Plant Substances as Potential Medicines - Lab 6d:
Purpose:
The purpose of this lab is to figure out whether some of the local plants in our field study areas have any active antimicrobial ingredients. In this lab we get to on our own and using our own skill test actual plants that we are familiar with. This is a cool way to connect what we are learning to our life.
Materials:
-Balance, weigh boat, lab scoops
-LB broth -Media bottles, 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, 60 x 15 mm, sterile -E. coli JM109 (stock plate) -Plant specimen -Mortar and pestle -Pipet, 10 mL and pump -Plastic funnels, short stemmed -Filter paper disks, 5 mm diameter -Beakers, 100 mL |
-Syringe, 10 mL and filter, 0.2 µm
-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:
Part II:
4.
-Grind 2 g of plant material and 10 mL of water using a mortar and pestle (This breaks all the plant cells open)
-Let the mixture sit for 3 minutes.
-Filter the mixture through a paper lined funnel. (Gravity pushes mixture through funnel)
-Using a syringe, push the mixture through a sterilize filter. Then put 1 mL of this into a microtube.
*keeping the mixture clean and sterilized is important because we are testing to see if bacteria develops or not with this plant, so we do not want any other bacteria infecting and contaminating our data*
-Repeat above steps but substituting 10 mL of methanol (MeOH) for the 10 mL of water (H2O). (Use gloves and safety goggles when dealing with the MeOH because it is metabolized into formic acid which is poisonous to the nervous system and can cause blindness, coma, and death.)
-Put methanol mixture in heat block with the lid open to evaporate the alcohol (a known antibacterial and would change our data).
-Add 1 mL of sterile water after the mixture goes through the heat block and vortex. This makes sure all molecules that were stuck to the sides after the evaporation process are incorporated into the mixture.
-Using flame sterilized forceps, put 2 discs into each tube to soak in the mixture.
-Again using flame sterilized forceps, add 1 disc to the microtube with the negative control (H2O).
-Again with the flame sterilized forceps, add 1 disc to the microtube with the positive control (ampicillin).
-Grind 2 g of plant material and 10 mL of water using a mortar and pestle (This breaks all the plant cells open)
-Let the mixture sit for 3 minutes.
-Filter the mixture through a paper lined funnel. (Gravity pushes mixture through funnel)
-Using a syringe, push the mixture through a sterilize filter. Then put 1 mL of this into a microtube.
*keeping the mixture clean and sterilized is important because we are testing to see if bacteria develops or not with this plant, so we do not want any other bacteria infecting and contaminating our data*
-Repeat above steps but substituting 10 mL of methanol (MeOH) for the 10 mL of water (H2O). (Use gloves and safety goggles when dealing with the MeOH because it is metabolized into formic acid which is poisonous to the nervous system and can cause blindness, coma, and death.)
-Put methanol mixture in heat block with the lid open to evaporate the alcohol (a known antibacterial and would change our data).
-Add 1 mL of sterile water after the mixture goes through the heat block and vortex. This makes sure all molecules that were stuck to the sides after the evaporation process are incorporated into the mixture.
-Using flame sterilized forceps, put 2 discs into each tube to soak in the mixture.
-Again using flame sterilized forceps, add 1 disc to the microtube with the negative control (H2O).
-Again with the flame sterilized forceps, add 1 disc to the microtube with the positive control (ampicillin).
Part III:
-Using a sterile pipet, move 1 mL of the E. coli broth to the center of each Petri dish. Flame sterilize a spreading loop and spread the bacterial culture around the dish. Cover for 15 minutes, allowing the culture to soak into the agar.
-Using the flame sterilized forceps, place a disc into the center of each quadrant of the Petri dish, about 2 cm away from outer edge. Keep separate the water and methanol samples in different dishes.
-Place a negative control disc in one the middle of each plate and a positive control disc in one quadrant of each plate.
-Record the quadrant of each sample.
-Give everything a few minutes to soak in.
-If discs are adhering properly to the dishes then invert the plates and incubate at 37˚C for 24 - 48 hours.
-Observe plates after incubation for any signs of inhibition. This will be a clear area formed around the disc from the antimicrobial substance in the disc if present.
-Using the flame sterilized forceps, place a disc into the center of each quadrant of the Petri dish, about 2 cm away from outer edge. Keep separate the water and methanol samples in different dishes.
-Place a negative control disc in one the middle of each plate and a positive control disc in one quadrant of each plate.
-Record the quadrant of each sample.
-Give everything a few minutes to soak in.
-If discs are adhering properly to the dishes then invert the plates and incubate at 37˚C for 24 - 48 hours.
-Observe plates after incubation for any signs of inhibition. This will be a clear area formed around the disc from the antimicrobial substance in the disc if present.
Results:
Plant samples:
-Methanol: weak positive
-uneven ring up to 4 mm around filter
-Water: positive
-uneven ring up to 3 mm around filter
Controls:
-Positive (Ampicillin): Successful
-Negative (Water): Successful
-Methanol: weak positive
-uneven ring up to 4 mm around filter
-Water: positive
-uneven ring up to 3 mm around filter
Controls:
-Positive (Ampicillin): Successful
-Negative (Water): Successful
Data Analysis/Conclusion:
Overall, I believe my experiment was successful. All of my extracts gave me positive results. My controls were successful as well. However, some errors could have occurred that affected the data. For example, when placing the filters on the agar and E. coli too much liquid on the filters could have diluted or washed away the bacteria around the filter making it seem like a weak positive.
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In the future, to decrease mixed results I would place each filter on it's own Petri dish and with it's own control filters. After that I would give more time to let the bacteria grow in the dish to give a more obvious result to tell if it is a positive or negative.
With the results I have now from this lab, I would first repeat this lab to double check my results; then I would create a lab to separate the antimicrobial ingredients in the plant and be able to test that ingredient further. |
Thinking Like a Biotechnician Discussion Questions:
If an extract gives a negative result in the antimicrobial assay, does that mean that the extract is not an antimicrobial agent?
Not always. If your end result is negative the extract could still be positive. The assay could have been misinterpreted or the dish could have been contaminated or some other experimental error.
Not always. If your end result is negative the extract could still be positive. The assay could have been misinterpreted or the dish could have been contaminated or some other experimental error.
In preparing the sample disks, some of the methanol extractions smell like alcohol. Why is that a problem?
Methanol is made of alcohol; which is a known antimicrobial. When testing to see if your sample has antimicrobial properties you do not want a different antimicrobial with the sample influencing the data. If your discs smell like alcohol then you did not burn off all the alcohol in the heat block and your data will be false.
Methanol is made of alcohol; which is a known antimicrobial. When testing to see if your sample has antimicrobial properties you do not want a different antimicrobial with the sample influencing the data. If your discs smell like alcohol then you did not burn off all the alcohol in the heat block and your data will be false.
Each extract may have one or more compounds in it. What should be done to begin to identify the exact compound in an extract that is causing that antimicrobial action?
To further identify the antimicrobial compounds in the plant you could use chromatography. This would separate all the compounds for easy identification.
To further identify the antimicrobial compounds in the plant you could use chromatography. This would separate all the compounds for easy identification.