Background: We are trying to find medicinal uses for plants by testing them for chemicals Purpose: What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria? Materials: Balance, weight boat, lab scoops, Inoculating loop, Ni/Cr wire, Syringe, LB broth base, Petri dishes, Media bottles, E. Coli
Procedure-
Preparing plant extracts:
Use a mortar and paste to grind up 2 grams 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-standard extract into a 17 microtube. Label the sample
Attach prefilter to syringe and rinse with water
Take to Laminar hood: Plant extract, Syringe/prefilter, Microfuge tube rack, Pipet
Label microfuge tube with initials and whether it is water/methanol and put in rack
Attach the sterile filter to the prefilter
Load 1.7 mL of extract to syringe using pipet
Put plunger in and depress it
Have at least 1 mL of sterilized extract
Snap on cap on microfuge tube
Evaporate methanol from methanol extracts by placing a tube, with a cap, upon a 65 degree heat-block overnight
Reconstitute methanol extract 1 mL sterile deionized water
Using sterile forceps place 3 sterile pieces of filter paper into the filtered extract 4 degree celsius
Store until ready to use
2) Preparing Agar Plates:
Draw a + on each plate bottom and number quadrants 1-4
Liquify sterile LB agar in the microwave
Using sterile technique pour approximately 20 mL of agar into Petri plate
Using sterile forceps add the appropriate number of sterile disks to each tube of filtered extract
Label both plates with either M for methanol or W for water
Place the disks into the appropriate solution
Plant extracts, syringe/ prefilter, pipet
Label microfuge tube: Water and Methanol
Attach sterile filter to pre filter
Load 1.7 ml of extract into syringe, using pipet
Depress plunger 1.0 ml filter sterilized extract
Label agar plates with a cross, label quadrants
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 in the appropriate solution
Sterile disks were added to microfuge tubes containing one 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
One mL of Ecoli 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: Water, plant extracts, ampicillin. Plants were left on lab bench for 20 min. to allow both bacteria and filter filter disks to adhere to the agar
Plants were incubated upside down, overnight at 37 degrees celsius. Plate were photographed and observed for clearance around the filter disks after 24 hours, 48 hours, and 72 hours
Results: Our results were as expected, on the first day, there was a bit of a ring around the paper. After that on the second day of incubation there was the clearance of the agar on the second day of incubation. In our water dish, the ampicillin did its job and there was a clearance. There was an obvious clearance of the agar. Our plant-water had a clearance as well and it was more obvious than the water. In our methanol, there was the same thing, on the first day there was a slight ring around the papers, but on the second day there was clearance ring around the papers. Overall the clearings around the filter disks for positive control and negative control was visible. The density of the bacterial lawn was not that dense but it was visible. There was no sign of contamination, and the petri dishes looked uncontaminated.