BISC209: Lab8

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Wellesley College-BISC 209 Microbiology -Spring 2010

Contents

Lab 8: Lab Practical

Today you will have a lab practical exam designed to assess your mastery of basic tools, techniques, and theoretical information on which the field of microbiology is based.

Culturable Bacteria Identification continued

Read last weeks tests; finalize testing

Assignment

Results section with figures/tables:
Characterization of Culturable Soil Bacteria: Functional roles and relationships by Physical and Metabolic Assessment

You will write part of the results section for your final paper as a graded assignment to turn in at the beginning of Lab 9. You have worked hard this semester to isolate, characterize and identify some of the thousands of microbes that contribute to the soil community of your habitat. You used DNA sequencing of the 16S rRNA gene to identify a not-completely random sample of bacteria (remember pcr bias and other unavoidable factors that contribute to sampling bias). You also used traditional culture and isolation methods, combined with biochemical and physical tests, to obtain morphologic and metabolic assessment of a few of those soil community bacteria. Now it's time to start the synthesis of all those results into a cogent and clear analysis of the evidence that answers or sheds some light on our original experimental question: What are the functional roles and relationships among soil bacteria in this community?

You spent many weeks just trying to coax some of these bacteria to grow outside of their soil habitat. You spent additional time separating them from other members of their community. Both of these tasks were difficult and time consuming. Why did we have to do all that when we know that these bacteria exist naturally, quite happily, as part of a large diverse community in the soil? One answer, possibly the most important and difficult goal, was that we needed to get the bacteria isolated to pure culture to be able to send off an adequate number of separated 16S rRNA genes to serve as the template for DNA sequencing to give us the position on the phylogenic J tree where our cultured bacteria belong. Taxonomic identification allows us to see phylogenetic and predict evolutionary relationships among bacteria.

Unlike the relative ease (if we forget how many times we had to repeat our experiments) of using expensive, sophisticated molecular tools (plasmid cloning vectors and genetically modified transformed bacteria) to separate and replicate enough copies of each of the amplified 16s rRNA genes from the culture-independent bacteria that we will study, we also wanted, in the case of our cultured bacteria, to know their "names". It may be possible to determine, not just our isolates' clad, but to get a genus and species id from our sequencing. If we do obtain that information, we can use it to evaluate whether or not our biochemical and physical test results are typical or atypical of the members of that group. That information belongs in your results section as part of your data analysis. However, as you now know, "species", "strain" and even "genus" can be debatable distinctions (remember the case of Shigella and E. coli)depending on your orientation (environmental or clinical). Since our orientation is more environmental than clinical, we want to use these comparisons of our metabolic and morphologic test results in the discussion, not so much for the purposes of proposing new strains, but rather to propose possible reasons for the acquisition of a new phenotype by horizontal gene transfer among members of a community. Might that unusual acquisition or loss be related a symbiosis or competition with community members that were identified through culture or culture independent means?

Another reason for presenting all the morphologic, physical and biochemical test results in the results section on your pure cultures is to have the evidence in your paper that will allow you to explore the possible functional roles of members of the community in your discussion. As you learned in lecture, phylogeny is not function; but function is important for a good discussion of the diversity of bacteria in your soil community. For example, if you found out that one or more of your bacteria secretes an antibiotic, what might that imply about at least one of that microbe's roles in its community and, possibly, in the larger world? If you found out that you have cellulolytic or nitrogen fixing bacteria in your community or that they are highly motile, you are supplying other evidence in your results section to prepare the ground for writing an interesting discussion section about the variety, diversity, and possible roles and relationships among the bacteria that were identified either by culture-dependent or culture-independent means.

You will not write a discussion section for this assignment, but a good results section is crafted to present all the evidence you have for the points you want to make or relationships you want to propose in your discussion. The results section will not propose conclusions as to roles or relationships among members of this soil community since we purposely worked hard to study these bacteria in isolation, therefore, don't know exactly how they work together. This results section will point out relevant test findings and observations that will allow you to discuss possible roles and relationships in your discussion, including putative symbioses.

Remember that any results section should start out with a restatement of the experimental goals of this part of the paper: "In order to investigate ....". Then it summarizes, BRIEFLY, what you did, without repeating the whole materials and methods section. The results narrative does not start describing and analyzing the data (evidence) until it BRIEFLY frames for the reader how the results were obtained and why. The most important findings are found in two forms in the results section: in a narrative description that describes and analyzes all important findings and in visual representations of some of those data: the figures or tables. There is extensive information about how to write a results section in the Guidelines to Science Writing in the Resources section of the wiki. Remember that you are preparing your reader for your discussion section. Make sure to limit or omit describing results that aren't pertinent to where you plan to go in your discussion of the diversity, functional roles, and phylogenetic relationships of bacteria in a particular soil community.

You will not get to analyze the sequencing results until next week. Nevertheless, you can write this partial results analysis without the complete id of your bacteria. You may, for this assignment, refer to your bacteria by their preliminary group names and by their code numbers. When we know more about the clads or "names" of our isolates after our sequences are analyzed, you can go back and expand this id information.

Finalize the classification of your culturable isolates. Start discarding your cultures. If you had a particularly interesting or rare isolate (violacein producing, antibiotic secreting, fruiting myxobacterium, cytophagia, methanol oxidizing or ammonia denitrifying, etc., or something unclassifiable, please give your lab instructor a pure culture plate of it with its code number and its identification information from your RDP work so we can start a strain collection of interesting soil bacteria. Thank you for your hard work and dedication this semester to this project. We hope you are as pleased and proud of the success of your hard work as we are!

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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