BISC314:Full Protocol

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'''Background'''<br>
'''Background'''<br>
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The morphological characteristics of bacteria, including size, shape, and arrangement, can be seen by staining a bacterial smear so that individual bacterial cells are distinguishedFor most species, morphologic characteristics are genetically determined and, thus, typical of the species. Generally, bacteria range in size from approximately 0.2 µm to 3.0 µm.  The basic shapes are spherical (coccus), rod-shaped (bacillus), curved (vibrio), or helical (spirillum, spirochete). However, there are some species of bacteria that show considerable within species variation (termed pleomorphism).  For example, both ''Mycoplasma'' (bacteria lacking the rigid cell wall of most bacteria) and ''Arthrobacter'' (a type of soil bacteria) show forms ranging from coccoid (round) to rodlike to filamentousSome pathogenic species such as ''Mycobacterium tuberculosis'' and ''Corynebacterium diptheriae'' are also pleomorphic.
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Bacteria come in many different shapes (see this [http://en.wikipedia.org/wiki/Bacteria#Morphology 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 fisheri''is a curved (vibrio) bacterial organism. <br>  
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Bacteria are often individual but some species take on a group arrangement based on the way cell division and subsequent separation of the daughter cells occurMost of the Gram-negative bacilli (rods) are found singly (''Escherichia coli'') or, sometimes, characteristically in pairs (''Klebsiella pneumoniae''). The cocci, ''Streptococcus pneumoniae'' and ''Neisseria'' are called diplococci because they tend to pair. Bacilli found in chains include some ''Bacillus'' species. The coccus genera that most often form chains are named ''Streptococcus'' for this defining arrangement. A few cocci make regular packets of four or eight (Micrococcus) and some are seen in irregular clumps that resemble bunches of grapes (Staphylococcus). Short rods that form parallel lines, called palisades, include the species that causes diptheria, ''Corynebacterium diptheriae''.  Chinese character formation describes a sharply angled bacterial arrangement.  Because of their waxy cell walls, ''Mycobacterium'' species are difficult to emulsify and tend to stick together in clumps. The pathogen in this group, ''Mycobacterium tuberculosis'', may form long cords of cells.<BR><BR>
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You will make smears of all of your isolatesFirst, 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. <br>
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Keep in mind that individual cells may show deviations from these standard forms.  For example, cocci of ''Neisseria'' show flattened sides, making them bean-shaped. The rods of ''Corynebacterium'' and ''Mycobacterium'' often appear club-shaped, with swollen ends or knobs.  Both groups may show irregular staining.  The diplococci of ''Streptococcus pneumoniae'' often appear slightly elongated and lancet-shaped (with one flattened end and one tapered end).<br>
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<b>Making a Smear</b>
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<b>Making a Bacterial Smear</b>
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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)<br>
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'''Activity 2:  Prepare a Bacterial Smear Slide of ''Serratia marcescens'' and ''Staphylococcus epidermidis'' and a mixture of both bacteria. '''<BR>
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2. Place a small loopful of deionized water on the slide as far from each other as possible. <br>
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This protocol is found below and at [[BISC209: Preparing a bacterial smear slide]] in the [[BISC209:Protocols | Protocols]] section of this wiki.
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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.<br>
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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.  
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'''Preparing a bacterial smear slide'''<BR>
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1. Label a clean, glass slide with a graphite pencil on the far left of the slide with SE, SM, MIX. (The decolorizer in the Gram stain can remove your labels if you use pen or wax pencil.)  By convention, labels (top to bottom) match smears (left to right).<BR>
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2. Place three very small loopfuls of deionized water on the slide as far from each other as possible. (You can use the deionized water bottle on your bench; remove the cover and dip your loop in since sterility is not required for this step.<br>
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3. Flame the loop, allow it to cool for a few seconds and touch the cooled loop to a colony of ''S. epidermidis'' , 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.<BR>
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4. Place the loop with the bacterial growth into the drop of water on far left of 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. Be sure to leave room for the adjacent drop of water to be spread to a similar size without mixing the two smears.<br>
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5. Reflame the loop.<br>
5. Reflame the loop.<br>
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6. Repeat step 4 with the ''Serratia marcescens'' in the middle drop of water and then, without flaming your loop, touch the loop to the drop of water on the far right and mix briefly. <BR>
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6. Add a coverslip to your slide and take it over to the microscope -it is ready for viewing!
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7. Reflame your loop and touch it to a ''Staphylococcus'' colony again. Place the loop in the far right drop of water mixing it with the ''Serratia'' and spread the drop as in step 4 to create a mixed smear.<BR>
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6. Allow the slide to air dry completely! Be sure ''all'' the water on the slide has evaporated before proceeding to heat fixation!!! This drying step is crucially important. If you are impatient, you will "explode" the cells in the next step . <br>
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7. Heat fix (to kill and attach organisms to the slide) by quickly passing the slide (smear side up) through a flame 3 times. Use a clothes pin or slide holder and avoid contact with hot glass.<BR><BR>
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Revision as of 13:44, 6 August 2010

BISC314: Environmental Microbiology

Home        People        Full Protocol        Laboratory Schedule        Lecture Syllabus       


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.


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. 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!

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