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CBIOL-1101 Microbiology Lab BACKGROUND We are surrounded by microbes. “Microbes” are tiny microscopic organisms, including bacteria, fungi, and single-celled eukaryotes like amoebae. Consider the bread that goes moldy when it has sat for too long – where did the mold come from? It started as a single-celled spore that drifted on the air and landed on the bread. You had no idea it was there until it had days and days to grow into a fuzzy blue mat. Your body is covered (and filled!) with microbes. Current estimates suggest that there are at least as many bacteria in your body as your own body cells, if not more! Many of these bacteria are helpful – they help you digest your food, stimulate your immune system, and even keep “bad” bacteria out. “Bad” bacteria cause problems when they are able to replicate into large populations that can cause damage. This happens when your immune system is compromised or you are exposed to a large number of bad bacteria (instead of just a few) during your first exposure. Our environment is also full of microbes. The ocean is teeming with bacteria. Likewise the soil – many of the bacteria in the soil are critical for decomposition and/or the growth of local plants. The things you regularly touch are also host to many bacteria: your phone, your laptop, your comb, and your water bottle are examples of items that have a variety of microbes on them. Most of these microbes are not actively growing, since there is no abundant food source on these items. But, if these microbes are transferred to a food source (such as your body or a nutrient agar petri dish in the lab), they will reproduce to produce many offspring. In this lab you will investigate the microbial load and diversity of various items in your environment and you will also investigate the ecological relationships between these microbes. QUESTIONS

1. What is the microbial load and microbial diversity of various items in your environment? 2. Is there evidence of antagonistic relationships between microbes in your samples, and if

so, what is the nature of the antagonistic relationship between microbes in your sample? OVERVIEW DAY 1. Your team will receive 3 petri dishes with sterile nutrient agar (“plates”). You will divide each plate into 2 sections, giving you 6 sections to grow microbes from different sources. You will use a cotton swab to collect microbes from 5 environmental sources, one for each section. The last section will not receive microbes from an environmental source and will act as a control. You will set the plates aside to incubate for 7 days DAY 2. For each sample, you will count how many colonies are present and estimate the number of different species. You will also look for potential cases of antagonistic relationships between species. For any potential antagonistic relationships you identify, you will re-inoculate fresh plates to investigate the relationship further. DAY 3. You will analyze the results of your re-inoculated plates to answer questions about the relationship between two species.

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PROTOCOL DAY 1

1. Collect your supplies:

• 3 nutrient agar plates • a sharpie marker • gloves for all team members • a foil packet of sterile Q-tips

2. As a team, select the 5 locations you will swab to collect microbes from your

environment. These locations can be on a body part, a food item, a thing you carry (phone, laptop, backpack, water bottle, etc) or a location in the classroom or anywhere in UHall. Write your 5 locations here, including your hypotheses about which location(s) will have the most/least microbes (microbial load) and the most/least microbial diversity (number of different species).

# Location Hypotheses:

1

2

3

4

5

6 negative control: Q-tip not swabbed on a surface

3. For your negative control, you will swab a sterile Q-tip on your agar plate without first

touching it to any other surface. What do the following possible results mean:

a. If your negative control shows microbial growth: _________________________

_________________________________________________________________

b. If your negative control shows no microbial growth: _______________________

_________________________________________________________________ 4. Using a sharpie marker, divide and label your plates on the “bottom” of each plate (the

part of the plate that has nutrient agar, not the lid). For each plate, also write the date. See the diagram on the next page.

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5. Use sterile technique to swab each of your specified locations: a. Wearing gloves, carefully pick up a sterile Q-

tip from your foil packet. Do not let the cotton-end of the Q-tip touch any surface other than your selected surface (if it accidentally touches something, throw it out and get another one).

b. Touch your sterile Q-tip end to your selected surface: for wet surfaces/objects, a single swipe should be sufficient; for dry surfaces, rub the Q-tip end vigorously across the surface of the item.

c. Then, streak your Q-tip carefully across the surface of the agar in the designated region of the plate, being careful not to puncture the Q-tip through the agar. The gray streak in the diagram represents how you should streak your sample on your agar plate. All of your agar plates should remain closed at all times except for the moment that you are streaking your sample onto the agar.

d. For your negative control, select a Q-tip as you did for the other samples but do not swab any surfaces – simply streak the sterile Q-tip directly on the agar as you did for the other samples.

6. Place your agar plates in the designated incubation area in the back of the room to

incubate for the next 7 days. DAY 2

1. Collect your plates. For each section, count the number of colonies and the number of different species. To identify different species, look for differences in color, shininess, and fuzziness between the colonies. You may choose to use a stereoscope (low-magnification microscope) to better view your samples. Often, different species will have different characteristics that are visible by eye. However, sometimes, different species are only identifiable through microscopic or molecular markers that you can’t see. So your estimate of species number is likely an underestimate. Write your colony counts and species count in the table below.

Location Microbial Load (number of colonies)

Microbial Diversity (number of species)

negative control

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2. In your notebook, make observations about the types of microbes that grow in each

location. Do some locations appear to have more fungi (fuzzy molds), whereas others appear to have more bacteria (flat shiny colonies)? In one section did one mold “take over” and rapidly cover the whole plate? Did some colony colors surprise you? This is not a cookie-cutter lab – your results depend entirely on the locations you chose to swab!

