BISC209/S11:Schedule: Difference between revisions

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| Mar. 7 || Mar. 8  <br>Lab 7 ||  Mar. 9 <br>Lab 7 || Mar. 10 || Mar. 11  
| Mar. 7 || Mar. 8  <br>Lab 7 ||  Mar. 9 <br>Lab 7 || Mar. 10 || Mar. 11  
|-
|-
| Mar. 14 || Mar. 15  <br>Lab 8<br>'''Lab Practical''' ||  Mar. 16 <br>Lab 8<BR>'''Lab Practical''' || Mar. 17<BR> '''Spring Break '''||  Mar. 18 <BR>'''Spring Break'''
| Mar. 14 || Mar. 15  <br>Lab 8 ||  Mar. 16 <br>Lab 8 || Mar. 17<BR> '''Spring Break '''||  Mar. 18 <BR>'''Spring Break'''
|-
|-
| Mar. 21 <br> '''Spring Break''' || Mar. 22 <br> '''Spring Break'''||  Mar. 23 <br>'''Spring Break''' || Mar. 24 <br>'''Spring Break'''||  Mar. 25 <br>'''Spring Break'''
| Mar. 21 <br> '''Spring Break''' || Mar. 22 <br> '''Spring Break'''||  Mar. 23 <br>'''Spring Break''' || Mar. 24 <br>'''Spring Break'''||  Mar. 25 <br>'''Spring Break'''
Line 30: Line 30:
| Mar. 28 || Mar. 29  <br>Lab 9 ||  Mar. 30<br> Lab 9  || Mar. 31||  Apr. 1
| Mar. 28 || Mar. 29  <br>Lab 9 ||  Mar. 30<br> Lab 9  || Mar. 31||  Apr. 1
|-
|-
| Apr. 4 || Apr. 5  <br>Lab 10 ||  Apr. 6 <br>Lab 10|| Apr. 7 ||  Apr. 8
| Apr. 4 || Apr. 5  <br>Lab 10<BR> '''Lab Practical''' ||  Apr. 6 <br>Lab 10<BR> '''Lab Practical'''|| Apr. 7 ||  Apr. 8
|-
|-
| Apr. 11 || Apr. 12  <br>Lab 11<br>'''Presentations''' ||  Apr. 13<br> Lab 11<br>'''Presentations'''|| Apr. 14 ||  Apr. 15
| Apr. 11 || Apr. 12  <br>Lab 11<br>'''Presentations''' ||  Apr. 13<br> Lab 11<br>'''Presentations'''|| Apr. 14 ||  Apr. 15
|-
|-
| Apr. 18 <br>'''Patriots' Day''' || Apr. 19  <br>'''Monday Schedule'''<BR> '''No Lab''' ||  Apr. 20 <br> '''Conferences'''  || Apr. 21||  Apr. 22
| Apr. 18 <br>'''Patriots' Day''' || Apr. 19  <br>'''Monday Schedule'''<BR> '''No Lab''' ||  Apr. 20 <BR>Lab 12<br> '''Conferences'''  || Apr. 21||  Apr. 22
|-
|-
| Apr. 25  || Apr. 26 <br>'''Conferences''' ||  Apr. 27 <br>'''Rhulman'''<br> '''NO Lab''' || Apr. 28 ||  April 29
| Apr. 25  || Apr. 26 <br>Lab 12<br>'''Conferences''' ||  Apr. 27 <br>'''Rhulman'''<br> '''NO Lab''' || Apr. 28 ||  April 29
|-
|-
| May 2  || May 3 <br>Lab 12<br>'''Paper Due''' ||  May 4 <br> Lab 12<br>'''Paper Due''' || May 5 ||  May 6<br> '''Last day of<br> classes'''  
| May 2  || May 3 <br>'''Paper Due''' ||  May 4 <br> Lab 12<br>'''Paper Due''' || May 5 ||  May 6<br> '''Last day of<br> classes'''  
|}
|}


