Testing Plant Substances as Potential Medicines
Purpose
- To discover what local plant materials contain active ingredients that will inhibit the growth of bacteria.
Materials
- Balance, weight boat, lab scoops - Inoculating loop, Ni/Cr wire - Syringe 10mL and filter
- LB broth base - 60X15mm sterile petri dishes - 1.7mL reaction tubes and
- 250mL media bottles - Stock plate rack
- Sterilizer/autoclave - Plant specimen - Absolute methanol
- 37°C water bath, shaking - Mortar and pestle - 1mL pipet and pump
- Sterile LB agar - 10mL and pump pipet - Fine-tipped forceps
- Laminar flow hood and dis disinfectant - Short-stemmed plastic funnels - Ampicillin
- Safety goggles - 5mm diameter filter paper disks - Glass spreader
- Bunsen burner/Gas lighter - 100mL beakers - Incubator oven 37°C
- LB broth base - 60X15mm sterile petri dishes - 1.7mL reaction tubes and
- 250mL media bottles - Stock plate rack
- Sterilizer/autoclave - Plant specimen - Absolute methanol
- 37°C water bath, shaking - Mortar and pestle - 1mL pipet and pump
- Sterile LB agar - 10mL and pump pipet - Fine-tipped forceps
- Laminar flow hood and dis disinfectant - Short-stemmed plastic funnels - Ampicillin
- Safety goggles - 5mm diameter filter paper disks - Glass spreader
- Bunsen burner/Gas lighter - 100mL beakers - Incubator oven 37°C
Procedure
Part II Preparing Plant Extracts
1). Grind up 2g of plant substances using a mortar and pestle with 10mL of deionized water. Let it rest for approximately 3 minuets. With a 11-cm paper funnel, filter the plant sample through. Using a syringe filter, filter sterilize the filtered sample extract. Collect 1mL of extract into a 1.7-mL microtube. Label sample
2). Repeat process but use metanol instead of deionized water. After this step, place 1.7-mL tube with 1mL of methanol extract in a 65°C heat block with caps open for a full day or more to evaporate the methanol. Vortex dry matter in the tube with 1mL of deionized water
3). Take forceps, dip them in alcohol, then run them through the flame so that they are sterilized. Put 2 disks in each tube.
4). Prepare negative control disk
5). Prepare positive control disk of ampicillin solution.
6). Close tubes and let them sit overnight at 4°C
Part III Setting up Antimicrobial Plant Extract
6). Divide bottom of petri dish into 4 quadrants: 2 labeled MeOH and 2 labeled H2O. Also into two of the corners of the intersection labeled a + for the positive control and a - for the negative control.
7). Use sterile 1mL pipet and transfer 1mL of E. coli culture to petri dish
8). With the glass spreader, sterilize it (same process as the forceps) and place one disk into the middle of each quadrant (2 cm from outer edge of petri dish). Be sure to kepp all methanol-extracted samples on the same dish and all water-extracted samples on the same dish.
9). Repeat step 8
10). Take one negative control disks (water or MeOH) in the center of the appropriate quadrant. Take positive control disk with ampicillin in another quadrant
11). There should be a negative control in the middle, a positive control, and 3 sample disks. Take note on which plant extracts with which solvent went into each quadrant.
12). Make sure the disks are adhering well to the surface of the agar. Invert the plates and incubate at 37*C for 1-2 days. Examine the plates with the plant extract disks for zones of inhibition
13). Collect data for positive and negative results
Part II Preparing Plant Extracts
1). Grind up 2g of plant substances using a mortar and pestle with 10mL of deionized water. Let it rest for approximately 3 minuets. With a 11-cm paper funnel, filter the plant sample through. Using a syringe filter, filter sterilize the filtered sample extract. Collect 1mL of extract into a 1.7-mL microtube. Label sample
2). Repeat process but use metanol instead of deionized water. After this step, place 1.7-mL tube with 1mL of methanol extract in a 65°C heat block with caps open for a full day or more to evaporate the methanol. Vortex dry matter in the tube with 1mL of deionized water
3). Take forceps, dip them in alcohol, then run them through the flame so that they are sterilized. Put 2 disks in each tube.
