||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.
Begin to research how to select/enrich for particular soil bacteria.
|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: .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 for culturable bacteria of specific groups.
Finish Plate Count & quantify cultured microbes;
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 enrichment and selection cultures; Assess your isolation streaking; check on your plate count plates and move any in danger of overgrowing to cold room; 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;|
Research likely bacterial members of your soil community, differentiating metabolic needs or capabilities, physicial differences, and other useful or differentiating characterisitics.
Graded Assignment: Make or Fill out a Table of the relevant morphologic, physical, and useful metabolic characteristics of expected genera of soil bacterial that you are attempting to find in your habitat. Be sure to read the directions for this assignment found at: Lab 2 Assignment: Assignment: Table of Cultured Soil Bacteria Characteristics.
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. View stained total soil community photomicrographs (provided by your instructor) and do calculations.
||Homework: Quantify the number microorganisms in your soil community from the photomicrographs prepared and stained by your instructors from your Lab 3 soil extract. Compare this estimation to the CFU/gram of soil (dry wt) calculated in LAB 2 and try to explain the disparity, WITHOUT criticizing your execution of the plate count protocols or other procedures involved.
|Isolation of Culturable Bacteria: Examine enrichment and selective media and pick unique isolated colonies of your soil bacteria to acquire pure cultures for each organism
Exoenzyme assessment; CLPP analysis and calculations of carbon source utilization
|Make sure you understand the CLPP analyses and calculations;
|Homework:Turn in calculations and graphs for CLPP analyses of carbon source testing.Introduction section of final paper. Read over the Introduction to the Project page in the wiki to identify the topic and experimental questions addressed. Include the history of the "Great Plate Count Anomaly" (the disparity between culturable and unculturable soil community bacteria). Use and cite references in journal Cell format. A reference that may be helpful is :| Uncultivated Microorganisms by Slava Epstein in Microbiology Monographs Vol. 10, 2009 DOI: 10.1007/978-3-540-85465-4 available as an e-book through Springerlink at the Wellesley College Library or as a pdf file in the Resources section of the lab Sakai site.
|Isolation & of Culturable Bacteria: Make new cultures from each of your pure cultures of your soil bacteria isolates.
||Homework: Write the M&M section (what you've done so far) for your final paper. Refer to the assignment directions in the BISC209/S12:Assignments section.
|Identification of soil community bacteria by 16S rDNA sequencing:Set up a pcr to amplified the 16s rRNA gene from 3-4 (per student) well-isolated, bacterial colonies in pure culture.
Isolation & of Cultured Bacteria: Perform physical characteristics tests: smear slide, Gram stain, Confirm Gram stain with selective media, start an antibiotic production test.
|Send away pcr products (amplified 16s rRNA gene) from pure cultures of bacteria isolated from your soil community for 16S rDNA sequencing and identification. Results should be back within 2 weeks.
||Homework: Write a brief summary of the theory behind using the 16s rRNA gene to identify our bacterial isolates. Your instructor will post appropriate reference papers to Resources in Sakai.
|Cultured bacteria assessment: Start SIMs test; Continue antibiotic production test; Read Gram stain confirmation by selective media, Start Bacterial interactions tests.
||Complete, read, or set up fresh cultures as needed.
||Homework: Write a partial Results section with figures/tables: Read more about this assignment at: Assignment: Partial Results section with Fig/Tables. Refer to the Results section (including the information on effective figure design and how to write figure legends in the "Guidelines for Science Writing" found in the Resources section of the wiki. Using other published journal articles as models is also an effective way to learn to write a good results analysis.
|Cont. Cultured bacteria assessment: Complete and read antibiotic production test, quorum sensing, interactions, SIMS tests. Confirm results with special stains or other motility tests as needed.
||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: Results section on isolates work and Graphical Abstract.
|Data Analysis & Science Writing Workshop I
||Homework:Analyze your 16S rDNA sequences and identify as narrowly as possible the bacteria that you isolated from your soil community. Make a table/figures or other displays of these data.
|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 "virtual" poster presentation
|Group "Virtual" Poster presentation
||End of lab