BISC314:Full Protocol

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BISC314: Environmental Microbiology

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Laboratory Protocols

LAB #1: Learning Sterile Technique and Field Trip to the Cheese Shop

Today we will be taking a trip in to Cambridge to visit Formagio Kitchen, a famous cheese shop in the area - they even have a cheese cave! Each of you will pick a cheese to be your microbial habitat for the next few weeks. We will sample a large variety and learn about where these cheeses came from. Let's be sure we have representative cheeses from these four main classes:

1. A blue cheese
2. A fresh cheese
3. A washed rind cheese
4. A soft, brie-like cheese

Back in the lab, we'll review sterile technique and inoculate media of various kinds from our cheese rinds. We will isolate a beautiful array of different microbes (both eukaryotic and bacterial) from these cheeses and in the next few weeks you'll be using them to investigate two major microbial community functions: growth interactions and chemical signaling. If you haven't taken BISC209, or if you feel rusty on your microbiology, you might want to peruse the following link: BISC209:Protocols

LAB #2: Macroscopic and Microscopic observation of Isolates

You will have many different colonies growing up on your plates from last week. In your lab notebook, take some time to look at your colonies and describe their morphology, color, and smells! Does your Camembert inoculum smell like Camembert? You may find the following link useful for colony morphology descriptions: ASM MicrobeLibrary

Let's also look at our isolates under the microscope. We will make smears of our organisms next. Before we get to that point, however, it's worth discussing cellular morphology a bit. For the most part, bacteria are much smaller (0.2 to 4 µm) than eukaryotes (~100 µm). We will be using the 100x objectives to see bacterial morphology under the scope. You may be able to see the shapes of many eukaryotes you've isolated under 40x magnification.

Bacteria come in many different shapes (see this Wikipedia article for a nice figure depicting different shapes). These shapes are not, however, good indicators of relatedness or even species type. Many bacteria can be multiple shapes (termed pleomorphism) depending on how you grow them or from what conditions they are isolated. However, traditional names for bacteria often elude to their shape: for example, Vibrio fisheriis a curved (vibrio) bacterial organism.

You will make smears of all of your isolates. First, look at all of your isolates and determine if they are bacterial or eukaryotic. Can you think of a way to test if you are correct? In your lab notebook, note the shape and size of each isolate.

Making a Smear

1. Label a clean, glass slide with a graphite pencil on the far left of the slide with the code name of the isolate. For example, my 2nd isolate will be my initials followed by the number 2 (IN-2)
2. Place a small loopful of deionized water on the slide as far from each other as possible.
3. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of your isolate, picking up a TINY bit of white growth from the bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony is fine.
4. Place the loop with the bacterial growth into the drop of water on the slide. Use a circular motion to make a smooth suspension of the bacteria in the water. Stop when there is a circle of emulsified bacteria about the size of a nickle on the slide. 5. Reflame the loop.
6. Add a coverslip to your slide and take it over to the microscope -it is ready for viewing!

Preserving DNA from your isolates

For each of your isolates you should save a small amount of DNA for later identification. The protocol is below:
1. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of your isolate, picking up a TINY bit of white growth from the bacterial colony. An invisible amount of growth obtained from just touching the cooled loop to the colony is fine.
2. Place the loop within a 1.5 ml eppendorf tube containing 200 µl of water.
3. Vortex the tube to fully resuspend the isolate.
4. Freeze the tube at -20C for later. We will be identifying the most interesting isolates using molecular methods after our lab assays

LAB #3: Antagonistic and Mutualistic Interactions

    • NOTE** You must come in the day prior to set up overnight cultures for 8 of your isolates!!!

The microbial community living within the cheese is a complex one with many different microorganisms. As is true of any environment, these microbes interact with each other - both metabolically and chemically. Today, you will be taking your isolates and testing them against each other for growth inhibition or growth benefit. Some of these organisms may prevent the growth of others through the production of chemicals we'll discuss in lecture; others might promote the growth of their neighbors by secreting metabolites that are needed.

Interaction Assay
1. Create a 96-well template for your isolates. Using the excel template found here Media:template.xls make up the map for this 96-well plate - you should know where each of your 8 chosen isolates will be.
2. Using sterile technique, take 50 µl of your isolate from the overnight culture and place it into the appropriate well. Add another 100 µl of sterile media to the well to dilute the culture
3. When you are finished loading the top and side wells, use the p 20 micropipette to move 10 µl from wells A2 - A8 into wells B2 - B8. Repeat this until H2 - H8 has 10 µl in it also.
4. Now we will add the organisms from wells B1 - H1 to the rest of the plate. Again, using the p 20, take 10 µl from wells B1 - H1 and mix into the wells B2 - H2. Repeat this process until you have mixed all of the possible combinations of your isolates on this plate.
5. Each of your wells now has isolates growing by themselves and isolates mixed together. We will inoculate from this plate onto a square agar tray in order to observe what these organisms look like when grown together. For this we will use the multichannel micropipette. Using the multichannel, take 5 µl of your inocula and deposit a drop on top of the square agar tray. Repeat this until you have completed the full array.
6. Wait for your spots to dry before placing your trays upside down.

You should come in to the lab a few times this week to check on your assay and note any interesting results

LAB #4: Quorum sensing: chemical signaling within our community

Many bacteria are able to secrete signals into their environment that they can then utilize to determine their own density. Since bacteria are single-celled organisms, why do you think they would they be interested in knowing how many of them are near? A very well studied example of the quorum sensing system comes from Vibrio fisheri, a bacterium that produces light only at high densities. If you think about it, the light produced by a single bacterium is unlikely to be seen by anyone so Vibrio wait until they have reached a "quorum" before turning on the metabolic pathway that creates light. In this way, a gene regulatory network is actually controlled by cell density. To hear more about it from another source, see this YouTube video of Bonnie Bassler.

Today we will be testing if any of your isolates are secreting Auto-Inducer (AI) into the surrounding media. Specifically, we will be using a strain of bacterium called Chromobacterium violaceum. This organism is gram-negative and normally found in the soil. It produces a very strong purple pigment (hence the name) in response to levels of AI. We will also use a violacein-negative, mini-Tn5 mutant of C. violaceum (CV026) which can produce pigment in response to the AI from other bacteria but can no longer secrete its own AI - this will be our biosensor.

1. Label your agar plates. You will draw a line down the middle of the plate and write CV026 on one side and your isolate code on the other side.
2. Flame your loop. Use your loops to dip into an overnight culture of CV026. Streak 1/2 of an agar plate with this organism (the side with the CV026 label).
3. Flame your loop. Grab a relatively large amount of your isolates and streak them near to the CV026 streaks. Re-flame your loop.
4. Place your plates upside down till next week.

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