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.
Many plants develop specialized chemicals to succeed against their competitors for space and sunlight. These chemicals can be used as medicines for humans, such as antimicrobial ingredients. These must be identified and extracted, and tested extensively.
Purpose:
What plants materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
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.
Many plants develop specialized chemicals to succeed against their competitors for space and sunlight. These chemicals can be used as medicines for humans, such as antimicrobial ingredients. These must be identified and extracted, and tested extensively.
Purpose:
What plants materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials:
4 grams of buddleja ring stand funnel 10ml test tube thing filter paper deionized water methanol 1.7ml microtube mortar and pestle laminar hood syringe prefilter |
sterile filter rack pipet heat block sterile forceps agar plates sharpie/labeling marker sterile LB agar e. coli colony spreading loop matches portable bunsen burner |
Procedure:
Preparing Plant Extracts
-(pokie note) before the procedure, make sure your plants are not poisonous by eating them.
-Using a mortar and pestle, grind up 2 g of plant tissue with 10 ml of deionized water
-Let it sit for 3 minutes
-Filter the sample through an 11 cm filter paper/funnel
-Filter sterilize the extract using a syringe filter
-Collect 1 ml of the filter-sterilized extract into a 1.7 microtube. Label the sample.
Filter Sterilization Steps
-Attach the prefilter to the syringe and rinse with water.
-Take to Laminar Hood: plant extract, syringe/prefilter, rack, pipet
-Label microfuge tube (H^2O and Me)
-Attach sterile filter to prefilter and load 1.7 mL of extract into syringe using your pipet.
-depress plunger ---> at least 1.0 mL filter-sterilized extract
-Snap on cap on microfuge
Preparing
-Evaporate methanol from methanol extracts by placing tube, with cap open, on 65° C 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 tubes at 4° C until ready to use.
Preparing Agar Plates
-Draw a + on each plate bottom and number the quadrants 1-4.
-Liquify sterile LB agar in the microwave.
-Using sterile technique, pour approximately 20 mL agar into Petri plate.
-Using sterile forceps, add the appropriate number of sterile disks to each tube of filtered extract
-prepare negative control disks: 2 - sterile water, 2 - ampicillin
-Place the disks into the appropriate solution
-Sterile disks were added to microfuge tubes containing 1 mL sterile water.
-10-20 mL of warmed nutrient agar was poured into 2 petri dishes using sterile technique.
-After allowing agar to solidify, plates were turned upside down and stored at 4 degrees Celsius overnight.
-1 mL of E. coli colony was added to each plate. A flame-sterilized spreading loop was used to spread the bacteria throughout the surface of the agar.
-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. Plates were left on lab bench for 20 minutes to allow both bacteria and filter disks to adhere to the agar.
-Plates were left incubated upside down, overnight at 37°C. Plates were photographed and observed for clearance around the filter disks after 24 hours, 48 hours, and 72 hours.
Preparing Plant Extracts
-(pokie note) before the procedure, make sure your plants are not poisonous by eating them.
-Using a mortar and pestle, grind up 2 g of plant tissue with 10 ml of deionized water
-Let it sit for 3 minutes
-Filter the sample through an 11 cm filter paper/funnel
-Filter sterilize the extract using a syringe filter
-Collect 1 ml of the filter-sterilized extract into a 1.7 microtube. Label the sample.
Filter Sterilization Steps
-Attach the prefilter to the syringe and rinse with water.
-Take to Laminar Hood: plant extract, syringe/prefilter, rack, pipet
-Label microfuge tube (H^2O and Me)
-Attach sterile filter to prefilter and load 1.7 mL of extract into syringe using your pipet.
-depress plunger ---> at least 1.0 mL filter-sterilized extract
-Snap on cap on microfuge
Preparing
-Evaporate methanol from methanol extracts by placing tube, with cap open, on 65° C 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 tubes at 4° C until ready to use.
Preparing Agar Plates
-Draw a + on each plate bottom and number the quadrants 1-4.
-Liquify sterile LB agar in the microwave.
-Using sterile technique, pour approximately 20 mL agar into Petri plate.
-Using sterile forceps, add the appropriate number of sterile disks to each tube of filtered extract
-prepare negative control disks: 2 - sterile water, 2 - ampicillin
-Place the disks into the appropriate solution
-Sterile disks were added to microfuge tubes containing 1 mL sterile water.
-10-20 mL of warmed nutrient agar was poured into 2 petri dishes using sterile technique.
-After allowing agar to solidify, plates were turned upside down and stored at 4 degrees Celsius overnight.
-1 mL of E. coli colony was added to each plate. A flame-sterilized spreading loop was used to spread the bacteria throughout the surface of the agar.
-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. Plates were left on lab bench for 20 minutes to allow both bacteria and filter disks to adhere to the agar.
-Plates were left incubated upside down, overnight at 37°C. Plates were photographed and observed for clearance around the filter disks after 24 hours, 48 hours, and 72 hours.
Results:
On the first day, the methanol plate showed very slight changes, with a ring around each of the filter papers.
density of bacterial lawn, presence of clearings around filter disks for positive control, negative control, negative control and experimental disks, signs of contamination, any other observations
On the first day, the methanol plate showed very slight changes, with a ring around each of the filter papers.
density of bacterial lawn, presence of clearings around filter disks for positive control, negative control, negative control and experimental disks, signs of contamination, any other observations
The images below were taken on February 9, when first placed.
The images below were taken on February 10, 24 hours after placed.
The images below were taken on February 11, 48 hours after placed.