== ''' Assignments ''' ==
== ''' Weekly Planner ''' ==


BISC209 Assignments/ Weekly Lab Planner
BISC209 Weekly Lab Planner


{| border=1px
{| border=1px
Line 58: Line 58:
|Introduction to Microbiology Lab<br>
|Introduction to Microbiology Lab<br>
Lab Safety<br>
Lab Safety<br>
Boot camp:  Tools and Techniques of Microbiologist: Aseptic Transfer,<br>  
Tools and Techniques of Microbiologist: Aseptic Transfer,<br>  
Intro to Microbial Diversity Project:Soil sampling in Greenhouse habitat;Begin culture of soil organisms; <BR> Start Plate Count of Culturable Soil Organisms<BR><br>
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''; <BR> Start Plate Count of Culturable Soil Organisms<BR><br>
 
| 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 [[BISC209/S11:Resources | Resources]]  section of wiki for information about organizing your lab notebook.<BR>'''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: [[BISC209/S11: Assignment_209_Lab1 | Assignment: Enrichment for culturable bacteria of specific groups]].
| '''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 [[BISC209/S11:Protocols | Protocols]] section of this wiki.<br> Read all of Lab 2 & outline or make flow diagrams of your lab work in your lab notebook. Check [[BISC209/S11:Resources | Resources]]  section of wiki for information about organizing your lab notebook.'''
|--
|--
| 2
| 2
| Tues. 2/1-<br>Wed. 2/2
| Tues. 2/1-<br>Wed. 2/2
|  Calibrate Micropipets; Genomic DNA isolation for Non-Culturable Bacterial ID;<BR> Finish Plate Count & quantify cultured microbes;<BR>Practice Streaking for Isolation
|  Calibrate Micropipets; Genomic DNA isolation for Culture-Independent Bacteria ID;<BR> Finish Plate Count & quantify cultured microbes;<BR>Practice Streaking for Isolation: Make soil extract from dried soil and set up isolation of spore-forming bacteria; <BR>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
| 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;<BR> 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.<BR>
|'''Homework''' Search the web for photos of colonies of desired bacteria;<BR> Research likely bacterial members of your soil community, differentiating metabolic needs or capabilities, physicial differences, and other useful or differentiating characterisitics. <BR>
'''Graded Assignment: Make a Table summarizing morphologic and function characteristics of groups of soil bacteria likely to be found in your habitat.'''
'''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: [[BISC209/S11: Assignment_209_Lab2 | Assignment: Table of Cultured Soil Bacteria Characteristics]].  
 
|--
|--
| 3
| 3
| Tues. 2/8-<br>Wed. 2/9
| Tues. 2/8-<br>Wed. 2/9
| '''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; <BR> PCR Amplification of 16S rDNA with "universal" bacterial primers and proofreading polymerase<BR> PCR product clean-up <BR><BR> 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<BR>
|  
'''Isolation of Culturable Bacteria:''' Complete Standard Plate Count counting & calculation<BR> Continue selection, isolation, identification of desired bacteria
'''Isolation of Culturable Bacteria:''' Evaluate your success at streaking for isolation;<BR>'''Start CLPP: Community Level Physiological Profiling: Carbon source utilization & nitrogen cycling analysis;'''<BR>Make another soil extract and serial dilution to '''evaluate carbon source utilization;'''<BR> Continue selection & isolation, of desired bacterial groups; <BR> Start '''community exoexyzme profiling''' (starch & cellulose digesters, phosphate solubilizers).
| Check on cultures and continue isolation
| 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''': 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 [[BISC209/S11:Resources | 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.  
| '''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.  
|--
|--
| 4
| 4
| Tues. 2/14-<br>Wed. 2/15
| Tues. 2/14-<br>Wed. 2/15
| '''Identification of Unculturable Bacterial by 16S rDNA sequencing:''' Clone 16s rDNA from successful pcr products into cloning vector<BR> Transform cloning vector into ''E. coli'' and select for transformants on selective media.<BR>
| '''Identification of soil community bacteria by 16S rDNA sequencing:''' PCR Amplification of 16S rDNA with "universal" bacterial primers and proofreading polymerase<BR> PCR product clean-up <BR><BR> 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;<BR>  
'''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.<BR> Continue selection, isolation, identification of desired bacteria<BR> Do simple, Gram, capsule, flagella, and endospore stains on control organisms and soil bacteria.  
'''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.<BR> Exoenzyme assessment; CLPP analysis and calculations of carbon source utilization and nitrogen cycling.  
| Continue isolation and identification of soil bacteria;<BR> Check to see if you have ''E. ooli'' transformants on your selection plates. If not, contact your instructor.
| Make sure you understand the CLPP analyses and calculations;<BR>  
| '''Homework''': Write the Introduction section of your final paper. Use the Introduction section of the "Guidelines for Science Writing" handout found in the [[BISC209/S11:Resources| 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.  
| '''Homework''':Turn in calculations and graphs for CLPP analyses of carbon source testing.'''Introduction section of final paper.''' Read over the [[BISC209/S11:Project1 | 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 :[http://www.springerlink.com/content/978-3-540-85464-7#section=183950&page=1&locus=2 | 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.  
|--
|--
| 5
| 5
| Tues. 2/22-<br> Wed. 2/23
| Tues. 2/22-<br> Wed. 2/23
| '''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. <BR>
| '''Identification of culture-independent soil community bacteria by 16S rDNA sequencing:''' Clone 16s rDNA from successful pcr products into cloning vector<BR> Transform cloning vector into ''E. coli'' and select for transformants on selective media.<BR>
'''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.   
'''Isolation & of Culturable Bacteria:''' Make new cultures from each of your pure cultures of your soil bacteria isolates.   
| Prepare glycerol stocks of overnight cultures of transformed bacteria to send away frozen for 16S rDNA sequencing. Results should be back within 2 weeks.  
| Check to see if you have ''E. coli'' transformants on your selection plates. If not, contact your instructor. <BR>
| '''Homework''': Write the following protocols as M&M sections for your final paper: <BR> 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).  
| '''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.
And revise and add to the methods for:  Identification of bacteria by 16S rRNA gene sequencing from soil genomic DNA
 
