BISC209: Lab7

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(Culturable Bacteria Characterization continued)
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(Culturable Bacteria Characterization continued)
 
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=='''LAB 7: ID of culturable soil bacteria: Agarose Gel Electrophoresis & Clean-UP of PCR Amplified 16S rDNA from pure colonies of soil bacteria'''==
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=='''LAB 7: Characterization of culturable soil bacteria'''==
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'''Agarose Gel Electrophoresis'''<BR>
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Before we get to work on characterizing our isolates, be aware of where are in indentification of those bacteria by 16s rDNA sequencing.<BR><BR>
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You will run your cleaned-up pcr products on a gel of 1.0 agarose solution (w/v) in 0.5x TBE buffer (10x=0.25 Tris, 1.9M Glycine, 13mM EDTA) with SybrSafe™ stain to assess your success at amplifying 16s rDNA from each of your soil bacteria that you want to identify.<BR><BR>
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DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA. <BR><BR>
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We have sent out all the amplicons that you prepared last week for DNA sequencing of the 16s rRNA gene. We must also send an appropriate primer. What's the sequence of such an appropriate primer? What's going on in those automatic sequencing machines in Danvers? Make sure you understand how Sanger sequencing works. There will be some theoretical questions in the lab practical.  We should have the sequencing data back by LAB 9 when we are scheduled to meet in a computer lab and learn to analyze the data generated so we can find out, we hope, a lot about the diversity of the soil bacteria in our habitat of interest.
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[[Image:BISC110_gel2.jpg]]<BR><BR>
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'''RUNNING THE GEL'''<BR>
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Keep your pcr products on ice until your instructor tells you that the gel is ready to load.<BR>
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1. Pipet 1μL of loading dye (0.25 XC, 30% glycerol, 0.1mg/mlRNase) for each of your pcr products on a piece of parafilm (use a P10 and space out the drops so they are not near each other). You should have 5 pcr products per person: 4 bacteria to id and one neg (water) control. <BR>
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2. Using a new tip, pipet 5μL of a pcr product into one of the drops of loading dye, mix with the pipet tip and then draw up all of it and dispense it into a well of the gel.<BR>
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3. Fill out the gel template which identifies which bacterial strain is in each well.(Use the unique code names we devised previously). Additionally, please label the gel template with the group of numbers for your isolates as, example: Tues.; Red; Student initials-S301-S306. Make a copy of your habitat team's part of the template in your lab notebook and make sure you know which code number corresponds with which isolate you have been gathering test results.<BR>
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4. Your instructor will add the ladder marker and a positive control and start the current when all students have loaded their samples.<BR>
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5. When the gel has finished (usually 45 min. at 100volts), your instructor will photograph it under UV light, label the lanes from the template, and post the photo to the data folder on the conference. We will use these same code identifiers when we send off the pcr products for 16S sequencing so your pcr product microfuge tubes MUST have these code identifiers and they must be clear.<BR><BR>
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How will you judge a successful amplification? How many fragments and of what size do you expect to see? Remember that you used the same "universal" bacteria primer pairs that we used in our other amplification of 16s rDNA from the genomic soil DNA extract but you used a less "picky" DNA polymerase, Taq, rather than an expensive proof-reading polymerase.  <BR><BR>
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We will send out all the successful amplicons for DNA sequencing of the 16s rRNA gene. We must also send an appropriate primer. What's the sequence of such an appropriate primer? Your instructor will give you more instructions on whether you or she will load your successful amplicons (pcr products)into a 96well plate for freezing and shipping to Agencourt/Beckman.
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==Culturable Bacteria Characterization continued==
==Culturable Bacteria Characterization continued==
Continue following the protocols for [[BISC209: Roles of soil Microbes |Tests to determine the role of a soil isolate]]<BR><BR>
Continue following the protocols for [[BISC209: Roles of soil Microbes |Tests to determine the role of a soil isolate]]<BR><BR>
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Read and assess tests (use the appropriate protocols-refer to lab 5 and 6).<BR><BR>
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Concentrate on completing and evaluating the tests you have in process on your isolates (use the appropriate protocols-refer to LABS 5 and 6; in the Protocols section: Enzymes, Motility, and Carbohydrate Fermentation sections, addition to the Roles protocols.<BR><BR>
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Continue with/add new differential ID test (see the appropriate protocols-refer to lab 5 and 6)<BR>
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Continue with/add new differential ID tests such as an SPECIAL STAINS (see the Special Stains section in the Protocols) that seem appropriate. For example, all Gram positive bacilli or any bacteria that showed unstained area in the cells when Gram stained should be stained for endospores. <BR><BR>
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All bacteria that were positive or ambiguous for motility in SIMs medium should be looked at in a wet mount and if positive in the wet mount, you could try the Flagella stain. All the "swarmers" (those bacteria that spread all over the plate)should be looked at in a wet mount too.<BR><BR>
 +
Highly mucoid (sticky and wet) colonies should be tested for the presence of a capsule using the capsule stain protocol. <Br><BR>
 +
If your Gram stain results were ambiguous or not what you expected from the descriptions of the bacteria that grow on the Enrichment media you used, you should probably repeat those Gram stains. Getting the Gram reaction right is crucial to the discussion of where these bacteria belong phylogenically and functionally. <BR><BR>
 +
 
