Francisella LD50 in Blaptica dubia roaches

Today I started an experiment for the high-throughput study of Francisella tularensis virulence using Blaptica dubia roaches. The purpose of this study is to ultimately develop a novel insect model that can be more applicable to long term studies.

The following protocol was used today.

1. Request blinded strains from dawn on Modified Mueller Hinton plates with appropriate times.
2. Suspend a colony of each bacterial strain in 1mL PBS.
3. Serial dilute 10-fold to 10^-6.
• Inject a set of 10 roaches for each dilution 10⁰ 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.
4. Create a control group by injecting 10 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 Mueller-Hinton plates with 2% isovitalex.
6. Store roaches at 37°C for 10 days—record any deaths.
7.  Calculate LD₅₀ of each strain using titer and survival of roaches.

//BEE