water in mortar and pestle, and let sit for 3 minutes. Filter in 11 cm funnel, sterilize extract with syringe filter, and collect 1 mL of extract in labelled 1.7 mL microtube.
5. Repeat Step 4, except replace deionized water with methanol. Place 1.7 mL tube with 1 mL of methanol extract in 65*C heat block (caps open) for 24 hours to evaporate methanol. Reconstitute dry matter in microtube with 1 mL of deionized water.
6. Repeat Step 4 and 5 for six samples and label them.
7. Drop filter paper disks in each filtered extract tube using sterile forceps (sterilized by being flamed in alcohol).
8. Prepare three negative control disks of only methanol and sterile and distilled water.
9. Prepare six positive control disks of ampicillin solution.
10. Allow disks to be saturated with the extract (perhaps overnight).
11. Close tubes. Store all samples at 4*C until ready to use.
Part III
12. Transfer 1 mL of the E. Coli broth to middle of Petri dish with sterile pipet. Sterilize spreading loop with alcohol and flame and spread bacteria culture in Petri dish. Cover and let culture soak in agar for at least 15 minutes.
13. Separating the methanol-extracted samples and the water-extracted samples in different dishes, place one disk on each quadrant at least 2 cm away from edge of dish. Block excess liquid before placing disks in.
14. Repeat step 13 twice. (3 methanol extraction replicates and 3 deionized water extraction replicates)
15. Place one of negative control disks in center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
16. Finish with 6 Petri plates with a negative control in the center, a positive control, and 3 sample disks. Record which solvents and plant extracts are in each quadrant. Let soak for a few minutes.
17. Ensure disks adhere to surface of agar. Invert the plates and incubate at 37*C for 24 to 48 hours.
18. After incubation, look for at the plates with plant extract disks for zones of inhibition, clear area formed by inhibitory (decrease in action) action of a substance in the plant material around the disk. Photograph the plates, labeling any inhibition of bacterial growth.
19. Create a data table for the replicates and averages. Include descriptions of the bacterial lawn around each disk. Record the diameter and clarity of any cleared areas around the disks in quantitative measurements.
Results:
Qualitative:
MeOH (Methanol) Extract - both positive - there is a clear ring around the filter, so the bacteria isn't growing right off from the filter.
H2O (Water) Extract - both positive - the filter is only surrounded by a clear ring while the bacteria grows outside of the clear ring.
+ Control - positive - very wide clear ring surrounds positive control filter in the agar plate growing bacteria.
- Control - negative - bacteria ring grows directly touching the filter. Then there is a thin clear ring around this bacteria. Finally, the clear ring is surrounded by bacteria.
Quantitative:
MeOH (Methanol) Extract - both positive - The clear ring around the filter is 0.25 mm thick.
H2O (Water) Extract - both positive - The clear ring around the filter is 0.4 mm thick.
+ control - positive - The clear ring around the filter is 0.5 mm thick.
- control - negative - The bacteria ring directly touching the filter is 0.2 mm thick. The clear ring around the filter is 0.2 mm thick.
Qualitative:
MeOH (Methanol) Extract - both positive - there is a clear ring around the filter, so the bacteria isn't growing right off from the filter.
H2O (Water) Extract - both positive - the filter is only surrounded by a clear ring while the bacteria grows outside of the clear ring.
+ Control - positive - very wide clear ring surrounds positive control filter in the agar plate growing bacteria.
- Control - negative - bacteria ring grows directly touching the filter. Then there is a thin clear ring around this bacteria. Finally, the clear ring is surrounded by bacteria.
Quantitative:
MeOH (Methanol) Extract - both positive - The clear ring around the filter is 0.25 mm thick.
H2O (Water) Extract - both positive - The clear ring around the filter is 0.4 mm thick.
+ control - positive - The clear ring around the filter is 0.5 mm thick.
- control - negative - The bacteria ring directly touching the filter is 0.2 mm thick. The clear ring around the filter is 0.2 mm thick.
