||IN LAB WORK__________________
||OUTSIDE OF LAB WORK________
|Introduction to Microbiology Lab
Tools and Techniques of Microbiologist: Aseptic Transfer,
Intro to Soil Microbial Community Project:Soil sampling in Greenhouse habitat;Begin culture of soil organisms: make soil extract and begin enrichment for selected bacteria;
Start Plate Count of Culturable Soil Organisms
|Visit the greenhouse and make notes on your selected habitat. Research how to select/enrich for particular soil bacteria. View stained total soil community photomicrographs (provided by your instructor) and do calculations to enumerate microorganisms/g wet soil through a culture independent direct count.
||Homework: Read all of Lab 2 & outline or make flow diagrams of your lab work in your lab notebook. |
Check Resources section of wiki for information about organizing your lab notebook.
Graded Assignment: Turn in at the beginning of Lab 2, a Discussion with References of how the enrichment culture techniques and media you will use will select soil bacteria of the specific groups we seek and differentiate them from other microbes in the community. Be sure to read the directions for this assignment found at: Assignment: Enrichment/Selection/Differentiation of Culturable Bacteria.
|Finish Plate Count & quantify cultured microbes by culture dependent method; Compare to culture independent method
Practice Streaking for Isolation: Make soil extract from dried soil and set up isolation of spore-forming bacteria;
Continue enrichment & isolation of selected groups of bacteria
|Check on your soil bacterial cultures; Assess your isolation streaking; BRING TO LAB 3 A NEW SOIL SAMPLE Collected from your group's sampling site. (Materials available for pick up in the lab.) Do not collect it until the day of lab.
||Homework Search the web for photos of colonies of desired bacteria or to match colonies you've found;|
Graded Assignment: Compare your culture dependent and culture dependent estimations of the CFUs/gram of soil (dry wt) calculated in LAB 2 and think about the disparity. (DO NOT explain the discrepancy by criticizing your execution of the experiments!) Write a draft Introduction section of your final paper that includes a discussion of the "Great Plate Count Anomaly". Be sure to read the full directions for this assignment found at: Lab 2 Assignment: Assignment: Introduction.
Isolation of Culturable Bacteria: Evaluate your success at streaking for isolation;
Start CLPP: Community Level Physiological Profiling: Carbon source utilization;
Make another soil extract and serial dilution to evaluate carbon source utilization;
Continue selection & isolation, of desired bacterial groups;
Start community exoexyzme profiling (starch & cellulose digesters, phosphate solubilizers).
|Collect data from BIOLOG ECO plates. Check on cultures and continue isolation.
||Homework: Construct a table (with properly formatted legend) of your experimental evidence for the abundance of microorganisms in your soil community. Write a draft results section. Consult the full directions for this assignment found at: Lab 3 Assignment: Colony Count vs. Direct Count Enumeration
|Isolation of Culturable Bacteria: Examine cultures and pick unique isolated colonies of your soil bacteria
Exoenzyme assessment; CLPP analysis and calculations of carbon source utilization
|Make sure you understand the CLPP analyses and calculations;
|Homework:Analyze prevalence of microbial starch & cellulose digesters and phosphate solubilizers in the soil community. Analyze CLPP data for carbon source utilization; turn in spreadsheet with calculations and make graphs turned into figures with legends. More information about how to use your data in these calculations can be found at BISC209/S12: Assignment_209_BIOLOG.
|Isolation of Culturable Bacteria: Make new cultures from each of your pure cultures of your soil bacteria isolates.
Use a pure culture of each isolate to its amplify 16srRNA gene by pcr. Clean up pcr product. Perform agarose gel electrophoresis of pcr product to assess success of amplification.
|Send away successfully amplified 16s rDNA for DNA sequencing at a commercial sequencing facility.
||Homework: M&M: Compose a draft of your Materials and Methods section of your final paper with the following three general sections:|
1)Community level physiological testing: carbon source profiling, exoenzymes profiling;
2) Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA;
3) Selection and isolation of soil community bacteria to pure culture. More information can be found at Lab 5 Assignment: Materials & Methods
|Isolate Testing: Perform physical characteristics tests: smear slide, Gram stain, confirm Gram stain with selective media, start an antibiotic production test.
||Homework: Write a brief summary of the theory behind the following techniques that we used to identify our bacterial species by molecular tools: polymerase chain amplification of the 16srRNA gene and DNA sequencing by the newer fluorescent-labeled ddNPTs chain -termination (Sanger) method. Directions found at: Lab 6 Assignment: Assignment: Theory Summary
|Isolates Testing: Start motility test and MNM tests; Continue antibiotic production test; Read Gram stain confirmation by selective media, Start Bacterial interactions test.
||Complete, read, or set up fresh cultures as needed.
||Homework: Annotated Bibliography of appropriate references for the discussion section of your paper|
Graphical abstract: See models in research reports found in recent issues of the journal Cell. A description and examples of Graphical Abstracts can be found at http://www.elsevier.com/wps/find/authorsview.authors/graphicalabstracts . More information on this assignment found at: Assignment: Annotated Bibliography/Graphical Abstract
|Cont. Isolates testing: Complete and read antibiotic production test, interactions, MNM tests. Special Staining as indicated.
||Homework: Study for your Lab Practical. Your instructor will give you more instructions about what that test will include and how to study.
|Make sure you have signed up for an account on the RDB and received a username and password. Link to the RDB:
|Homework: Write a draft results section of your work on your isolates. Construct a table of the tests performed on the isolates from your soil community (your isolates and your teammates from the same sampling site) and write a complete results narrative. You should include in the table: Gram stain, description of the colony morphology, description of the individual bacteria and any characteristic arrangement (cocci, rods, with descriptors ie., large, small, bullet shaped, in chains, etc.), evidence of spores (either endospore stain positive or visualization of empty areas in vegetative cells on Gram stain), motility, evidence for nitrogen cycling capabilities, sole carbon source (citrate or mannitol) . Make a separate figure from a photograph of your interactions assay and your antibiotic production assays and analyze those data.
|Data Analysis & Science Writing Workshop I
||Homework: Analyze your 16s rRNA gene sequences identification of your isolates and make a table of bacteria isolated from your soil community. Construct a tree from your group's data showing taxonomic diversity among these bacteria. We will use your figures in a Science Writing Workshop next time.
|Science Writing Workshop II
||Homework: Write your final paper in the form of a scientific paper. See the Resources section for an extensive handout on How to Write in Scientific Style. |
Information on your final paper found at: Assignment: Final Paper
|Final Paper Due by 4pm uploaded to Sakai AND in hard copy to your instructor's office.
||Homework: Prepare your group presentation. See information in Resources in Sakai lab site
||End of lab