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 Formaggio 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.

Background
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.

Making smears of your isolates

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!

Lab Questions: 1. 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? 2. In your lab notebook, note the shape and size of each isolate.
3. Is your organism swimming around or are you observing brownian motion? How can you tell? Could you think of a way to test for motility?

The Gram Stain

Bacteria have been classically divided into two main groups based on a simple staining protocol: the Gram stain. It is named after its inventor (hence the capitalization) and is used primarily in clinical microbiology. The importance of the Gram stain is that it can distinguish many bacteria based on the architecture of their cell walls. However, not all bacteria can be classified by the Gram stain and many are what is known as "Gram variable" depending on what stage of growth they are in. Gram-positive and Gram-negative bacteria are both stained purple by the crystal violet (primary) stain. Addition of the iodine leads to the formation of a crystal violet-iodine complex within the cell wall. The decolorizer extracts lipid from the cell wall of Gram-negative bacteria, so the crystal violet-iodine complex diffuses from these cells and loses color. The crystal violet-iodine complex remains within the Gram-positive bacteria because their cell walls are think and lack the lipid-rich outer membrane of Gram-negative bacteria. Due to the increase in porosity of the Gram negative cells after lipid loss from decolorization, safranin (counterstain) is able to permeate the cell wall of Gram-negative bacteria. Purple (primary stain retaining) indicates Gram positive and red (counter stain uptake) indicates Gram negative. Some organisms and dead or dying cells do not take up or lose the stain appropriately and can not be classified as either Gram positive or Gram negative.

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. Allow the liquid on the slide to dry.
4. Be sure all the liquid on the slide has evaporated before proceeding to heat fixation (or you will explode the cells in the next step).
5. Heat fix (to kill and attach organisms to slide) by rapidly passing the slide (smear side up) through a flame 3 times. Use a slide holder and avoid contact with hot glass.

To Gram stain the desired organism(s):
Use the staining trays and sink area. Be sure you evenly cover all the smears on the slide throughout this procedure.

1. Place your smear on the staining tray. It is important that the slide be level during staining so use paper towels under the tray to get it leveled. If you do, it is much easier to prevent dye from running off the slide during the staining process and to be sure that your smears are evenly covered with each reagent.
2. Dispense just enough Crystal Violet solution (0.5% crystal violet, 12% ethanol, 0.1% phenol) to completely cover each smear and stain for 1 minute. (Crystal violet is the primary stain.)
3. Rinse the slide by lifting it at a 45 degree angle (using gloves or a clothes pin or slide holder) in a very gentle stream of water that is directed above the top smear until the waste water coming off the bottom is relatively clear; drain off excess water by touching the edge of the slide to a paper towel.
4. Dispense just enough Gram's Iodine (mordant)to completely cover each smear. Let stand for 1 minute, and rinse thoroughly with a gentle stream of water as in Step 1.
5. Lift the slide at a 45 degree angle and drip Decolorizing Reagent (80% isopropyl alcohol, 20% acetone) down the length of the slide making sure it comes in contact with all three smears. This step is tricky as it is easy to over- or under-decolorize. Do this for 10 seconds and IMMEDIATELY rinse, as in step 3, with a gentle stream of water.
6. Place the slide flat on the staining tray and dispense just enough Counterstain Solution (0.6% safranin in 20% ethanol) to cover each smear. Let stand for 2 minutes; rinse with water as in step 3.
7. Blot dry using the bibulous paper package found in your orange drawer. Do not tear out the pages, just insert your slide and pat it dry.
8. Clean up your area; rise your staining tray. Leave it upside down by the sink on paper towels
9. Observe your stained microbes microscopically following carefully the procedure for using the the oil immersion objective on your compound brightfield microscope BISC209: Microscopy described in the protocol pages of BISC209.

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 remember to set up overnight cultures for 8 of your isolates the prior Friday!

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. Also label a plate for our positive control, the parent strain to CV026: Chromobacterium violaceum ATCC 12472.
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. Dip the loop into an overnight culture of ATCC 12472 and streak it near to the CV026 streak.
5. Place your plates upside down till next week. If your isolates are producing AI sensed by CV026, you will see a purple pigment.

LAB #5 and field trip #2: Colony PCR and identification of our isolates

The organisms you've been working with for your cheese exhibit interesting properties. At this point, you should know what they look like, what their gram stain is, whether or not they interact with each other via growth inhibition or promotion and if they produce AI detectable by CV026. Today we will use a molecular tool to identify what genus your organism is from. We will be amplifying the 16S rRNA gene from the frozen aliquots you prepared in lab 2. This gene is well conserved among all cellular life forms - so much so that you can build a phylogenetic tree of all cellular life using an alignment of the sequences from this gene (or its homolog). To see and learn more about this tree read the primary paper by Carl Woese here.

Boiling lysis of our isolates

1. Take your 1.5 ml tubes containing your isolates in water out of the freezer and place on ice.
2. Cap each of the tubes securely with a cap lock.
3. Place them in a floating rack and add them to the boiling water. Set a timer for 5 minutes.
4. After the time is done, take your rack out of the water and place on ice immediately.
This is the lysate we will use for PCR below

PCR
We are utilizing "universal" primers to amplify the 16S rRNA gene from the isolates. Why are these primers called "universal"? Can you think of how they were designed? What organisms may they detect? Which may be excluded?

**ALL ON ICE**
For each PCR reaction you will need to generate the following mix:

Reagent Quantity Calculated Total
Phusion 2x Mix 10 ul
10uM F primer 0.5 ul
10uM R primer 0.5 ul
Water 8 ul
lysate 1 ul ----

You'll be doing one PCR reaction for each of your isolates. So, for example, if I had 10 isolates, I would make up what's called a master mix. I'd need 11 total reaction (one for a "blank" that won't have DNA template added). Add 1 to this number so that you end up with enough total mix. That leaves me with 12. I'd multiply each of the amounts above by 12 to get this table:

Reagent Quantity Calculated Total
Phusion 2x Mix 10 ul 120 ul
10uM F primer 0.5 ul 6 ul
10uM R primer 6 ul
Water 8 ul 48
lysate 1 ul ----

Remember to keep this all on ice! and Note that I do not multiply my lysate by 12!

1. Make your master mix following the calculated amounts above.
2. Gently mix the reaction tubes by flicking - do not pipette up and down!
3. The mix is a total of 19 ul per reaction. Label your PCR tubes according to the isolate lysate that will be added. To each tube add 19 ul of master mix. If there are bubbles in your tubes, spin them down before adding your lysate.
4. Add 1 ul of your lysate to each tube.
5. Take the tubes over to the PCR machine and we'll start the reaction. It should take about 1 hour to complete.

While the PCR machine is working, we will travel on our second field trip - to the Oak Knoll Dairy. There we will hear about how they raise their cows and how they process their milk for cheese making. When we return, we'll use the LAB media to select for Lactic Acid Bacteria in the milk.

LAB #6:

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