 +
 
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If there are extra test media available you may repeat any other tests that were ambiguous and/or add any you think might be useful for identifying your organism.  You can also make a list of tests that would allow you to identify your isolates to species that were not available this year. 
   
   
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If you have an interesting new isolate it is not too late to check its Gram stain reaction and morphology, motility, and a few simple "role" tests performed over the last few weeksSelect tests that can be completed within 2 more weeksBy this point, you should be wrapping up collecting test results.
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By this point, you should be wrapping up the testing, beginning to discard your plates and checking with the instructor about freezing glycerol stocks of some of the isolates.   
 +
 
 +
Pleaes start using the data sheet (located in the Data folder of the lab conference) or one of your own design to leave an electronic record of the isolates you have worked with this termOrganization of all this information is key. Be sure that you have added the unique code numbers for your isolates so we can correlate identity from sequencing with your functional, morphologic, and metabolic tests.  If you are pretty sure you know the genus of an isolate from our characterization tests, please add that as well.
==CLEAN UP==
==CLEAN UP==
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==Assignment==
==Assignment==
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Study for your Lab Practical<BR><BR>
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Study for your '''Lab Practical'''. <BR><BR>
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Begin to wind down and finalize the characterization of your culturable isolates.
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In Lab 8 you will have a LAB PRACTICAL, an exam that tests your ability to function as a microbiologist in the lab. There will be two main categories of assessment: testing practical skills such as your ability to perform crucial techniques (aseptic transfer, streaking for isolation, performing a Gram stain, micropipetting, etc.) and testing for your understanding of the biological basis behind the work that you've done this semester. For example, you assess the OF-glucose test for yellow or blue color in certain regions or tubes to distinguish facultative fermentors of glucose from bacteria that have only an aerobic pathway for catabolism of glucose or no way to break down that nutrient; you also learn from that test whether or not the bacteria can catabolize peptone. What the practical will assess on the OF-glucose test is not that you can perform the test without access to the directions, but that you understand how to evaluate the results in terms of the general biological mechanism of the test (pH change) and the metabolic capabilities implied from the results (such as my description of glucose and peptone catabolism above). Another example: we might give you a starch plate flooded with iodine and ask you which, if any, of the bacteria forming colonies on this plate are capable of digesting starch and/or what is the ingredient in the medium that allows this evaluation? <BR><BR>
 +
 
 +
Wondering how to study? A lot of what we will evaluate you on, you already know how to do in your sleep, such as streaking for isolation using proper technique. You do not have to memorize the procedures in the wiki. You will have access to the directions that are printed in the wiki for performing techniques, such as Gram staining; however, you will ''not'' have access to how to read those tests or to the theoretical background information about those tests, such as the cell structural differences that the Gram stain uses as its differentiating principle. You should know that sort of information by studying the background information and protocols found in the Protocols section of the wiki for all the work that have you performed this semester. The protocols in the wiki that describe tests that you have not done (like Enterotubes) are NOT included in the practical. However, even if you didn't do or get a positive test from those tests performed on your isolates, you should know how to evaluate the tests we all set up, such as the Oxyswab test or the SIMS medium and to recognize a positive vs. a negative.<BR><BR>
 +
 
