||IN LAB WORK________
||OUTSIDE OF LAB WORK________
|Introduction to Microbiology Lab
Boot camp: Tools and Techniques of Microbiologist: Aseptic Transfer, Streaking for Isolation, Making a bacterial Smear, Gram Stain, Microscopy
Intro to Microbial Diversity Project:
Tour greenhouse, pick habitat
||Homework: Visit the greenhouse and make notes on your selected habitat (see Lab 2 Part A for how to focus your observations). Begin to research how to select/enrich for particular soil bacteria. References posted to the Lab First Class conference (Reference Folder) and lots of information in the Protocols section of this wiki.|
Read all of Lab 2 & outline or make flow diagrams for your lab work
Answer the Questions found at the end of LAB 1 and turn them in at the beginning of Lab 2
|Soil sampling in Greenhouse habitat
Begin culture of soil organisms on selective, differential and enrichment media
Standard Plate Count of Culturable Soil Organisms
Genomic DNA isolation for Non-Culturable Bacterial ID
|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
||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 11 groups that you are attempting to find in your habitat.
|Identification of Unculturable Bacterial by 16S rDNA sequencing: Assess success of soil DNA isolation using Nanodropper to measure DNA conc.; Dilute or conc. DNA isolate to 100ng/μL;
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 amplification. Instructor will finish, photograph it and post the results to the data file in First Class Lab conference
Isolation of Culturable Bacteria: Complete Standard Plate Count counting & calculation
Continue selection, isolation, identification of desired bacteria
|Check on cultures and continue isolation
||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 Unculturable Bacterial 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: Examine enrichment and selective media and pick unique isolated colonies of your soil bacteria to make 2 fresh slants per organism.
Continue selection, isolation, identification of desired bacteria
Do simple, Gram, capsule, flagella, and endospore stains on control organisms and soil bacteria.
|Continue isolation and identification of soil bacteria;
Check to see if you have E. ooli transformants on your selection plates. If not, contact your instructor.
|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 Unculturable Bacterial by 16S rDNA sequencing:Select 48/per pair (96 per soil habitat) well-isolated, transformants from selective media and grow in broth overnight.
Isolation & Id of Culturable Bacteria: Make new stock slants from each of your pure cultures of your soil bacteria. Perform stains, physical, metabolic and morphologic tests to differentiate soil bacteria.
|Prepare glycerol stocks of overnight cultures of transformed bacteria to send away frozen for 16S rDNA sequencing. Results should be back within 2 weeks.
||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
|Isolation & Id of Culturable Bacteria: Perform physical characteristics tests: start a motility test by inoculating SIM tube or a soft agar deep; perform other differentiating tests as needed.
Start the ID of Cultured Bacteria by 16srDNA Sequencing and Analysis: Prepare lysates from pure colonies of your soil organisms of interest; pcr amplification of lysate DNA using "universal" bacterial primers and Taq polymerase.
|Complete or set up differential tests for bacterial id as needed.
||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
|Final Paper due on your lab day.
When & where at discretion
of your instructor.
||End of lab