Background:
In nature, organisms are constantly battling for resources and survival. All organisms are infected by viruses or threatened by bacterial disease. Many organisms can produce substances to combat infections and viruses. Finding these substances can be used to potentially be turned into medicine. It is a long and difficult process to extract, process, and test these samples. Technicians have to deal with the extracts and then purify them enough in order for them to be used.
Materials:
-balance, weigh boat, lab scoops -inoculating loop, Ni/Cr wire -reaction tubes and rack, 1.7 mL
-LB broth base -petri dishes, 60x15 mm, sterile -methanol, absolute
-media bottles, 250 mL -E. coli JM109 (stock plate) -pipet, 1 mL and pump
-sterilizer, autoclave -plant specimen -dry block heater/heat block
-water bath, 37° C, shaking -mortar and pestle -forceps, fine-tipped
-laminar flow hood and disinfectant -pipet, 10 mL and pump -ampicillin
-glasses, safety, plastic -plastic funnels, short-stemmed -glass spreader
-Bunsen burner and gas lighter -filter paper disks, 5 mm diameter -incubator over, 37°C
-beakers, 100 mL
Procedure:
1. Prepare a nutrient or LB culture for the E. coli at least 24 hours in advance. Using sterile technique, add a colony of E. coli culture to the broth medium and incubate, shaking at 37 C for 24 hours.
2. Each lab group need 2 petri plates. Draw a "+" on each plate bottom to divide the pate into quadrants (four sections). Label the quadrants No. 1 through 4. Also, label the dish with your initials and the date.
3. Liquefy sterile LB agar in the microwave at 50% power. Using sterile technique, pour approximately 20 mL of sterile, liquid LB agar into each Petri plate. Let the agar solidify for 15 minutes. Let it dry for at least 24 hours.
4. Using a mortar and pestle, grind up 2 g of plant tissue (leaves or bark) with 10 mL of deionized water. Lwt it sit for 3 minutes. Filter and sample through an 11 cm filter paper funnel. Filter sterilize the filtered sample extract using a syringe filter, as demonstrated by the instructor. Collect with 1 mL of extract into a 1.7 mL microtube. Label the sample.
5. Repeat Step 4, but replace the water with methanol as as the extracting solvent. After the methanol extraction, place the 1.7 mL tube with the 1 mL of methanol extract in a 65 C heat block (caps open) for 24 hours or more, if necessary to evaporate the methanol. Reconstitute dry matter in the tube with 1 mL of deionized water.
6. For each of the other samples, repeat steps 4 and 5. Label all samples. There should be six tubes of samples.
7. Using sterile forceps (that have been flamed in alcohol) drop three filter paper disks into each tube of the filtered extract.
8. Prepare negative control disks, three each, of only methanol and only sterile distilled water.
9. Prepare six positive control disks of ampicillin solution.
10. Allow the disks sufficient time to soak up enough extract to be saturated (perhaps overnight).
11. Close the tubes. Store all samples at 4 C until ready to use.
12. Using a sterile pipette transfer 1 mL of the E. coli broth
(made at step 1) to the middle of each Petri dish. Sterilize a spreading loop (using alcohol and a flame) , and evenly spread the bacterial culture around the Petri plate. Quickly cover, and allow the culture to soak into the agar for at least 15 minutes.
13. Using sterile forceps carefully place one disk into the middle of each quadrant, about 2 cm from the outer edge of the Petri dish. Blot any excess liquid before placing the disk on the Petri dish. Keep all the methanol-extracted samples on the same dish and all the water-extracted samples on the same dish.
14. Repeat step 13 twice so that you have three replicates of the methanol extraction and three replicates of the deionized water extractions.
15. Place one of the negative control disks, either sterile distilled water or methanol, in the center of the appropriate plate. Place a positive control disk with ampicillin in another quadrant of each plate.
16. You should end up with six Petri plates, each containing a negative control in the middle, a positive control , and three sample disks. Make sure you have recorded exactly which plant extracts and which solvent went into each quadrant.
17. Make sure the disks are adhering well to the surface of the agar. For incubation, invert the plates and incubate at 37 C for 24 to 48 hours.
18. After incubation, examine the plates with the plant extract disks for zones of inhibition. This is a clear area formed around the disk by the inhibitory action of a substance(s) in the plant material. Photograph or draw the plates, labeling any inhibition of bacterial growth.
19. Create a data table to collect and present data of all the replicates as well as the averages. include descriptions of the bacterial lawn around each disk. Measure and record the diameter and clarity of any cleared areas around the disks. Give quantitative measurements of your observations.