LD50s of Listeria monocytogenes in Blaptica dubia roaches

Last Friday (10/24/14) I started an experiment for the high-throughput study of Listeria monocytogenes virulence using Blaptica dubia roaches.  I had previously determined the LD50 values of several mutants of Listeria from Dr. Teresa Bergholz lab in Galleria mellonella (wax worms). I using the roaches for add information about the isolates and to further develop the roach model.

The following protocol was used on Friday.

1. 2 days before: Request cultures from Becky and streak E.coli DH5alpha on an LB plate
2. 1 day before: Create overnight (20 hour) cultures in BHI broth and DH5alpha in LB broth and incubate at 37deg C, shaking >225rmp
3. Day of Injections: Serial dilute 10-fold to 10^-7.
• Inject a set of 8 roaches for each dilution 10^-1 to 10^-5.Wipe roach with isopropyl alcohol before injection
• Using sharpened pipette tips, pierce the 2^nd to last segment of the abdomen on the dorsal side.
• Inject 20μl of bacterial solution.
• Place 4 roaches in a sterile plastic petri dish for storage.
4. Create a control group by injecting 8 roaches with PBS in the same way.
5. Make a titer by dividing a plate into 4 quadrants, and spotting five 20μl drops of each 10-fold dilution on BHI plates (LB for E. coli).
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

Sharpening of extra-long gel loading tips.

Recording of roach survival

//BEE