Testing Plant Substances as Potential Medicines
Purpose:
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials:
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:
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 plant.
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 results were negative.
Data Analysis/Conclusion:
All results were negative. None of my extracts showed any anti microbial activity. Our experiment yielded many opportunities for error, such as the possibilities of mixed up plants and contamination during the filtering process.
1. If an extract comes out negative then it could mean that that extract could not work with that type of E. Coli.
2. Alcohol kills bacteria which would defeat the purpose of growing bacteria.
3. You can use chromatography to separate the different compounds out of the extract.
Purpose:
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials:
- Balance, weight boat, lab scoops
- LB broth base
- Media bottles, 250 mL
- Sterilizer/autoclave
- Water bath, 37 degrees C, shaking
- Sterile LB agar
- Laminar flow hood and disinfectant
- Glasses, safety, plastic
- Bunsen burner and gas light
- Syringe, 10 mL and filter. 0.2 um
- Piper, 1 mL and pump
- Dry dock heater/heat block
- Ampicillin
- Incubator oven, 37 degrees C
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15 mm, sterile
- E. Coli JM109 (stock plate)
- Plant specimen
- Mortar and pestle
- Pipet, 10 mL and pump
- Plastic funnels, short-stemmed
- Filter paper disks, 5 mm diameter
- Beakers, 100 mL
- Reaction tubes and rack, 1.7 mL
- Methanol, absolute
- Forceps, fine-tipped
- Glass spreader
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:
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 plant.
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 results were negative.
Data Analysis/Conclusion:
All results were negative. None of my extracts showed any anti microbial activity. Our experiment yielded many opportunities for error, such as the possibilities of mixed up plants and contamination during the filtering process.
1. If an extract comes out negative then it could mean that that extract could not work with that type of E. Coli.
2. Alcohol kills bacteria which would defeat the purpose of growing bacteria.
3. You can use chromatography to separate the different compounds out of the extract.