 +
Don't forget that understanding of the molecular tools we used for culture independent 16srRNA analysis and what we can learn from sequencing this gene ARE practical material. Your homework assignment for last week should be a good study guide for that material as is all of the background information in the wiki in LABS 1-6 and in the general information page for our project in the main menu.<BR><BR>
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'''Begin to finalize the characterization of your culturable isolates and start to analyze your data''' in an attempt to organize your thinking about our experimental question: What bacteria comprise the soil community in the particular habitat I am investigating and, more importantly, what are the functional and phylogenetic relationships among those bacteria that I have studied directly or that I know are there from our culture independent sequencing? We have investigated directly in your roles tests (starch digestion, antibiotic production and sensitivity, cellulose digestion, ammonification, carbohydrate and other nutrient breakdown pathways, etc. ) and we have tested directly only a few of the thousands of enzymes these bacteria may possess. We have tried to find out some things about their structure, their ability to do self directed motility, and we have looked for capabilities involved in protecting themselves from adverse environmental conditions (capsules, endospores) or from competition (antibiotic production). Don't forget to analyze the enrichment media we used. The idea of "selecting" particular bacterial group on special enrichment media is based on the ability of members of these groups to use certain nutrients for carbon, electrons, or energy that other competitors can't use or to provide growth conditions that members of these groups prefer or can tolerate that other competitors don't like. You can learn a lot about the unique properties of your cultured bacteria but don't forget to think about how those capabilities complement those of other bacteria found in your habitat. We are particularly interested in potential symbioses or how competitors in the soil carve out a niche and thrive. <BR><BR>
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You should not expect to get complete answers to our questions from your data, but you should get more than enough information to be able to propose from the evidence you have gathered some interesting functional roles and relationships amoung the bacteria you have identified and characterized.
==Links to Labs==
==Links to Labs==

Current revision

Wellesley College-BISC 209 Microbiology -Spring 2010



Contents

LAB 7: Characterization of culturable soil bacteria

Before we get to work on characterizing our isolates, be aware of where are in indentification of those bacteria by 16s rDNA sequencing.

We have sent out all the amplicons that you prepared last week for DNA sequencing of the 16s rRNA gene. We must also send an appropriate primer. What's the sequence of such an appropriate primer? What's going on in those automatic sequencing machines in Danvers? Make sure you understand how Sanger sequencing works. There will be some theoretical questions in the lab practical. We should have the sequencing data back by LAB 9 when we are scheduled to meet in a computer lab and learn to analyze the data generated so we can find out, we hope, a lot about the diversity of the soil bacteria in our habitat of interest.

Culturable Bacteria Characterization continued

Continue following the protocols for Tests to determine the role of a soil isolate

Concentrate on completing and evaluating the tests you have in process on your isolates (use the appropriate protocols-refer to LABS 5 and 6; in the Protocols section: Enzymes, Motility, and Carbohydrate Fermentation sections, addition to the Roles protocols.

Continue with/add new differential ID tests such as an SPECIAL STAINS (see the Special Stains section in the Protocols) that seem appropriate. For example, all Gram positive bacilli or any bacteria that showed unstained area in the cells when Gram stained should be stained for endospores.

All bacteria that were positive or ambiguous for motility in SIMs medium should be looked at in a wet mount and if positive in the wet mount, you could try the Flagella stain. All the "swarmers" (those bacteria that spread all over the plate)should be looked at in a wet mount too.

Highly mucoid (sticky and wet) colonies should be tested for the presence of a capsule using the capsule stain protocol.

If your Gram stain results were ambiguous or not what you expected from the descriptions of the bacteria that grow on the Enrichment media you used, you should probably repeat those Gram stains. Getting the Gram reaction right is crucial to the discussion of where these bacteria belong phylogenically and functionally.


If there are extra test media available you may repeat any other tests that were ambiguous and/or add any you think might be useful for identifying your organism. You can also make a list of tests that would allow you to identify your isolates to species that were not available this year.

By this point, you should be wrapping up the testing, beginning to discard your plates and checking with the instructor about freezing glycerol stocks of some of the isolates.