|--
|--
| 6
| 6
| Tues.3/1. - <br> Wed. 3/2
| Tues.3/1. - <br> Wed. 3/2
| '''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.<BR>
| '''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. <BR>
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.
'''Isolation & of Cultured Bacteria:''' Perform physical characteristics tests: smear slide, Gram stain, Confirm Gram stain with selective media, start an antibiotic production test.<BR>
|  Send away frozen glycerol stocks of overnight cultures of transformed bacteria for 16S rDNA sequencing. Results should be back within 2 weeks.  
|  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.  
| '''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.  
|--
|--
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| 7
| 7
| Tues. 3/8- <br> Wed. 3/9  
| Tues. 3/8- <br> Wed. 3/9  
| '''16S rDNA sequencing:''' Run a gel and Clean-up pcr products of successful amplifications of 16s rDNA from your soil bacteria isolates. <BR> '''Cultured bacteria ID''': Read last weeks tests; Continue with new differential ID tests; Start
| '''Cultured bacteria assessment''': Start SIMs test; Continue antibiotic production test; Read Gram stain confirmation by selective media, Start Quorum Sensing & Bacterial interactions tests.
Antibiotic sensitivity and nitogen recycling tests;
| Complete, read, or set up fresh cultures as needed.
add other "role" tests as needed.
| '''Homework''': Write a partial Results section with figures/tables: Read more about this assignment at: [[BISC209/S11: Assignment_209_Lab7 | 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 [[BISC209/S11:Resources| 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.
| 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.
|--
|--
| 8
| 8
| Tues. 3/15- <br> Wed. 3/16
| Tues. 3/15- <br> Wed. 3/16
| '''Lab Practical'''<BR> Cont. '''Cultured bacteria ID:''' cont. with tests to differentiate and id your cultured bacteria.  
| 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:<BR>
| '''Homework''': Write a Results section with figures/tables:
[http://rdp.cme.msu.edu/index.jsp].
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 [[BISC209/S11:Resources| 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.
| '''Homework''': Results section on isolates work and Graphical Abstract.
<BR>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 [http://0-www.springerlink.com.luna.wellesley.edu/content/?k=title%3a%28the+prokaryotes%29&sortorder=asc&Content+Type=Reference+Works | The Prokaryotes]through Springer ebooks;<BR>  
Link to the electronic edition of [http://0-www.springerlink.com.luna.wellesley.edu/content/?k=title%3a%28bergey%27s%29&sortorder=asc&Content+Type=Books | Bergey's Manuals]through Springer ebooks
|--
|--
| 9
| 9
| Tues. 3/28- <br> Wed. 3/29
| Tues. 3/28- <br> Wed. 3/29
| Meet in a computer lab (TBA) for data analysis of your 16S rDNA sequencing results.   
| Meet in a computer lab (TBA) for data analysis of your 16S rDNA sequencing results.   
| Complete tests to ID cultured bacteria
| Complete all work on cultured bacterial isolates to provide evidence for functional co-operation and/or competition in your soil community
| '''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)
| '''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.<BR>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 .
|--
|--
| 10
| 10
| Tues. 4/5- <br> Wed. 4/6
| Tues. 4/5- <br> Wed. 4/6
| Conference with your instructor to discuss your data analysis, poster presentation or your progress on your final paper.
| '''Lab Practical'''<BR>
|
| Conference with your instructor to discuss your data analysis& poster presentation.
| '''Homework''': Prepare your group (4 students- 1 habitat) "virtual" poster presentation to be presented in LAB 11
| '''Homework''': Prepare your group (4 students- 1 soil community) "virtual" poster presentation to be presented in LAB 11


