21: ANTIBIOTIC PRODUCING MICROBES IN SOIL
- Page ID
- 157090
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Differentiate between normal microbial colony growth and colonies that possibly produce antimicrobial compounds.
- Evaluate the effectiveness of antimicrobial compounds produced by soil microbes on known pathogens.
BACKGROUND
You might remember that the very first antibiotic, penicillin, was discovered by Alexander Fleming in 1928. Penicillin is made by a fungus in the genus Penicillium. Today, many antibiotics come from a group of thread-like bacteria called Actinomycetes (a group of bacteria that form branching filaments and are commonly found in soil). These bacteria are responsible for producing several well-known antibiotics, including streptomycin and neomycin.
Infections caused by bacterial strains that are resistant to antibiotics are becoming an increasingly serious global health concern. These antibiotic-resistant infections can be difficult—or even impossible—to treat with conventional medications. As a result, scientists are working hard to discover new types (or classes) of antibiotics and identify new organisms that produce them. Interestingly, some of the most promising sources of new antibiotics are quite unexpected! Researchers are now investigating tropical plants, marine organisms (like certain sponges and even sharks), and other unique environments to find microorganisms that naturally produce antibiotic compounds.
Today you will explore a more familiar environment—soil—in search of an antibiotic-producing organism. Soil is a rich and diverse ecosystem filled with bacteria, fungi, and other microorganisms—many of which engage in chemical warfare to survive. That chemical warfare includes producing antibiotics to inhibit the growth of nearby competing microbes. Because of this, soil is one of the most productive environments antibiotic-producing organisms.
In this exercise, you will collect a soil sample, culture it on nutrient agar plates, and observe whether any colonies are producing substances that inhibit the growth of other bacteria. If so, you may have found a natural antibiotic producer!
MATERIALS
Day 1
100 mL bottle (pre-measured for you)
1 gram soil sample (student-collected)
2 Nutrient agar plates
1 Alcohol burner
1 Cell spreader
1 Jar of alcohol (for sterilizing metal rods)
1 Turntable
Balance
Weighing paper or boats
Day 2
Soil culture plate (from previous lab)
2 Nutrient agar plates
Day 3
Isolated colony from soil culture (previous lab)
Test organisms:
Escherichia coli
Pseudomonas fluorescens
Staphylococcus aureus
METHODS/PROCEDURES
Day 1
1. Collect a soil sample from anywhere on campus.
👉 Tip: Typically slightly damp soil contains a higher number of microorganisms compared to very dry soil.
- Use the balance to weigh approximately 1 gram of your soil sample.
3. Place the 1.0g of soil in to the 100 mL bottle of water.
4. Secure the cap and shake vigorously for at least 30 seconds to mix well.
👉 This creates a soil suspension (a mixture of soil particles and microorganisms evenly dispersed in water).
5. Allow the particles to settle at the bottom of the bottle (about 10 minutes).
6. Label one of the plates with your name date, dilution amount (1.0 mL or 0.1 mL), and the source location of your soil sample.
(Your partner will label the other plate. Decide which dilution you will plate and which your partner will plate before labeling).
7. Student A: Transfer 1.0 mL of the liquid from the top of the suspension onto the nutrient agar plate labeled 1.0 ml.
8. Use a sterilized cell spreader to spread the sample evenly across the surface of the agar.
👉 Tip: Let the cell spreader cool for a few seconds after flaming to avoid killing the bacteria.
9. Student B: Transfer 0.1 mL of the suspension onto the second plate.
10. Use a sterilized cell spreader to spread the sample evenly across the surface of the agar.
👉 Tip: Let the cell spreader cool for a few seconds after flaming to avoid killing the bacteria.
- Incubate the plates agar down at room temperature until the next lab session.
Day 2
- Examine your soil plates carefully for colonies that seem to inhibit the growth of surrounding colonies.
👉 Remember: you are not looking for isolated colonies but rather colonies that appear to be hindering the growth of colonies around it. See
example below.
Draw your plate in the results section then indicate and label the colony that appears to be possibly producing an antibiotic.
1. Draw your plate in the results section then label the colony that appears to be possibly producing an antibiotic.
2. Show your drawing and plate to your instructor for approval of your colony selection.
3. Once approved use a sterile cooled loop to scrape the top of the chosen colony and inoculate a fresh plate by making a single streak down the
center.
4. Label plate and incubate at room temperature for at least 2 days.
Day 3
1. After the streak line has grown, add the provided known cultures to the plate by making short streaks that are perpendicular
on each side of the main streak line. Use the diagram below as a guide.
👉 Do not actually touch the original growth line - get as close to it as you safely can without touching.
2. Incubate at 30⁰C for 2-5 days.
3. Observe the test cultures for inhibition of growth where they are nearest to the original streak.
4. Draw what you see on the plate in the results section.
Print a hard copy to bring to lab (PDF).
👉 If you are filling this out on a digital iPad or tablet please note put your name here and take a screen shot.
You are also welcome to print the PDF and turn in a physical copy of the following.
Exercise #27 Antibiotic Producing Microbes in Soil
NAME ______________________
EXPECTATIONS
If the colony you choose secrets an antibiotic should you anticipate all test cultures to be inhibited equally? Explain your reasoning.
Describe what the growth might look like on the plate if a microorganism is producing an antibiotic?
RESULTS
Draw your soil plate after incubation Draw your plate after incubation with the know pathogens
CONCLUSIONS
1. Did you find any inhibition by your chosen soil organism? If so, which test bacteria were inhibited?
2. Producing and releasing antibiotics requires significant energy and resources from a bacterial cell. What evolutionary advantage does this ability provide? In other words, what benefit does the antibiotic-producing organism gain from investing in this process?
3. Compare the bacterial growth on your original soil plate and the 10⁻³ dilution plate. What does the difference in colony density tell you about the effectiveness of dilution in isolating individual colonies?
4. If no zones of inhibition were observed around any colonies, what are some possible explanations for this result? How would you modify the experiment to increase your chances of finding an antibiotic-producing organism?