BISC314:Lab2

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BISC314: Environmental Microbiology

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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

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