|--
|--
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| Tues. 5/3-<br> Wed. 5/4
| Tues. 5/3-<br> Wed. 5/4
| Final Paper due on your lab day.<BR> When & where at discretion<BR> of your instructor.
| Final Paper due on your lab day.<BR> When & where at discretion<BR> of your instructor.
 
| Information on your final paper found at: [[BISC209/S11: Assignment_209_Lab12 | Assignment: Final Paper]]
|  
| End of lab
| End of lab
|--
|--

Latest revision as of 14:16, 28 March 2011

Wellesley College-BISC 209 Microbiology -Spring 2011


BISC209 S11 Lab Calendar

Monday Tuesday Wednesday Thursday Friday
Jan.24 Jan. 25
Lab 1		 Jan. 26
Lab 1		 Jan. 27 Jan. 28
Jan. 31 Feb. 1
Lab 2		 Feb. 2
Lab 2		 Feb. 3 Feb. 4
Feb. 7 Feb. 8
Lab 3		 Feb. 9
Lab 3		 Feb. 10 Feb. 11
Feb. 14 Feb. 15
Lab 4		 Feb. 16
Lab 4		 Feb. 17 Feb. 18
Feb. 21
Presidents'
Day		 Feb. 22
Lab 5		 Feb. 23
Lab 5		 Feb. 24 Feb. 25
Feb. 28 Mar. 1
Lab 6		 Mar. 2
Lab 6		 Mar. 3 Mar. 4
Mar. 7 Mar. 8
Lab 7		 Mar. 9
Lab 7		 Mar. 10 Mar. 11
Mar. 14 Mar. 15
Lab 8		 Mar. 16
Lab 8		 Mar. 17
Spring Break 		Mar. 18
Spring Break
Mar. 21
Spring Break		 Mar. 22
Spring Break		 Mar. 23
Spring Break		 Mar. 24
Spring Break		 Mar. 25
Spring Break
Mar. 28 Mar. 29
Lab 9		 Mar. 30
Lab 9		 Mar. 31 Apr. 1
Apr. 4 Apr. 5
Lab 10
Lab Practical		 Apr. 6
Lab 10
Lab Practical		 Apr. 7 Apr. 8
Apr. 11 Apr. 12
Lab 11
Presentations		 Apr. 13
Lab 11
Presentations		 Apr. 14 Apr. 15
Apr. 18
Patriots' Day		 Apr. 19
Monday Schedule
No Lab		 Apr. 20
Lab 12
Conferences		 Apr. 21 Apr. 22
Apr. 25 Apr. 26
Lab 12
Conferences		 Apr. 27
Rhulman
NO Lab		 Apr. 28 April 29
May 2 May 3
Paper Due		 May 4
Lab 12
Paper Due		 May 5 May 6
Last day of
classes

Weekly Planner

BISC209 Weekly Lab Planner

Lab # LAB DATES__ IN LAB WORK________ OUTSIDE OF LAB WORK________ Assignment/Notes
1 Tues. 1/25-
Wed. 1/26 		Introduction to Microbiology Lab

Lab Safety
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.
2 Tues. 2/1-
Wed. 2/2 		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.

3 Tues. 2/8-
Wed. 2/9

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.
4 Tues. 2/14-
Wed. 2/15 		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.
5 Tues. 2/22-
Wed. 2/23 		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.
6 Tues.3/1. -
Wed. 3/2 		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.
7 Tues. 3/8-
Wed. 3/9 		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.
8 Tues. 3/15-
Wed. 3/16 		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:

[1].

Homework: Results section on isolates work and Graphical Abstract.
9 Tues. 3/28-
Wed. 3/29 		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 .
10 Tues. 4/5-
Wed. 4/6 		Lab Practical
 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
11 Tues. 4/12-
Wed. 4/13 		"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
Conferences Wed. 4/20-
Tues. 4/26 		Meet with your instructor to discuss your final paper.
When & where at discretion
of your instructor.
12 Tues. 5/3-
Wed. 5/4 		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


Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12

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