Testing Plant Substances as Potential Medicines
Purpose: In this lab, we took extracts from plant samples at our sites to distinguish if they had medicinal qualities. By plating the extracts on a petri dish with E. coli, we observed if the plant was able to kill off bacteria. Two extracts were used -- one with methanol and the other with water -- in order to account for both polar and non-polar molecules. This type of research has become extremely important to biotech companies and doctors. Scientists can now look back on medicinal plants, discover the key components that make them helpful to ailing patients, and isolate those genes; they can then be transformed into vaccines or forms of medicine that help millions of people.
Materials:
- balance, weight boat, lab scoops - LB broth base - media bottles, 250 mL - sterilizer/autoclave - water bath, 37 degrees Celsius - sterile LB agar - laminar flow hood and disinfectant - plastic safety glasses - Bunsen burner and gas lighter - inoculating loop, Ni/Cr wire |
- petri dishes, 60 x 15 mm, sterile - E. coli JM109 (stock plate) - plant specimen - mortar and pestle - pipet, 10 mL and pump - short-stemmed plastic funnels - filter paper disks, 5 mm diameter - Beakers, 100 mL - syringe, 10 mL and filter, 0.2 microliters - reaction tubes and rack, 1.7 mL |
- absolute methanol - pipet, 1 mL and pump - dry block heater/heat block - fine-tipped forceps - ampicillin - glass spreader - incubator oven, 37 degrees Celsius - forceps - alcohol - clamp |
Procedure:
Part I. 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. Repeat the step, 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 tube with 1 mL of deionized water. Use sterile forceps to drop two filter paper disks into each tube of filtered extract. Prepare a negative control disk of only methanol and only sterile distilled water. Prepare positive control disk of ampicillin solution. Allow the disks sufficient time to soak up enough extract to be saturated. Close tubes. |
Part II. Add 1 mL of filter sterilized water to evaporated methanol extract. Vortex. Retrieve water extract from rack. Acquire two sterilized tubes, one with 0.5 mL of ampicillin, and the other with 0.5 mL of filter sterilized water. These are the controls of the experiment, the amp being negative because it will kill all the bacteria, and the water being positive, because bacteria will grow. Using sterilized forceps, drop 2 discs in each extract tube, and one disc in both the control and negative solutions. Part III. Use a sterile 1 mL pipet to transfer 1 mL of the E. Coli bacteria culture onto a petri dish, which should be divided into four quadrants. Spread the culture with a glass, flame-sterilized spreader. Cover and let it soak. Then, use sterile forceps to place discs on each plate. Incubate at 47 degrees Celsius for 24 hours. Then observe bacteria activity on the culture. |
Results: The plant sample produced negative results. The methanol disc had larger rings around it (1.0 cm), which seemed to have killed off some bacteria. However, there are still signs of bacterial activity around the inner rims, which suggests that the disc did not kill off any bacteria but instead pushed the bacteria farther out because it was still wet when we applied it to the agar. The plant sample that had been doused in water clearly provided negative results. These disks were only 0.5 - 0.6 cm in width, and were most likely due to water presence.
Analysis: None of the extracts gave positive results. This was to my expectation, since my plant sample was common grass. No antibacterial quality has been found in it so far, so I wasn't surprised that my experiment showed that too. This being said, there could have been experimental errors that muddled my results, including human error and contamination. There may be ways to further improve this experiment. For example, grinding up the plant material in a blender instead of hand grinding would be more effective, and we would have had more plant extract to work with. Because it was so hard to break down the cellulose of grass, I don't think that I was able to break out many important cells from my plant material; by the time I ran it through the filter, there was still a plethora of chunky plant material that I wasn't able to grind down to a mush.
TLAB Questions: If an extract gives a negative result in the antimicrobial assay, it does not necessarily mean that the extract does not have an antimicrobial agent. The extract could possibly be potent for a very specific type of bacteria not present in the culture. / In preparing some of the disks, some of the methanol extractions smelled like alcohol. This could be a significant problem because alcohol is known to disinfect and kill bacteria, and this would compromise the experiment. You wouldn't know if it was the alcohol or the extract that eliminated the bacteria. / Each compound may have one or more compounds in it. In order to identify the exact compound in an extract that is causing microbial action, scientists can use chromatography. By utilizing this method, scientists can filter and separate compounds through "thin chromatography," where individual molecules are separated based on polarity.