Testing Plant Substances as Potential Medicines
Background
Organisms in nature battle for resources and survival. They compete with each other for light and water. Viruses and threats of bacterial disease affect all plants. Antimicrobial agents used in plants for defense could lead to a potential therapeutic medicine. To do this, samples must be extracted and processed. After testing, the antimicrobial agents have to be proven safe to be used as medicine.
Purpose
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials
Balance, weight boat, lab scoops
LB broth base
Media bottles, 250 mL
Sterilizer/autoclave
Water bath, 37 degrees, shaking
Sterile LB agar
Laminar flow hood and disinfectant
Glasses, safety, plastic
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 μL
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 degrees
Procedure
Preparing Plant Extracts:
-Using a mortar and pestle, grind of 2g of plant tissue with 10mL of deionized water
-Let sit for 3 minutes.
-Filter the sample through an 11 cm paper/funnel
-Filter sterilize the extract using a syringe filter
-Collect 1 mL of sterilized extract into a 1.7 microtube. Label the sample.
Filter sterilization steps:
-Attach the pre-filter to the syringe and rinse with water.
-Take to Laminar Hood: plant extract, syringe/pre-filter, rack, pipet
-Label microfuge tube (W and M)
-Attach sterile filter to pre-filter and load 1.7 mL of extract into syringe using your pipet.
-Depress plunger and press at least 1.0 mL of filter-sterilized extract
-Snap on cap of microfuge.
-Evaporate methanol from methanol extracts by placing tube, with cap open, on a 65 degree Celsius heat block overnight.
-Reconstitute methanol extract with 1.0 mL sterile deionized water.
-Using sterile forceps, place 3 sterile pieces of filter paper in each extract tube.
-Store at 4 degrees Celsius until ready to use.
Preparing Agar Plates:
-Liquefy sterile LB agar in the microwave.
-Using sterile technique, pour approximately 20 mL agar into Petri plate.
-Label agar plates with a cross, label quadrants
-Using sterile forceps, add the appropriate # of sterile disks to each tube of filtered extract.
-Prepare 2 negative control disks: Sterile water and Ampicillin
-Place the disks into the appropriate solution
-Add sterile disks to microfuge tubes containing 1mL sterile water.
-Add 10-20 mL of warmed nutrient agar into 2 petri dishes using sterile technique.
-Turn plates upside down and store at 4 degrees Celsius.
-Add 1 mL of E. coli colony to each plate and spread throughout surface.
-Add filter disks into separate quadrants.
-Leave plates upside down to incubate and observe after 24, 48, and 72 hours.
Organisms in nature battle for resources and survival. They compete with each other for light and water. Viruses and threats of bacterial disease affect all plants. Antimicrobial agents used in plants for defense could lead to a potential therapeutic medicine. To do this, samples must be extracted and processed. After testing, the antimicrobial agents have to be proven safe to be used as medicine.
Purpose
What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials
Balance, weight boat, lab scoops
LB broth base
Media bottles, 250 mL
Sterilizer/autoclave
Water bath, 37 degrees, shaking
Sterile LB agar
Laminar flow hood and disinfectant
Glasses, safety, plastic
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 μL
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 degrees
Procedure
Preparing Plant Extracts:
-Using a mortar and pestle, grind of 2g of plant tissue with 10mL of deionized water
-Let sit for 3 minutes.
-Filter the sample through an 11 cm paper/funnel
-Filter sterilize the extract using a syringe filter
-Collect 1 mL of sterilized extract into a 1.7 microtube. Label the sample.
Filter sterilization steps:
-Attach the pre-filter to the syringe and rinse with water.
-Take to Laminar Hood: plant extract, syringe/pre-filter, rack, pipet
-Label microfuge tube (W and M)
-Attach sterile filter to pre-filter and load 1.7 mL of extract into syringe using your pipet.
-Depress plunger and press at least 1.0 mL of filter-sterilized extract
-Snap on cap of microfuge.
-Evaporate methanol from methanol extracts by placing tube, with cap open, on a 65 degree Celsius heat block overnight.
-Reconstitute methanol extract with 1.0 mL sterile deionized water.
-Using sterile forceps, place 3 sterile pieces of filter paper in each extract tube.
-Store at 4 degrees Celsius until ready to use.
Preparing Agar Plates:
-Liquefy sterile LB agar in the microwave.
-Using sterile technique, pour approximately 20 mL agar into Petri plate.
-Label agar plates with a cross, label quadrants
-Using sterile forceps, add the appropriate # of sterile disks to each tube of filtered extract.
-Prepare 2 negative control disks: Sterile water and Ampicillin
-Place the disks into the appropriate solution
-Add sterile disks to microfuge tubes containing 1mL sterile water.
-Add 10-20 mL of warmed nutrient agar into 2 petri dishes using sterile technique.
-Turn plates upside down and store at 4 degrees Celsius.
-Add 1 mL of E. coli colony to each plate and spread throughout surface.
-Add filter disks into separate quadrants.
-Leave plates upside down to incubate and observe after 24, 48, and 72 hours.
Results
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Analysis
For the positive control, there was a clearance that was wider than the clearance of the other disks, but not by a lot. There may have been some contamination due to sharing forceps with another group. The ampicillin might have not been concentrated in the filter enough to show clearer results. In the negative control, the bacteria lawn grew completely onto the filter disk. Water works well as a negative control in this experiment because water isn’t antimicrobial.