Testing Plant Substances as Potential Medicines
Background: In the outside world, it's a constant battle for existing resources. There are many characteristics that organisms possess that can help them survive in the constant competition for light, water, and food. Some of these characteristics would include big leaves and large root systems.
All living organisms run the risk of getting infected by blight or disease. Many organisms/species have ways of defending themselves against such dangers. Antimicrobial agents that fight microbes are extremely common. Antimicrobial agents can be used in medicines for daily use and use in the general medical field.
Isolating these agents is hard work that takes time. Samples need to be collected, and not all these samples can be found locally. The samples must be processed and scientists must determine an extraction technique that is appropriate to use. The samples have to be tested in order to see what microbes they can kill. They must also be tested to see if they could cause any harm to someone ingesting this future medicine.
In order to test this, scientists add extract-soaked filter paper disks to bacteria cultures that are spread on Petri dishes. Plant extracts that contain compounds effective against bacteria leave clear halos (which is a result of bacterial death or inhibited bacterial growth) around the soaked disks in the bacterial lawns. These extracts then must be further tested and purified and screened for ingredients that are the cause of bacterial death. These compounds can include but are not limited to: antiseptics, astringents, and toxins.
Purpose: What plant materials, found locally, contain active ingredients what will inhibit the growth of bacteria?
Materials List:
-Balance, weigh boat, lab scoops
- LB broth base
- Media Bottles, 250 ML
- Sterilizer/autoclave
- Water bath
- Sterile LB Agar
- Laminar flow hood and disinfectant
- Plastic safety glasses
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15 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 um
- 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
- Incunbator oven, 37 degrees Celsius
Procedure:
1. 2 grams of plant tissue were ground up using a mortar and pestle.
2. Using sterile forceps, 3 sterile filter disks were added to each filtrate.
3. Using sterile forceps, sterile filter disks were added to the tubes containing 1 mL sterile water and 1 mL ampicillin.
4. 10-20 mLs of warmed, pressure sterilized nutrient agar was poured into 2 petri dishes, using sterile technique.
5. After allowing the agar to solidify, plates were turned upside down and stored at 4 degrees Celsius overnight.
6. 1 mL of E. coli colony was added to each plate.
7. A flame-sterilized spreading loop was used to spread the bacteria evenly over the surface of the agar.
8. Using flame-sterilized forceps, filter disks were placed in separate quadrants onto the plate in the following sequence: 1) Water 2) Plant extracts 3) Ampicillin
9. Plates were left on the lab bench for 20 minutes to allow the bacteria and filter disks to adhere to the agar.
10. Plates were then incubated upside down, overnight in a 37 degree Celsius incubator.
11. Plates were photographed and observed, for clearings around the filter disks after 24 hours, 48 hours, and 72 hours.
12. Any bacterial or fungal contamination was noted.
Analysis/Conclusions:
Our ampicillin control for water had a massive bacterial lawn. It was very, very surprising. We are unclear as to what might have caused this occurrence.
Around the negative controls, there was a very limited area of clearance. On one of the negative controls in the first quadrant, it seems like there is no bacterial lawn at all. On the other controls, there is at least a minor amount of bacterial growth after 24, 48, and 72 hours.
Around the positive control we observed a clearing almost immediately. In fact, there was a bigger clearing around the ampicillin control than the other disks. There were contamination spots fairly close to the ampicillin control, although I don't know that this means anything significant.
The negative controls had a very limited area of clearance from our results. The area of clearance around these disks grew right up against the negative control disks. Water would not be expected to have antimicrobial activity, therefore this makes our negative controls very good for this experiment.
All living organisms run the risk of getting infected by blight or disease. Many organisms/species have ways of defending themselves against such dangers. Antimicrobial agents that fight microbes are extremely common. Antimicrobial agents can be used in medicines for daily use and use in the general medical field.
Isolating these agents is hard work that takes time. Samples need to be collected, and not all these samples can be found locally. The samples must be processed and scientists must determine an extraction technique that is appropriate to use. The samples have to be tested in order to see what microbes they can kill. They must also be tested to see if they could cause any harm to someone ingesting this future medicine.
In order to test this, scientists add extract-soaked filter paper disks to bacteria cultures that are spread on Petri dishes. Plant extracts that contain compounds effective against bacteria leave clear halos (which is a result of bacterial death or inhibited bacterial growth) around the soaked disks in the bacterial lawns. These extracts then must be further tested and purified and screened for ingredients that are the cause of bacterial death. These compounds can include but are not limited to: antiseptics, astringents, and toxins.
Purpose: What plant materials, found locally, contain active ingredients what will inhibit the growth of bacteria?
Materials List:
-Balance, weigh boat, lab scoops
- LB broth base
- Media Bottles, 250 ML
- Sterilizer/autoclave
- Water bath
- Sterile LB Agar
- Laminar flow hood and disinfectant
- Plastic safety glasses
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15 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 um
- 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
- Incunbator oven, 37 degrees Celsius
Procedure:
1. 2 grams of plant tissue were ground up using a mortar and pestle.
2. Using sterile forceps, 3 sterile filter disks were added to each filtrate.
3. Using sterile forceps, sterile filter disks were added to the tubes containing 1 mL sterile water and 1 mL ampicillin.
4. 10-20 mLs of warmed, pressure sterilized nutrient agar was poured into 2 petri dishes, using sterile technique.
5. After allowing the agar to solidify, plates were turned upside down and stored at 4 degrees Celsius overnight.
6. 1 mL of E. coli colony was added to each plate.
7. A flame-sterilized spreading loop was used to spread the bacteria evenly over the surface of the agar.
8. Using flame-sterilized forceps, filter disks were placed in separate quadrants onto the plate in the following sequence: 1) Water 2) Plant extracts 3) Ampicillin
9. Plates were left on the lab bench for 20 minutes to allow the bacteria and filter disks to adhere to the agar.
10. Plates were then incubated upside down, overnight in a 37 degree Celsius incubator.
11. Plates were photographed and observed, for clearings around the filter disks after 24 hours, 48 hours, and 72 hours.
12. Any bacterial or fungal contamination was noted.
Analysis/Conclusions:
Our ampicillin control for water had a massive bacterial lawn. It was very, very surprising. We are unclear as to what might have caused this occurrence.
Around the negative controls, there was a very limited area of clearance. On one of the negative controls in the first quadrant, it seems like there is no bacterial lawn at all. On the other controls, there is at least a minor amount of bacterial growth after 24, 48, and 72 hours.
Around the positive control we observed a clearing almost immediately. In fact, there was a bigger clearing around the ampicillin control than the other disks. There were contamination spots fairly close to the ampicillin control, although I don't know that this means anything significant.
The negative controls had a very limited area of clearance from our results. The area of clearance around these disks grew right up against the negative control disks. Water would not be expected to have antimicrobial activity, therefore this makes our negative controls very good for this experiment.