lab 6d - testing plant substances
Purpose: What local plant materials 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 Celsius
- sterile LB agar
- laminar flow hood and disinfectant
- plastic safety glasses
- Bunsen burner and gas lighter
- inoculating loop, Ni/Cr wire
- petri dishes, 60 x 15 mm, sterile
- E. coli JM109 (stock plate)
- plant specimen
- mortar and pestle
- pipet, 10 mL and pump
- short-stemmed plastic funnels
- filter paper disks, 5 mm diameter
- Beakers, 100 mL
- syringe, 10 mL and filter, 0.2 microliters
- reaction tubes and rack, 1.7 mL
- absolute methanol
- pipet, 1 mL and pump
- dry block heater/heat block
- fine-tipped forceps
- ampicillin
- glass spreader
- incubator oven, 37 degrees Celsius
- forceps
- alcohol
Procedure:
Part 2:
-Grind up 2 g of plant leaf material using a mortar and pestle with 10 ml of deionized water. Then filter the sample through a 11 cm filter paper funnel. After, filter sterilize the sample extract using a syringe filter. Collect 1ml of extract into a 1.7 ml microtube. Label the sample.
-REPEAT THE LAST STEP, but using methonal instead of water. Place 1 ml of methonal extraction into a 1.7 ml tube and place the tube with the cap open into a heat block set at 65 degrees F. Using sterile forceps, drop three filter paper discs into each tube filled with extracts. Let sit overnight at room temperature until ready to test.
Part 3
-Use a sterile pipet to transfer 1 ml of E. coli to the middle of a petri dish. Use a sterile loop to pread the ecoli evenly on the surface. Let ecoli soak into petri dish for about 15 minutes. Using sterile forceps, take each tube and place the discs on the petri dish about 2 cm from the edge. Place two discs on the top part of the dish for methonal and two discs on the botton part of the dish for the water extract. Place the positive and negative controls seperately but close to the center of the petri dish.
Results:
Description: Together as a class we did this experiment to test if there was any active ingredients to promote the growth of bacteria. The plant samples were collected from our field study site. This lab took about 3 days to fully complete. When doing this lab, I made some errors in placing the discs into the tubes filled with extract. I only had the methonal and the negative and positive control discs on the petri dish. I had a negative on the methanol disk for no bacteria growth. There is not an active antimicrobial enzyme in my plants because no visible bacteria growth was visible close to the methanol disks.
Data Analysis/Conclusions:
1. Did any extract give you positive results?
- I had no positive results for any bacteria growth on my plant substance.
2. Did your controls worked as expected?
- Yes, my negative control had no bacteria growth but my positive control discs had evidence of bacterial growth.
3. Discuss errors that could give you false results?
- This lab is supposed to be a sterile procedure with no foreign bacteria introduced into this lab. There could have been a chance of a procedure that was supposed to be sterile that bacteria was mixed in with the extracts. If the tube smelled like alcohol, then you wouldn't know what killed the bacteria, the alcohol or the molecules.
4. Discuss further experimentations to improve this experiiment.
- I would recommend using gloves and or protective shirts to protect the extracts from being mixed with foreign bacteria.
Thinking like a biotechnician:
1. In preparing the sample disks, some of the methanol extracts smell like alcohol. Why is this a problem?
- Alcohol is known for killing bacteria, we cant test the substance if the bacteria is dead.
Reflection: This lab was a very important step in our field study. Our field study goal is to observe and monitor the biotic and abiotic activity within our site. My results show no evidence of bacteria growth on the plants I collected. This lab taught me alot about how to extract plant materials and do micro procedures to transfer the plant substance onto a sterilized disk to test for the presence of bacterial growth. These labs are important because they help us gather resources and evidence about our field site, therefore, helping us better understand how our environment works.
Description: Together as a class we did this experiment to test if there was any active ingredients to promote the growth of bacteria. The plant samples were collected from our field study site. This lab took about 3 days to fully complete. When doing this lab, I made some errors in placing the discs into the tubes filled with extract. I only had the methonal and the negative and positive control discs on the petri dish. I had a negative on the methanol disk for no bacteria growth. There is not an active antimicrobial enzyme in my plants because no visible bacteria growth was visible close to the methanol disks.
Data Analysis/Conclusions:
1. Did any extract give you positive results?
- I had no positive results for any bacteria growth on my plant substance.
2. Did your controls worked as expected?
- Yes, my negative control had no bacteria growth but my positive control discs had evidence of bacterial growth.
3. Discuss errors that could give you false results?
- This lab is supposed to be a sterile procedure with no foreign bacteria introduced into this lab. There could have been a chance of a procedure that was supposed to be sterile that bacteria was mixed in with the extracts. If the tube smelled like alcohol, then you wouldn't know what killed the bacteria, the alcohol or the molecules.
4. Discuss further experimentations to improve this experiiment.
- I would recommend using gloves and or protective shirts to protect the extracts from being mixed with foreign bacteria.
Thinking like a biotechnician:
1. In preparing the sample disks, some of the methanol extracts smell like alcohol. Why is this a problem?
- Alcohol is known for killing bacteria, we cant test the substance if the bacteria is dead.
Reflection: This lab was a very important step in our field study. Our field study goal is to observe and monitor the biotic and abiotic activity within our site. My results show no evidence of bacteria growth on the plants I collected. This lab taught me alot about how to extract plant materials and do micro procedures to transfer the plant substance onto a sterilized disk to test for the presence of bacterial growth. These labs are important because they help us gather resources and evidence about our field site, therefore, helping us better understand how our environment works.