||IN LAB WORK________
||OUTSIDE OF LAB WORK________
|Introduction to Microbiology Lab
Tools and Techniques of Microbiologist: Aseptic Transfer,
Intro to Microbial Diversity Project:Soil sampling in Greenhouse habitat;Begin culture of soil organisms: make soil extract and begin enrichment for Azotobacter and Hypomicrobia 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.
|Calibrate Micropipets; Genomic DNA isolation for Culture-Independent Bacteria ID;
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 denitrifying and nitrogen cycling 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 & nitrogen cycling analysis;
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.
|Identification of soil community bacteria by 16S rDNA sequencing: PCR Amplification of 16S rDNA with "universal" bacterial primers and proofreading polymerase
PCR product clean-up
Run a gel of your cleaned-up pcr product to assess the success of your 16s rRNA gene amplification. Instructor will finish, photograph gel and post the labeled image to the data file;
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 and nitrogen cycling.
|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.
|Identification of culture-independent soil community bacteria by 16S rDNA sequencing: Clone 16s rDNA from successful pcr products into cloning vector
Transform cloning vector into E. coli and select for transformants on selective media.
Isolation & of Culturable Bacteria: Make new cultures from each of your pure cultures of your soil bacteria isolates.
|Check to see if you have E. coli transformants on your selection plates. If not, contact your instructor.
|Homework: Write the M&M section (what you've done so far) for your final paper. Refer to the assignment directions in the BISC209/S11:Assignments section.
|Identification of culture-independent soil community bacteria by 16S rDNA sequencing:Select 48/per pair (96 per soil habitat) well-isolated, transformants from selective media and grow in broth overnight.
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 frozen glycerol stocks of overnight cultures of transformed bacteria for 16S rDNA sequencing. Results should be back within 2 weeks.
||Homework: Write a brief summary of the theory behind the following techniques that we used to identify our bacterial species by molecular tools: genomic DNA isolation, polymerase chain amplification of part of the 16s rRNA genes, use of the Zero Blunt® TOPO® PCR Cloning Kit to create a library of unique plasmid vector with our 16S rRNA gene inserts and then select, One Shot® TOP10 Competent E. coli Cells that allowed us to select and separate our 16S rRNA genes for sequencing, and DNA sequencing by the Sanger method.
|Cultured bacteria assessment: Start SIMs test; Continue antibiotic production test; Read Gram stain confirmation by selective media, Start Quorum Sensing & 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.
||Make sure you have signed up for an account on the RDB and received a username and password before you come to RDP Lab (9). Link to the RDB:
|Homework: Results section on isolates work and Graphical Abstract.
|Meet in a computer lab (TBA) for data analysis of your 16S rDNA sequencing results.
||Complete all work on cultured bacterial isolates to provide evidence for functional co-operation and/or competition in your soil community
||Homework: Study for your Lab Practical that will be given in Lab 10. Your instructor will give you more instructions about what that test will include and how to study.|
Analyze your sequencing data from 16S rDNA sequencing of the soil genomic DNA sequences and start preparing phylogenic trees with identification information for the bacteria in your soil community. Make figures or other displays of these data to supply evidence for the diversity and evolutionary relationships of some of the bacteria in your soil community .
|Conference with your instructor to discuss your data analysis& poster presentation.
||Homework: Prepare your group (4 students- 1 soil community) "virtual" poster presentation to be presented in LAB 11
|"Virtual" Poster presentation in groups by habitat
||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 and a link to Wellesley Library information
|Meet with your instructor to discuss your final paper.|
When & where at discretion
of your instructor.
|Final Paper due on your lab day.
When & where at discretion
of your instructor.
|Information on your final paper found at: Assignment: Final Paper
||End of lab