Natural Product Development lab
Purpose: To find out if the plants in my site contain ingredients that inhibit the growth of bacteria.
Materials:
Balance
weigh boat
lab scoops
LB broth base
Media bottles, 250 ml
sterilizer
water bath, 37 degrees C
Laminar flow hood
safety glasses
Bunsen burner
matches
petri dishes
plants
mortar and pestle
pipet, 10 ml
glass funnels
filter paper disks
beakers
reaction tubes and rack
methanol
1 ml pipet
dry block heater
foreceps, fine-tipped
ampicillin
glass spreader
incubator oven, 37 degrees C
Procedure
Part II:
1. Divide the plant matter in half then grind up one half of the plants with deionized water. Filter the liquid through a funnel with paper and collect what comes through in a tube.
2. Repeat this step with the other half of the plants but use methanol instead of water. Then place the methanol solution in a heating unit to evaporate the methanol. Then add 1ml of deionized water to reconstitute the dry matter.
3. Prepare 2 controls, one with water, and one with ampicillin. Drop paper disks to soak over night in each of the tubes using sterile foreceps.
Part III:
1. Use a sterile pipet to transfer 1ml of E. coli broth onto a petri dish. Distribute evenly using a sterile spreading rod. Using foreceps, place one disk of each tube into one quadrant of the petri dish and label. Let sit over night.
2. Analyze the discs to see if your plant grew alongside bacteria.
Results:
+ control: Showed positive results that bacteria wouldn't grow on it.
- control: Showed results that bacteria can grow on it.
MeOH: One of the discs had a large enough ring to be positive.
H2O: These were also positive with moderate disc size.
Data Analysis:
The extracts all turned out positive by the end of the lab. The controls did work with ampicillin being positive and the water being negative as I had hoped they would. Although everything looked right, there might have been cross contamination in one of my H2O quadrants where my partner and I accidentally put one of his discs in mine, so I might have gotten his results as well and messed it up a little. We can further the experiment by testing for different types of bacteria that are on the plants. Now the next step is to continue testing the plants to make sure it's done right and is accurate in its data.
TLAB Questions:
If an extract shows negative results in the antimicrobial assay, this does not mean that it can't be an antimicrobial agent. It could still be an agent for something that isn't being tested in this lab. If some of the methanol extractions smell like alcohol it would be an issue because the alcohol would then kill the bacteria and ruin the experiment. To identify the exact compound of an extract we can use chromatography to seperate molecules and find out what it is.
Materials:
Balance
weigh boat
lab scoops
LB broth base
Media bottles, 250 ml
sterilizer
water bath, 37 degrees C
Laminar flow hood
safety glasses
Bunsen burner
matches
petri dishes
plants
mortar and pestle
pipet, 10 ml
glass funnels
filter paper disks
beakers
reaction tubes and rack
methanol
1 ml pipet
dry block heater
foreceps, fine-tipped
ampicillin
glass spreader
incubator oven, 37 degrees C
Procedure
Part II:
1. Divide the plant matter in half then grind up one half of the plants with deionized water. Filter the liquid through a funnel with paper and collect what comes through in a tube.
2. Repeat this step with the other half of the plants but use methanol instead of water. Then place the methanol solution in a heating unit to evaporate the methanol. Then add 1ml of deionized water to reconstitute the dry matter.
3. Prepare 2 controls, one with water, and one with ampicillin. Drop paper disks to soak over night in each of the tubes using sterile foreceps.
Part III:
1. Use a sterile pipet to transfer 1ml of E. coli broth onto a petri dish. Distribute evenly using a sterile spreading rod. Using foreceps, place one disk of each tube into one quadrant of the petri dish and label. Let sit over night.
2. Analyze the discs to see if your plant grew alongside bacteria.
Results:
+ control: Showed positive results that bacteria wouldn't grow on it.
- control: Showed results that bacteria can grow on it.
MeOH: One of the discs had a large enough ring to be positive.
H2O: These were also positive with moderate disc size.
Data Analysis:
The extracts all turned out positive by the end of the lab. The controls did work with ampicillin being positive and the water being negative as I had hoped they would. Although everything looked right, there might have been cross contamination in one of my H2O quadrants where my partner and I accidentally put one of his discs in mine, so I might have gotten his results as well and messed it up a little. We can further the experiment by testing for different types of bacteria that are on the plants. Now the next step is to continue testing the plants to make sure it's done right and is accurate in its data.
TLAB Questions:
If an extract shows negative results in the antimicrobial assay, this does not mean that it can't be an antimicrobial agent. It could still be an agent for something that isn't being tested in this lab. If some of the methanol extractions smell like alcohol it would be an issue because the alcohol would then kill the bacteria and ruin the experiment. To identify the exact compound of an extract we can use chromatography to seperate molecules and find out what it is.