Background
After watching many videos about finding rare plant medicines in the rainforest, we decided to see if we could find some of our own.
Materials
Plant leaves as source materials
Bunsen Burner and matches for sterilization
Alcohol for sterilization
Sterile Agar plate
Safety goggles and gloves
Mortar and pestle
Filter paper disks
Plastic funnels
10 mL syringe
Reaction tubes and rack
Fine tipped
Methanol
Ampicillin
Glass spreader
Procedures
Results
We did not observe a clearance around our plant, so our plant did not hold an e-coli medicine.
Analysis/Confusion... I mean Conclusion
We watched the bacteria grow very slowly, and when it did grow it showed minimal results. This means that our pllant did not hold an e-coli medicine, but it may have possibly held a medicine for a different illness or bacteria.
After watching many videos about finding rare plant medicines in the rainforest, we decided to see if we could find some of our own.
Materials
Plant leaves as source materials
Bunsen Burner and matches for sterilization
Alcohol for sterilization
Sterile Agar plate
Safety goggles and gloves
Mortar and pestle
Filter paper disks
Plastic funnels
10 mL syringe
Reaction tubes and rack
Fine tipped
Methanol
Ampicillin
Glass spreader
Procedures
- Grind up 2 grams of plant tissue from leaves or bark with your mortar and pestle with 10 mL of de-ionized water. Rest for 3 minutes. Then filter the sample through the 11 cm filter paper funnel. With a syringe filter, sterilize the filtered sample extract. Collect 1 mL of the extract into a 1.7 mL microbe, and be sure to label it. Do step 4 again, but replaced the deionized water with methanol. After you extract the methanol, place the 1.7 mL tube with the extract in a 65*C heat block with caps open for 24+ hours to evaporate the methanol.
- Sterilization: Attach prefilter to syringe and rinse with water. Take to Laminar Hood (plant extract syringe/pre-filter, pipet). Label microfuge tube. (initials, W or M). Attach sterile filter to pre-filter. Load 1.7 ml of extract into syringe using pipet. Depress plunger- at least 1 ml. Snap on cap without touching inside. For the rest of your samples, do steps 4 and 5 again, making sure to label all samples. There should be 2 tubes in total.
- Evaporate methanol from methanol extract by placing tube, with cap open, on a 65 C heat block overnight.
- Reconstitute methanol extract with 1.0 ml sterile deionized water. With sterilized forceps that have been flamed in alcohol, drop 3 filter paper disks into every tube of filtered extract.
- Make the negative control disks: three each of only the methanol and only the distilled water. Make 2 positive control disks of the ampicillin solution.
- Let the disks soak up enough extract to be saturated. This may have to happen overnight.
- Sterile disks were added to microfuge tubes containing 1 mL sterile water and 1 mL ampicillin.
- 10-20 mL of warmed nutrient agar was poured into 2 petri dishes using sterile technique. Close all tubes, and store the samples at 4*C until time to use.
- With a sterile pipet, transfer 1 mL of the prepared E. coli broth to the middle of the Petri dish. Then get a glass spreader, sterilize it using alcohol and flame, and spread the broth evenly across the dish. Cover and allow the culture to soak into the agar for at least 15 minutes.
- With sterile forceps (alcohol and flame), place one disk into the middle of each quadrant (which you should have drawn in). Blot out the extra liquid on the disks before you place them on the 2 Petri dishes. Keep the methanol-extracted samples in one dish and the water in the other.
- Put one of the negative control disks (with methanol or distilled water) in the marked area. Do the same with the positive ampicillin soaked one. Incubate the petri dish at 37*C overnight upside down. Examine each quadrant and the controls for areas of inhibition. Photograph and draw the results.
Results
We did not observe a clearance around our plant, so our plant did not hold an e-coli medicine.
Analysis/Confusion... I mean Conclusion
We watched the bacteria grow very slowly, and when it did grow it showed minimal results. This means that our pllant did not hold an e-coli medicine, but it may have possibly held a medicine for a different illness or bacteria.