Data Analysis and Conclusion:
1. Most of the extracts gave me a positive result. Both of the MeOH extracts and both of the H2O extracts gave me positive answers. The MeOH extracts were weaker positives than the H2O extracts.
2. My controls did work as expected. The positive control had a clear ring around it. The negative control was a weak negative because the bacteria grew touching the filter but just outside of that was a thin clear ring with no bacteria.
3. One of the errors that could have given me false results was with the H2O extract. I'm not sure how since the two filters were separated when I placed the Petri dish in the box, but the plate probably got tossed around or knocked by another Petri dish and the two filters clung together in one of the quadrants. Together, they created a positive result, but that might have been different if the filters were separate. However, we used two filters in case there was error so there is a possibility the results would have been positive anyway.
4. In the future, I would test more than two filters per extract because there are many factors that could have manipulated my results. For example, maybe the clear ring appeared because my filters were wet and the bacteria was cleared.
5. The next steps that need to be taken is to compare my results with the plant I test. What do these positive results tell me bout my plant in my site? Can this plant in my site be used in medicine and possibly provide pain relievers and antibiotics? Does this mean we can use this plant to fight bacterial diseases? We should test if this plant provides antibiotics that bacteria are not immune to, due to the evolving bacteria.
1. Most of the extracts gave me a positive result. Both of the MeOH extracts and both of the H2O extracts gave me positive answers. The MeOH extracts were weaker positives than the H2O extracts.
2. My controls did work as expected. The positive control had a clear ring around it. The negative control was a weak negative because the bacteria grew touching the filter but just outside of that was a thin clear ring with no bacteria.
3. One of the errors that could have given me false results was with the H2O extract. I'm not sure how since the two filters were separated when I placed the Petri dish in the box, but the plate probably got tossed around or knocked by another Petri dish and the two filters clung together in one of the quadrants. Together, they created a positive result, but that might have been different if the filters were separate. However, we used two filters in case there was error so there is a possibility the results would have been positive anyway.
4. In the future, I would test more than two filters per extract because there are many factors that could have manipulated my results. For example, maybe the clear ring appeared because my filters were wet and the bacteria was cleared.
5. The next steps that need to be taken is to compare my results with the plant I test. What do these positive results tell me bout my plant in my site? Can this plant in my site be used in medicine and possibly provide pain relievers and antibiotics? Does this mean we can use this plant to fight bacterial diseases? We should test if this plant provides antibiotics that bacteria are not immune to, due to the evolving bacteria.
Thinking Like a Biotechnician (TLAB) Questions:
1. If an extract gives a negative result in the antimicrobial assay, the extract is not an antimicrobial assay because the bacteria grows right off of the filter. This means it cannot fight off the bacteria. If it were a medicine, the bacteria would continue to attack.
2. In preparing the sample disks, some of the methanol extractions smell like alcohol. This is a problem because alcohol sterilizes substances, therefore it damages the experiment. The methanol extract would not be the one that is tested if it was sterilized.
3. Each extract may have one or more compounds in it. We can identify the exact compound in an extract that is causing the antimicrobial action by isolating each compound. We could use chromatography to separate each compound in order to test each compound in the extract for antimicrobial action. After isolating each compound, we would do the same experiment above to test for positive and negative results.
1. If an extract gives a negative result in the antimicrobial assay, the extract is not an antimicrobial assay because the bacteria grows right off of the filter. This means it cannot fight off the bacteria. If it were a medicine, the bacteria would continue to attack.
2. In preparing the sample disks, some of the methanol extractions smell like alcohol. This is a problem because alcohol sterilizes substances, therefore it damages the experiment. The methanol extract would not be the one that is tested if it was sterilized.
3. Each extract may have one or more compounds in it. We can identify the exact compound in an extract that is causing the antimicrobial action by isolating each compound. We could use chromatography to separate each compound in order to test each compound in the extract for antimicrobial action. After isolating each compound, we would do the same experiment above to test for positive and negative results.