LD50s of Francisella LVS in Blaptica dubia roaches

Today I started an experiment to assay the virulence of the attenuated live vaccine strain (LVS) of Francisella tularensis. I am using Blaptica dubia roaches as the host organism.
The following protocol was used on today:

CHOC II plate with LVS

1. Acclamate roaches (each 0.7-0.9g) to 37degC at least 7 days prior to injections.
2. 2 days before: Streak CHOC II agar plates with bacterial cultures of LVS, dsbA, dipA, iglC, amd deoB. Plates were inverted and incubated at 37degC. 
3. E.coli DH5alpha was inoculated in LB broth and incubated at 37degC at 225 rpm.
4. 1 day before: From E.coli broth culture, perform an isolation streak on LB plate-incubate at 37deg C.
5. Day of Injections: 
• Using a sterile loop, scrape plate to obtain a large amount of bacterial colonies.
• Suspend in 1ml of sterile PBS to create a bacterial suspension (view images for quantity reference).



Amount of LVS that was used

• 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.
• Repeat for each bacterial strain.
6. Create a control group by injecting 8 roaches with PBS in the same way.
7. Place 8 roaches in petri plates for non-injected control.
8. Make a titer for each strain by dividing CHOC II plates (LB for E. coli) into 4 quadrants, and spotting five 10μl drops of the 10^-4 to  10^-7 10-fold dilutions; make two replicate plates.
9. Store all roaches at 37°C for 10 days—record any deaths.



Bacterial suspension of LVS in PBS

10.  Calculate LD₅₀ of each strain using titer and survival of roaches.

Storage of roaches in petri plates

//BEE