3. Re-inspect your plates and look for any cases of microbial antagonism. Some microbes produce toxins that kill other microbes. For example, Alexander Fleming originally identified the antibiotic penicillin by noticing that bacteria did not grow on agar plates if a specific mold (Penicillium) was growing nearby. The mold produces an antibacterial toxin that kills bacteria. Look for any cases where you see a potential “zone of inhibition” between microbes of different species. A zone of inhibition is a region where no growth occurs. In the cartoon to the right, a fuzzy mold is antagonizing the nearby colony of bacteria, resulting in a crescent-shaped zone of inhibition that allows only part of the bacterial colony to grow.

4. You may find multiple cases of possible antagonism. Or you may not. If you do not find

any cases of microbial antagonism, see if another group has found one. You can use their samples for the following experiments.

5. For each possible antagonistic relationship, label the two species A1 (for Antagonist 1)

and B1 (for Blocked 1). Every pair should get its own number, i.e. A1 blocks B1, A2 blocks B2, etc. Write your pair names here with a short description of each colony. Additionally, on the underside of your plates (the same side you labeled on Day 1), write directly over where each colony is with its name (A1, B1, etc). This way you can return back to these original plates and see what the original antagonistic colonies looked like. (They will be stored in the refrigerator, which will inhibit additional growth.)

Location Antagonists

(include a description of the colony)

Blocked Species (include a description of the

colony)

A1 B1

A2 B2

A3 B3

6. For each antagonistic pair, you will test the following:

a. Is the antagonistic relationship reproducible?

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To test this, you will transfer a few cells from each colony to a new plate and let them grow another 7 days. If you have isolated a real antagonistic pair of species, then you should see a zone of inhibition on your re-streaked plates. To re-streak your colonies, use a sterile Q-tip and touch just the tip of it to your first Blocked colony. Streak this in a straight line onto a fresh plate using sterile technique following the diagram to the right. Repeat this procedure for the cognate Antagonistic colony, except streak this in a T. Repeat for each antagonistic pair. b. Is the antagonistic relationship species-specific? To test this, repeat step a) above, but instead of using the cognate Blocked species, use another species from a different location. Choose a species that appears to be similar (i.e. if Blocked species B1 is a shiny flat colony, choose a shiny flat colony from another location). Name these non-cognate “Blocked” species C1, C2, etc according to the antagonist you have paired them with.

7. Place your agar plates in the designated incubation area in the back of the room to

incubate for the next 7 days. DAY 3

1. Collect your plates. Inspect each A/B and A/C pair for evidence of zones of inhibition. You may need to use a stereoscope for closer analysis. For each Antagonist, answer the following questions:

a. Is the zone of inhibition reproducible on your antagonist test plates against the cognate Blocked species?

b. If yes, how large is the zone of inhibition (in cm)? c. Does the Antagonist block growth of the non-cognate test species C?

d. Do you notice anything else noteworthy about your tests – anything surprising or unusual? If so, what do you observe? This is investigative science without a cookie-cutter answer. What do you observe that might lead you to ask new questions about your microbes?

2. Once you are finished Dispose of all your plates in the designated plate disposal area.

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LAB WRITE-UP

While you worked together with your teammates to collect data for this lab, the Lab Write-Up is a strictly individual project. Plagiarism is strictly prohibited. Every sentence in your Lab Write Up should be written only by you! Any evidence of plagiarism will result in a 0 for this assignment.

Your Lab Write-Up should include these sections. Additionally, remove page 3 & 4 (your raw data) from this handout and attach it to your Lab Write Up when you turn it in.

Background & Questions (1-2 paragraphs)

Provide sufficient background information that a peer (freshman or sophomore who has not taken CBIOL1101) could understand them. Restate the questions in your own words. Include in your questions which locations you chose to swab and why.

Hypotheses (1 paragraph)

Describe your hypotheses and your rationale for each.

Results (2-3 paragraphs)

For Question 1 (about microbial load and diversity of each swabbed location), create two bar charts to graph your results. One chart should have Location on the X-axis and Microbial load (# of colonies) on the Y-axis. The other chart should have Location on the X-axis and Microbial Diversity (# of species) on the Y-axis. Both graphs should have their own figure legend with an appropriate figure title and description. Briefly describe the results in the graphs. Include noteworthy observations about the microbes growing in each section. Finally, mention whether your negative control sections had or did not have microbial growth.

For Question 2 (about antagonistic relationships), describe the results from your Day 3 analysis. Mention how many A/B pairs you tested. For each pair answer the questions posed on page 5.

If there were any sources of potential human error (e.g. mis-labeled plates or difficulty counting separate colonies), describe them here.

Conclusions (1-2 paragraphs)

Restate the answers to Question 1. Speculate about why certain locations might have greater microbial load or microbial diversity. For Question 2, did you find evidence of an antagonistic relationship? Describe the key features of that relationship – was it reproducible, species-specific, etc? Finally, ask a “What Next” question – what is the next step you think would be interesting to pursue? This question should go beyond the scope of this lab (i.e. it is not sufficient to ask, “What is the microbial load/diversity of a different location?”).

GRADING

• Completeness (45) – Did you address all of the points for each section listed above? Did you include your raw data?

• Scientific Rigor (45) – Do your data make sense? Did you present the data appropriately? Do your conclusions match your data?

• Presentation (10) – Is your Lab Write-Up free of grammatical and formatting errors?