Pleaes start using the data sheet (located in the Data folder of the lab conference) or one of your own design to leave an electronic record of the isolates you have worked with this term. Organization of all this information is key. Be sure that you have added the unique code numbers for your isolates so we can correlate identity from sequencing with your functional, morphologic, and metabolic tests. If you are pretty sure you know the genus of an isolate from our characterization tests, please add that as well.

CLEAN UP

1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.

2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.

3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.

4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.

5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.

6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.

7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.

8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.

9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.

10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.

11. Take off your lab coat and store it in the blue cabinet with your microscope.

12. Wash your hands.


Assignment

Study for your Lab Practical.

In Lab 8 you will have a LAB PRACTICAL, an exam that tests your ability to function as a microbiologist in the lab. There will be two main categories of assessment: testing practical skills such as your ability to perform crucial techniques (aseptic transfer, streaking for isolation, performing a Gram stain, micropipetting, etc.) and testing for your understanding of the biological basis behind the work that you've done this semester. For example, you assess the OF-glucose test for yellow or blue color in certain regions or tubes to distinguish facultative fermentors of glucose from bacteria that have only an aerobic pathway for catabolism of glucose or no way to break down that nutrient; you also learn from that test whether or not the bacteria can catabolize peptone. What the practical will assess on the OF-glucose test is not that you can perform the test without access to the directions, but that you understand how to evaluate the results in terms of the general biological mechanism of the test (pH change) and the metabolic capabilities implied from the results (such as my description of glucose and peptone catabolism above). Another example: we might give you a starch plate flooded with iodine and ask you which, if any, of the bacteria forming colonies on this plate are capable of digesting starch and/or what is the ingredient in the medium that allows this evaluation?

Wondering how to study? A lot of what we will evaluate you on, you already know how to do in your sleep, such as streaking for isolation using proper technique. You do not have to memorize the procedures in the wiki. You will have access to the directions that are printed in the wiki for performing techniques, such as Gram staining; however, you will not have access to how to read those tests or to the theoretical background information about those tests, such as the cell structural differences that the Gram stain uses as its differentiating principle. You should know that sort of information by studying the background information and protocols found in the Protocols section of the wiki for all the work that have you performed this semester. The protocols in the wiki that describe tests that you have not done (like Enterotubes) are NOT included in the practical. However, even if you didn't do or get a positive test from those tests performed on your isolates, you should know how to evaluate the tests we all set up, such as the Oxyswab test or the SIMS medium and to recognize a positive vs. a negative.

Don't forget that understanding of the molecular tools we used for culture independent 16srRNA analysis and what we can learn from sequencing this gene ARE practical material. Your homework assignment for last week should be a good study guide for that material as is all of the background information in the wiki in LABS 1-6 and in the general information page for our project in the main menu.

Begin to finalize the characterization of your culturable isolates and start to analyze your data in an attempt to organize your thinking about our experimental question: What bacteria comprise the soil community in the particular habitat I am investigating and, more importantly, what are the functional and phylogenetic relationships among those bacteria that I have studied directly or that I know are there from our culture independent sequencing? We have investigated directly in your roles tests (starch digestion, antibiotic production and sensitivity, cellulose digestion, ammonification, carbohydrate and other nutrient breakdown pathways, etc. ) and we have tested directly only a few of the thousands of enzymes these bacteria may possess. We have tried to find out some things about their structure, their ability to do self directed motility, and we have looked for capabilities involved in protecting themselves from adverse environmental conditions (capsules, endospores) or from competition (antibiotic production). Don't forget to analyze the enrichment media we used. The idea of "selecting" particular bacterial group on special enrichment media is based on the ability of members of these groups to use certain nutrients for carbon, electrons, or energy that other competitors can't use or to provide growth conditions that members of these groups prefer or can tolerate that other competitors don't like. You can learn a lot about the unique properties of your cultured bacteria but don't forget to think about how those capabilities complement those of other bacteria found in your habitat. We are particularly interested in potential symbioses or how competitors in the soil carve out a niche and thrive.

You should not expect to get complete answers to our questions from your data, but you should get more than enough information to be able to propose from the evidence you have gathered some interesting functional roles and relationships amoung the bacteria you have identified and characterized.

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11

Lab 12
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