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==LAB 9 ==
==LAB 9 Analyzing Your DNA Sequencing data and Finishing the Project==
 
'''You will have a workshop to evaluate and analyze your 16S rRNA gene sequencing data'''


==Complete Testing for Antibiotic Producers Among Your Cultured Isolates==
'''Week 3 <BR>'''
<UL><LI>
Examine the plates you inoculated with known bacterial cultures last week and look for evidence of a zone of inhibition of growth near the antibiotic producer's midline streak  <LI>
Draw the results and evaluate whether or not there was evidence that an antibiotic was produced by your isolate and, if so, which of the known bacteria were sensitive to it and to what degree. If you found no inhibition of growth, does that mean that your isolate does not secrete an any antimicrobial compounds? Why or why not? </LI></UL>
[[Image:antibiotic.jpg]].


==Finishing the project==
==Finishing the project==
After you have recorded all your isolate test results on a lab Google Doc,  please discard all cultures.  You will receive 5 "clean-up points"  if we do not find any evidence of your cultures or tests you will receive 5 points toward your lab grade. If we have to discard and clean-up later after you, you will not receive any of those points.
After you have recorded all your isolate test results on a lab Google Doc,  please discard all your cultures in the lab and in the storage cold room.  You will receive 5 "clean-up points"  if we do not find any evidence of your cultures or tests you will receive 5 points toward your lab grade. If we have to discard and clean-up later after you, you will not receive any of those points.
The date that clean up must be completed will be announced by your instructor.
The date '''clean up must be completed is Friday, Dec 6 at 10am'''; however, it would be better to take care of it now.
 
==Freeze freshly cultured isolate protocol to make Stocks==
Please complete the Google doc prior to freezing your isolate.
<BR>
 
FREEZING '''PURE''' bacterial CULTURES
<UL><LI>microfuge tubes
<LI>1000µL Pipetman
<LI>microfuge tube labels
<LI>sterile 20% glycerol stock
<LI>sterile toothpicks
<LI>young culture (~24 hr) of student pure isolates grown on plates
<LI>Google Doc or Excel Spreadsheet for Strain Database</LI></UL>
<BR>
Remember that your frozen stocks are potentially the one and only source you have for that bacterial species.  Therefore be especially careful with your sterile technique when creating and working with your stocks, and make sure you are vigilant about keeping your strain list up-to-date and correct<BR>
 
Wear gloves to avoid contamination and work near flame (watch your hair and hands!)<BR>
 
Thoroughly wash your hands and decontaminate the bench before beginning<BR>
 
Make sure not to place tube cap on any surface (to keep it sterile)<BR>
 
'''Create a strain database file'''
 
# Give each isolate a strain name:  '''your initials followed by a number'''
# Use the 209 lab Google Doc spreadsheet and fill in all the information listed below<BR>
<UL><UL><UL>
      <LI> Strain name
      <LI> Bacterial species (if known)
      <LI> The source of the bacterial strain (person or environment)
<LI>Any known genotype information or mutations
<LI>The date the stock was made
<LI>Information about where in your notebook you first discuss this strain
        <LI>The source of the bacterial strain (person or environment)
<LI>Any known genotype information or mutations
<LI>The date the stock was made
<LI>Information about where in your notebook you first discuss this strain
</UL></UL></UL>
 
'''Preserving the Stock''' <BR>
1. Label the side of the cryo-tube with the cell strain name (and any additional info you want) <BR>
2. Add scotch tape over the label to ensure it doesn’t rub off <BR>
3. If the cryo-tube has a place to put a top label on, prepare that also <BR>
 
NOTE:  The cryo-tubes typically do not fit into the standard eppendorf racks; a good alternative is to use the styrofoam base that 15 ml conicals come in—those openings are large enough for the cryo-tubes to rest securely in but not so deep you cannot remove and replace the cap easily<BR>
 
4. Screw open the tube but leave the cap sitting loosely on top <BR>
5. Add 800 ul of 20% glycerol in LB to the cryo-tube using P1000 pipette (be careful to not set cap down or let it touch any surface)<BR>
6. Scrape up cells from the plate with a flat toothpick—try not to gouge agar, just remove cells<BR>
7. Open cap and swirl toothpick in the media/glycerol to remove cells<BR>
8. Place 2 – 3 good-sized scrapings into the glycerol media <BR>
9. Pipette gently with P1000 to break up chunks of cells and disperse them<BR>
10. Mixture should be opaque—if not, add more cells<BR>
11. Place in -80 degree freezer in labeled freezer box<BR>
 
Be sure to label both the lid and the tube of a glycerol stock before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube!
 
'''Tips and FAQ'''<BR>
 
The optimal concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol.<BR>
 
Try not to freeze/thaw your glycerol stock too many times.  If you need to thaw frequently than make a new glycerol stock replacement from a freshly grown culture.<BR>


==Assignment==
==Assignment==
'''Study for your Lab Practical'''<BR>
'''Study for your Lab Practical (45points)'''<BR>
More information at  [[BISC209/F13: Assignment 209 Lab9 | Lab Practical tips]] <BR><BR>
Information and Tips can be found at : [[BISC209/F13: Assignment 209 Lab9 | Lab Practical]].  <BR>
Your '''final paper is due on Tues. Dec. 10 by 4pm''' (the last day of classes) for all students. You have all the data you need to start working on it. Instructions can be found at: [[BISC209/F13: Assignment_209_Lab10 | Assignment: Final Paper]] <BR>


'''To Do:'''<BR>
'''To Do:'''<BR>
'''Discard and clean up any remaining test materials and old cultures of your isolates.''' Note that there are 5 bonus points awarded for perfect clean-up. Your instructor will explain the criteria for obtaining these points. '''<BR>
'''Discard and clean up any remaining test materials and old cultures of your isolates.''' <BR>
'''Work on  your final paper'''. Instructions at [[BISC209/F13: Assignment 209 Lab10 | Final Paper]]


==Links to Labs==
==Links to Labs==

Latest revision as of 06:05, 19 November 2013

Wellesley College- BISC209 Microbiology- Fall 2013

LAB 9 Analyzing Your DNA Sequencing data and Finishing the Project

You will have a workshop to evaluate and analyze your 16S rRNA gene sequencing data

Complete Testing for Antibiotic Producers Among Your Cultured Isolates

Week 3

  • Examine the plates you inoculated with known bacterial cultures last week and look for evidence of a zone of inhibition of growth near the antibiotic producer's midline streak
  • Draw the results and evaluate whether or not there was evidence that an antibiotic was produced by your isolate and, if so, which of the known bacteria were sensitive to it and to what degree. If you found no inhibition of growth, does that mean that your isolate does not secrete an any antimicrobial compounds? Why or why not?

.

Finishing the project

After you have recorded all your isolate test results on a lab Google Doc, please discard all your cultures in the lab and in the storage cold room. You will receive 5 "clean-up points" if we do not find any evidence of your cultures or tests you will receive 5 points toward your lab grade. If we have to discard and clean-up later after you, you will not receive any of those points. The date clean up must be completed is Friday, Dec 6 at 10am; however, it would be better to take care of it now.

Assignment

Study for your Lab Practical (45points)
Information and Tips can be found at : Lab Practical.
Your final paper is due on Tues. Dec. 10 by 4pm (the last day of classes) for all students. You have all the data you need to start working on it. Instructions can be found at: Assignment: Final Paper

To Do:
Discard and clean up any remaining test materials and old cultures of your isolates.

Links to Labs

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


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