Testing Plant Substances as Potential Medicines
Purpose: The purpose of this lab is to find what local plant materials contain active ingredients that will inhibit the growth of bacteria.
Materials: Balance, weight boat, lab scoops, LB broth base, Media bottlels 250 mL, Sterilizer/autoclave, Water bath 37*C shaking, Sterile LB agar, Laminar flow hood and disinfectant, Glasses safety plastic, 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 10mL and pump, Plastic funnels short stemmed, Filter paper disks 5 mm diameter, Beakers 100 mL, Syringe 10 mL and filter 0.2um, Reaction tubes and rack 1.7mL, Methanol absolute, Pipet 1mL and pump, Dry block heater/heat block, Forceps fine-tipped, Ampicillin, Glass spreader, Incubator oven 37*C
Procedure:
1. Get mortar and pestle and grind 2 grams of plant with 10 mL of di H20. Then let it sit for 3 minutes. After this let this paste filter through a piece of paper that is lined with a funnel. Let this filter sterilize (.2 micrometers using syringe) and put it in a 1 mL microtube.
2. Repeat step 1, but instead of distilled water use 10 mL of Methanol. It is very toxic so use gloves and goggles.Put this extract in a heat block and leave the lid open to evaporate and leave it overnight. The next day add 1 mL of sterile water and vortex.
3. Sterilize forceps by putting it in flames, then put 2 discs in each of the extract and leave it overnight for the H20 extract or 15 minutes for the Methanol extract.
4. Once again get sterilized forceps and put 2 discs in each microbe with H20 (negative control).
5. Use flame to sterilize forceps once last time and put 1 disc in microtube with ampicillin (positive control).
6. Sterilize 1 mL pipet and transfer 1 mL of E. coli culture to a petri dish. Before you do that part divide the petri dish into 4 quadrants. Then spread the E. coli into all four quadrants with a sterilized glass spreader. Then cover the petri dish and let it soak for 15 minutes.
7. Sterilize forceps and place discs on the plates in each of the four quadrants s shown on the diagram below.
8. Incubate this plate at 37 degrees Celsius for 24 hours.
9. Measure qualitative and quantitative data. Personally, my results were negative for inhibiting bacteria growth. The bacteria lawn around each disk was roughly 2mm. Around the positive disk the clearing was about 3mm, while at the negative control was about 2mm. Both of my MeOH tested negative because they both fostered bacteria growth. For the H20, it tested negative because bacteria also grew next to the disc. The reason it was negative was because bacteria grew around the disc and there was a clearing of no bacteria.
Materials: Balance, weight boat, lab scoops, LB broth base, Media bottlels 250 mL, Sterilizer/autoclave, Water bath 37*C shaking, Sterile LB agar, Laminar flow hood and disinfectant, Glasses safety plastic, 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 10mL and pump, Plastic funnels short stemmed, Filter paper disks 5 mm diameter, Beakers 100 mL, Syringe 10 mL and filter 0.2um, Reaction tubes and rack 1.7mL, Methanol absolute, Pipet 1mL and pump, Dry block heater/heat block, Forceps fine-tipped, Ampicillin, Glass spreader, Incubator oven 37*C
Procedure:
1. Get mortar and pestle and grind 2 grams of plant with 10 mL of di H20. Then let it sit for 3 minutes. After this let this paste filter through a piece of paper that is lined with a funnel. Let this filter sterilize (.2 micrometers using syringe) and put it in a 1 mL microtube.
2. Repeat step 1, but instead of distilled water use 10 mL of Methanol. It is very toxic so use gloves and goggles.Put this extract in a heat block and leave the lid open to evaporate and leave it overnight. The next day add 1 mL of sterile water and vortex.
3. Sterilize forceps by putting it in flames, then put 2 discs in each of the extract and leave it overnight for the H20 extract or 15 minutes for the Methanol extract.
4. Once again get sterilized forceps and put 2 discs in each microbe with H20 (negative control).
5. Use flame to sterilize forceps once last time and put 1 disc in microtube with ampicillin (positive control).
6. Sterilize 1 mL pipet and transfer 1 mL of E. coli culture to a petri dish. Before you do that part divide the petri dish into 4 quadrants. Then spread the E. coli into all four quadrants with a sterilized glass spreader. Then cover the petri dish and let it soak for 15 minutes.
7. Sterilize forceps and place discs on the plates in each of the four quadrants s shown on the diagram below.
8. Incubate this plate at 37 degrees Celsius for 24 hours.
9. Measure qualitative and quantitative data. Personally, my results were negative for inhibiting bacteria growth. The bacteria lawn around each disk was roughly 2mm. Around the positive disk the clearing was about 3mm, while at the negative control was about 2mm. Both of my MeOH tested negative because they both fostered bacteria growth. For the H20, it tested negative because bacteria also grew next to the disc. The reason it was negative was because bacteria grew around the disc and there was a clearing of no bacteria.
Data Analysis/Conclusion:
Results: My plant sample produced negative results. The methanol disc had larger rings around it, which most likely killed off some of the bacteria. There are still signs of bacteria activity near the inner rims, which shows that the disc did not kill off any of the bacteria, but actually pushed the bacteria farther out because it was still wet when we applied it to the agar.
Analysis/Conclusion:
None of the extracts gave positive results. No antibacterial quality was found in it, so I was not surprised that my experiment showed that. Experimental errors could have messed up my results a little bit, including human error. There may be some other ways in order to improve this experiment. For example, if we grinded up the plant material in a blender instead of hand grinding the plants would have worked better, and we would have had more plant extract to work with. Because it was so hard to break down the cellulose of the plants, I don't think that I was able to grind up many important cells from my plants.
Thinking Like a Biotech questions
1. No it doesn't mean that the extract is a microbial agent because there are many errors that could have happened. But if the lab was conducted perfectly then the extract wouldn't be a microbial agent because of bacteria grew.
2. If the methanol extractions smell too much like alcohol, then when conducting the experiment you wouldn't know if the molecules killed the bacteria, or if the alcohol did it by itself.
3. We would use some type of chromatography to extract each individual molecule to be able to identify the exact compound.
Results: My plant sample produced negative results. The methanol disc had larger rings around it, which most likely killed off some of the bacteria. There are still signs of bacteria activity near the inner rims, which shows that the disc did not kill off any of the bacteria, but actually pushed the bacteria farther out because it was still wet when we applied it to the agar.
Analysis/Conclusion:
None of the extracts gave positive results. No antibacterial quality was found in it, so I was not surprised that my experiment showed that. Experimental errors could have messed up my results a little bit, including human error. There may be some other ways in order to improve this experiment. For example, if we grinded up the plant material in a blender instead of hand grinding the plants would have worked better, and we would have had more plant extract to work with. Because it was so hard to break down the cellulose of the plants, I don't think that I was able to grind up many important cells from my plants.
Thinking Like a Biotech questions
1. No it doesn't mean that the extract is a microbial agent because there are many errors that could have happened. But if the lab was conducted perfectly then the extract wouldn't be a microbial agent because of bacteria grew.
2. If the methanol extractions smell too much like alcohol, then when conducting the experiment you wouldn't know if the molecules killed the bacteria, or if the alcohol did it by itself.
3. We would use some type of chromatography to extract each individual molecule to be able to identify the exact compound.