4). Prepare negative control disk
5). Prepare positive control disk of ampicillin solution.
6). Close tubes and let them sit overnight at 4°C
Part III Setting up Antimicrobial Plant Extract
6). Divide bottom of petri dish into 4 quadrants: 2 labeled MeOH and 2 labeled H2O. Also into two of the corners of the intersection labeled a + for the positive control and a - for the negative control.
7). Use sterile 1mL pipet and transfer 1mL of E. coli culture to petri dish
8). With the glass spreader, sterilize it (same process as the forceps) and place one disk into the middle of each quadrant (2 cm from outer edge of petri dish). Be sure to kepp all methanol-extracted samples on the same dish and all water-extracted samples on the same dish.
9). Repeat step 8
10). Take one negative control disks (water or MeOH) in the center of the appropriate quadrant. Take positive control disk with ampicillin in another quadrant
11). There should be a negative control in the middle, a positive control, and 3 sample disks. Take note on which plant extracts with which solvent went into each quadrant.
12). Make sure the disks are adhering well to the surface of the agar. Invert the plates and incubate at 37*C for 1-2 days. Examine the plates with the plant extract disks for zones of inhibition
13). Collect data for positive and negative results
Data Analysis and Conclusion
1). I did not receive any strong positive results. My positive control was close to having a positive result, however there were still some bacteria surrounding it.
2). My controls did not work completely as expected. My negative control did show as negative, but my positive control looked more negative than positive. In terms of water and methanol tests, both water samples and methanol samples showed negative. So it was a good sign to see each sample share symmetrical results.
3). Some errors that could have gave false results were cross contamination of substances, such as using the same forceps with two different test tubes. Another possible error could have been that my parent and I used different a plant for the water and methanol tests. Lastly, there could have been a chance of placing the wrong disk in the wrong quadrant of the petri dish.
4). If I were to repeat this experiment some improvements I would hope to accomplish are collecting enough of the same plant to ensure more accurate results, enhance my precision with placing the disks into the tubes, and really focus on transferring the different substances into the right places.
5). Because I didn't receive any strong positive results, my next steps would be to repeat the experiment.
1). I did not receive any strong positive results. My positive control was close to having a positive result, however there were still some bacteria surrounding it.
2). My controls did not work completely as expected. My negative control did show as negative, but my positive control looked more negative than positive. In terms of water and methanol tests, both water samples and methanol samples showed negative. So it was a good sign to see each sample share symmetrical results.
3). Some errors that could have gave false results were cross contamination of substances, such as using the same forceps with two different test tubes. Another possible error could have been that my parent and I used different a plant for the water and methanol tests. Lastly, there could have been a chance of placing the wrong disk in the wrong quadrant of the petri dish.
4). If I were to repeat this experiment some improvements I would hope to accomplish are collecting enough of the same plant to ensure more accurate results, enhance my precision with placing the disks into the tubes, and really focus on transferring the different substances into the right places.
5). Because I didn't receive any strong positive results, my next steps would be to repeat the experiment.
Thinking Like a Biotechnician
1). If an extract gives a negative result in the antimicrobial assay, that means that the extract is not an antimicrobial agent because the bacterial will continue to grow.
2). It is a problem that some of the methanol extractions smell like alcohol because alcohol is a known substance that kills bacteria, and with a test like this where you are testing bacteria, it may lead to lack of results.
3). To begin identifying the exact compound in an extract that is causing the antimicrobial action, you start with extraction, then isolation and characterization, followed by chromotography. Chromotography allows you to figure out how many components are in a mixture, as well as to support the identity of a compound in a mixture.
1). If an extract gives a negative result in the antimicrobial assay, that means that the extract is not an antimicrobial agent because the bacterial will continue to grow.
2). It is a problem that some of the methanol extractions smell like alcohol because alcohol is a known substance that kills bacteria, and with a test like this where you are testing bacteria, it may lead to lack of results.
3). To begin identifying the exact compound in an extract that is causing the antimicrobial action, you start with extraction, then isolation and characterization, followed by chromotography. Chromotography allows you to figure out how many components are in a mixture, as well as to support the identity of a compound in a mixture.