Testing Plant Substances as Potential Medicines
Purpose:
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials:
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Procedure:
Part II: Preparing Plant Extracts
1. Grind up 2 grams of plant tissue (leaves or bark) with 10 mL of deionized water. Use a mortar and pestle. Let sit for 3 minutes, before filtering the sample through an 11-cm filter paper funnel. Filter sterilize the sample extract and collect 1 mL of extract into a 1.7-mL microtube.
2. Repeat, but replace the water with methanol as the extracting solvent. After methanol extraction, place the 1.7-mL tube with the 1 mL of methanol extract in a 65 degree Celsius heat block (caps open) for 24 hours or more. Reconstitute dry matter in the tube with 1 mL of deionized water.
3. Use sterile forceps to drop two filter paper disks into each tube of filtered extract.
4. Prepare a negative control disk of only methanol and only sterile distilled water.
5. Prepare six positive control disk of ampicillin solution. Allow the disks sufficient time to soak up enough extract to be saturated. (Recommended: overnight) Then, close the tubes and store all samples at 4 degrees Celsius until it is ready to use.
Part III: Setting up Antimicrobial Plant Extract Assay
1. Using sterile pipet transfer 1 mL of the E. coli to the middle of each Petri dish. Sterilize a spreading loop, and evenly spread the bacterial culture around the Petri plate. Cover quickly, and allow the culture to soak into the agar for atleast 15 minutes.
2. Use sterile forceps to carefully place one disk into the middle of each quadrant (about 2cm from the outer edge of the dish). Blot excess liquid before placing the disk on the dish. Keep all methanol- extracted sample on the same dish and same with the water-extracted samples.
3. Repeat step 2 twice so you have three replicates of the methanol and three of the water extractions.
4. Place one of the negative control disks, either sterile distilled water or methanol, in the center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
5. Make sure all the disks are adhering well to the surface of the agar. For incubation, invert the plates and incubate at 37 degrees C for 24 to 48 hours
6. After incubation, examine the plant extract disks for zones of inhibition. This is a clear area formed around the disk by the inhibitory action of a substance in the plant material. Photograph or draw the plates
Part II: Preparing Plant Extracts
1. Grind up 2 grams of plant tissue (leaves or bark) with 10 mL of deionized water. Use a mortar and pestle. Let sit for 3 minutes, before filtering the sample through an 11-cm filter paper funnel. Filter sterilize the sample extract and collect 1 mL of extract into a 1.7-mL microtube.
2. Repeat, but replace the water with methanol as the extracting solvent. After methanol extraction, place the 1.7-mL tube with the 1 mL of methanol extract in a 65 degree Celsius heat block (caps open) for 24 hours or more. Reconstitute dry matter in the tube with 1 mL of deionized water.
3. Use sterile forceps to drop two filter paper disks into each tube of filtered extract.
4. Prepare a negative control disk of only methanol and only sterile distilled water.
5. Prepare six positive control disk of ampicillin solution. Allow the disks sufficient time to soak up enough extract to be saturated. (Recommended: overnight) Then, close the tubes and store all samples at 4 degrees Celsius until it is ready to use.
Part III: Setting up Antimicrobial Plant Extract Assay
1. Using sterile pipet transfer 1 mL of the E. coli to the middle of each Petri dish. Sterilize a spreading loop, and evenly spread the bacterial culture around the Petri plate. Cover quickly, and allow the culture to soak into the agar for atleast 15 minutes.
2. Use sterile forceps to carefully place one disk into the middle of each quadrant (about 2cm from the outer edge of the dish). Blot excess liquid before placing the disk on the dish. Keep all methanol- extracted sample on the same dish and same with the water-extracted samples.
3. Repeat step 2 twice so you have three replicates of the methanol and three of the water extractions.
4. Place one of the negative control disks, either sterile distilled water or methanol, in the center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
5. Make sure all the disks are adhering well to the surface of the agar. For incubation, invert the plates and incubate at 37 degrees C for 24 to 48 hours
6. After incubation, examine the plant extract disks for zones of inhibition. This is a clear area formed around the disk by the inhibitory action of a substance in the plant material. Photograph or draw the plates
Results:
All my extracts turned out negative
All my extracts turned out negative
Data Analysis/Conclusion:
None of my plant extracts gave me positive results. All were negative and had "halo's" around the disks. Neither one of my controls worked as I expected. Many errors could have occur that would have effected my results such as contamination or error in making my extracts.
None of my plant extracts gave me positive results. All were negative and had "halo's" around the disks. Neither one of my controls worked as I expected. Many errors could have occur that would have effected my results such as contamination or error in making my extracts.
Questions:
1) If an extract gives a negative result in the antimicrobial assay, that means the extract may not work for that type of E. coli but could work for others.
2) In preparing the sample disks, some of the methanol extractions smell like alcohol. This is a problem because alcohol kills bacteria which defeats the purpose of trying to grow antimicrobial bacteria.
3) Each extract may have one or more compounds in it. To begin identifying the exact compound in an extract you would use chromatography to separate molecules.
1) If an extract gives a negative result in the antimicrobial assay, that means the extract may not work for that type of E. coli but could work for others.
2) In preparing the sample disks, some of the methanol extractions smell like alcohol. This is a problem because alcohol kills bacteria which defeats the purpose of trying to grow antimicrobial bacteria.
3) Each extract may have one or more compounds in it. To begin identifying the exact compound in an extract you would use chromatography to separate molecules.