Testing Plant Substance as Potential Medicines
Background: Plants are always battling for survival in the wild especially against diseases. Due to this they have developed antimicrobial agents to defend against the microbes. If isolated these antimicrobial agents can betested against many viruses. Then if proven safe for humans can be used to help kill the microbes that have infected the body.Purpose/Objective: The purpose of our project is to test whether the plant will use defensive antimicrobials. Then we will see what specific antimicrobials it will use to fend off the bacteria growth.
Materials:
● Balance, weigh boat, lab scoops
● LB broth base
● Media bottles, 250 mL
● Sterilizer/ autoclave
● Water bath, 3 degrees celcius 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, 10mL and pump
● Plastic funnels short-stemmed
● Filter paper disks, 5 mm diameter
● Beakers, 100mL
● Syringe, 10 mL and filter, 0.2 um
● Reaction tubes and rack, 1.7mL
● Methanol, absolute
● Pipet, 1 mL and pump
● Dry block heater/heat block
● Forceps, fine-tipped
● Ampicillin
● Glass spreader
● Incubator oven, 37 degrees celcius
● Lemon leaves
Procedure:
● measure 2 grams of plant material
● using a mortar and pestle, grind up 2 grams of plant tissue with 10milliliters of deionized water
● let sit for 3 minutes
● filter the sample through an 11 centimeter filter paper funnel
● filter sterilize the extract using a syringe filter
● collect 1 milliliter of extract in a 1.7 milliliter microtube. Label the tube
● Repeat the steps using methanol instead of water
● Filter Sterilization(procedure)
● Attach pre-fitter to syringe and “rinse” with H2O
● Carefully open sterilization filter, keeping the filter in its plastic covering
● Load approx 1.5 mL of H2O-based filtrate using a pipet
● Depress the plunger, Collecting the sterile-filtered filtrate into microfuge tube
● Quickly close microfuge cap until a “snap” sound is heard
● use flame-sterilization forces to transfer filter squares into H2O control and ampicillin control
● label plate(bottom)
● dispense 1 mL E.coli in each plate
● Flame sterile spreading loop
● place filter discs in plate
Methanol Extract 24 then 48 hours:
Water Extract 24 then 48 hours:
Methanol Extract after 24 hours: Ampicillin paper dischas an anti-microbial ring around it and the plant extracts don’t have the ring
Methanol Extract after 48 hours: Ampicillin paper disc still has anti-microbial ring around it and the plant extracts are starting to have anti-microbial ring.There are white dots on our solution because of cross contamination.
Water Extract after 24 hours: Ampicillin paper dischas an anti-microbial ring around it and the plant extracts don’t have the ring
Water Extract after 48 hours: Ampicillin paper disc still has anti-microbial ring around it and the plant extracts are starting to have anti-microbial ring.There are white dots on our solution because of cross contamination.
Methanol Extract after 48 hours: Ampicillin paper disc still has anti-microbial ring around it and the plant extracts are starting to have anti-microbial ring.There are white dots on our solution because of cross contamination.
Water Extract after 24 hours: Ampicillin paper dischas an anti-microbial ring around it and the plant extracts don’t have the ring
Water Extract after 48 hours: Ampicillin paper disc still has anti-microbial ring around it and the plant extracts are starting to have anti-microbial ring.There are white dots on our solution because of cross contamination.
Analysis:
Our methanol extract and our plant extract both ended up having an antimicrobial ring around it. Our ampicillin ring also had an antimicrobial ring around it. We could tell because there was a clear circle around the pieces of paper. Unfortunately our trays got a little contaminated during the lab. We had white dots on the bacteria field that was in the tray, but that didn’t affect the end results of the project.
Our methanol extract and our plant extract both ended up having an antimicrobial ring around it. Our ampicillin ring also had an antimicrobial ring around it. We could tell because there was a clear circle around the pieces of paper. Unfortunately our trays got a little contaminated during the lab. We had white dots on the bacteria field that was in the tray, but that didn’t affect the end results of the project.