Testing Plant Substances as Potential Medicines
Background- Plants are always battling for survival, and because of this, they can have defence systems to battle foreign invaders. Humans can extract these defences (antimicrobial agents) to use in their own medicines.
Objective/Purpose- What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials List-
- Balance, weigh boat, lab scoops
- LB broth base
- Media bottles, 250 mL
- Sterilizer/ autoclaves
- Water bath, 37*C, shaking
- Sterile LB agar
- Laminar flow hood and disinfectant
- Plastic safety glasses
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15mm, sterile
- E. coli JM109 (stock plate)
- Plant specimen
- Mortar and pestle
- Pipet, 10 mL and pump
- Plastic funnels, short-stemmed
- Filter paper disks, 5mm diameter
- 100 mL beakers
- Syringe, 10 mL and filter, 0.2 micrometers
- Reaction tubes and rack, 1.7 mL
- Methanol, absolute
- Pipet, 1 mL and pump
- Dry block heater/heat block
- Forceps, fine-tipped
- Ampicillin
- Glass spreader
- Incubator oven, 37*C
Procedure-
- 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 de-ionized 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 pre-filter 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 de-ionized 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 upsidown.
- Examine each quadrant and the controls for areas of inhibition. Photograph or draw your results.
- Depending on your results, make qualitative and quantitative observations and record.
Results-
No microbial action was shown. Our extracts had a small ring around them, but we concluded that water wetness caused a fake no bacteria ring around a smaller ring of bacteria. To be classified as microbial action, there must be an obvious centimeter of clearance around the extracts, and we had maybe 0.5 cm in clearance. Here are the results:
Day 1 of 37*C:
Day 2:
Analysis:
There was a clearing around our positive control. We believe that we had a complete field of bacteria, but we aren’t positive. If we didn’t have a complete lawn of bacteria, the disk may not have absorbed enough of the ampicillin solution. The bacteria did grow around the negative control disks. Water is not expected to have a negative control disk or antimicrobial activity because water doesn’t kill bacteria, which means there is not any clearance around the paper. There was a very little clearance around our methanol plant extracts, and none that we could tell around the water plant extracts. Something that could have affected our results was a chance of contamination because of the bacteria on the desks or on our hands. Our plant could have antimicrobial activity, but we don’t know because we haven’t tested it. According to our results, lemon leaves don’t have any antimicrobial activity, because there was no or very little clearance around the extracts.
There was a clearing around our positive control. We believe that we had a complete field of bacteria, but we aren’t positive. If we didn’t have a complete lawn of bacteria, the disk may not have absorbed enough of the ampicillin solution. The bacteria did grow around the negative control disks. Water is not expected to have a negative control disk or antimicrobial activity because water doesn’t kill bacteria, which means there is not any clearance around the paper. There was a very little clearance around our methanol plant extracts, and none that we could tell around the water plant extracts. Something that could have affected our results was a chance of contamination because of the bacteria on the desks or on our hands. Our plant could have antimicrobial activity, but we don’t know because we haven’t tested it. According to our results, lemon leaves don’t have any antimicrobial activity, because there was no or very little clearance around the extracts.