||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
||Homework: Visit the greenhouse and make notes on your selected habitat. Begin to research how to select/enrich for particular soil bacteria. References information in the Protocols section of this wiki.|
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: Explanation of selective, differential, and enrichment media and their use in our project.
|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 morphology of likely members found in your soil community, differentiating metabolic needs or capabilities used for culture, habitat range, and role in the soil. Write a summary of the relevant morphologic (shape, arrangement, Gram stain, motility etc) and useful metabolic characteristics (antibiotic producers, cellulose or nitrogen recyclers, etc)of expected soil bacterial genera from each of the groups that you are attempting to find in your habitat.
Graded Assignment: Make a Table summarizing morphologic and function characteristics of groups of soil bacteria likely to be found in your habitat.
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 start a MPN analysis of aerobic heterotropic nitrifiers & 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 and for nitrate, ammonia, nitrite. Read about MPN calculations and be prepared to calculate MPN for the nitrogen cycling organisms in your soil community. Check on cultures and continue isolation. View stained total soil community photomicrographs (provided by your instructor) and do calculations.
||Homework: Write up your procedure for genomic DNA isolation and amplification of 16S rDNA from a soil sample as a Materials & Methods section for your final scientific paper. Use the information in the Methods section in the "Guidelines for Science Writing" handout found in the Resources section of the wiki and/or model your protocol descriptions like those in the journal articles in the References folder on the First Class Lab Conference.
|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: Write the Introduction section of your final paper. Use the Introduction section of the "Guidelines for Science Writing" handout found in the Resources section of the wiki. You will need to use journal articles as sources for a hypothesis on the diversity of the soil bacterial community and the functional roles and relationships among the bacteria in your habitat. There are some articles in the References folder on the First Class Lab Conference to get you started. You will need to cite sources in the Name/Year style of the journal Cell. See the Wellesley library e-data base for recent issues of this journal to use as models for your citations.
|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 following protocols as M&M sections for your final paper: |
Isolation of soil bacteria to pure culture. (You may refer to your organisms by a code number or group name if you don't yet know the genus and species name).
And revise and add to the methods for: Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA
|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, start a motility test by inoculating SIM tube, 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.
|16S rDNA sequencing: Run a gel and Clean-up pcr products of successful amplifications of 16s rDNA from your soil bacteria isolates.
Cultured bacteria ID: Read last weeks tests; Continue with new differential ID tests; Start
Antibiotic sensitivity and nitogen recycling tests;
add other "role" tests as needed.
|Complete, read, or set up differential tests for bacterial id as needed.
||Homework: Study for your Lab Practical that will be given in the first part of Lab 8. Your instructor will give you more instructions about what that test will include and how to study.
Cont. Cultured bacteria ID: cont. with tests to differentiate and id your cultured bacteria.
||Homework: Write a Results section with figures/tables:
Characterization of Culturable Soil Bacteria: Functional Roles and Relationships. (You may refer to each organism with a group name or code number since the full id is still unknown .) 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.
Finish the characterization of your cultured soil bacteria by traditional physical, functional, and metabolic tests. Use the electronic version of The Prokaryotes and Bergey's Manuals to help you. Link to the electronic edition of | The Prokaryotesthrough Springer ebooks;
Link to the electronic edition of | Bergey's Manualsthrough Springer ebooks
|Meet in a computer lab (TBA) for data analysis of your 16S rDNA sequencing results.
||Complete tests to ID cultured bacteria
||Homework: Analyze your sequencing data and write a partial Results section with figures/tables on the Soil Community Diversity Results from 16S rDNA sequencing of the soil genomic DNA (you do not have to include the 16S rDNA sequencing results of the cultured bacteria)
|Conference with your instructor to discuss your data analysis, poster presentation or your progress on your final paper.
||Homework: Prepare your group (4 students- 1 habitat) "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.
||End of lab