This blog is the ONS (Open Notebook Science) record for the work that I personally perform in the lab. It is posted informally and without peer review. Please feel free to comment and contact me at bridget.eklund@ndsu.edu if there is something you're interested in. You can learn more about the lab on our wiki page (http://openwetware.org/wiki/Fisher). Thanks for visiting.

Friday, October 17, 2014

Lab Work Update

For the past month I have been working with Dr. Teresa Bergholz's lab. They have supplied me with Listeria monocytogenes and several mutants strains, and I have tested their virulence in Galleria mellonella (wax worms) to determine the lethal dose 50% (LD50). The following protocol was used for the 8 isolates being tested. A total of three replicates were done to find the final LD50s.

Listeria monocytogenes LD50 Protocol
  1. Obtain inoculated plates from Becky
  2. Give to Dawn for blinding of overnight broth cultures in BHI media 
    • Incubate at 37°C shaking for 20 hours 
  3. Remove 1 mL of bacterial suspention into a 2.0 mL epp tube 
  4. Wash cells to remove BHI media and resuspend with PBS 
    • Centrifuge at 13,000g for 1 minute in 2.0mL epp tube, wash twice. 
  5. Make serial 1:10 dilutions of each overnight culture to 10-7 using 2.0 mL epp tubes and PBS as diluent 
  6. Create a spot plate titer on BHI plates to determine CFU/mL of each dilution 
    • Using 5x10μL spots for -4 to -7 dilutions 
  7. With a Insulin syringe, inject 30μL into each worm 
    • Set of 10 wax worms for each dilution of each mutant 
  8. Create a control group by injecting 10 worms with PBS 
  9. Place worms in labeled petri plates and securely store them at 37°C for 9 days, observe and record day and number of deaths and pupate 
  10. Calculate LD50 for each mutant of Listeria using F020_LD50Gm excel file
Data was entered into the excel file and stored on my OneDrive account (Listeria Survival Data in wax worms) and found the following data:

Isolate    Rep 1          Rep 2         Rep 3         Average         St Dev
TB7        6.53E+03    3.39E+06    1.12E+04    1.14E+06    1.95E+06
TB10      4.18E+04    1.30E+03    1.13E+04    1.81E+04    2.11E+04
TB18      2.74E+04    1.62E+04    1.16E+04    1.84E+04    8.13E+03
TB19      4.77E+04    2.47E+03    1.10E+04    2.04E+04    2.40E+04
TB25      2.66E+05    5.56E+04    1.24E+05    1.48E+05    1.07E+05
TB26      7.07E+04    2.62E+04    6.18E+04    5.29E+04    2.35E+04
TB27      1.13E+04    1.53E+03    6.83E+03    6.55E+03    4.89E+03
TB133    4.18E+04    7.42E+04    8.25E+04    6.62E+04    2.15E+04
DH5a     3.39E+06    6.60E+04    1.47E+05    9.79E+05    1.61E+06
                                   3.13E+05   

Table can be viewed in the excel file here:Listeria LD50 Values     

//BEE 
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