LD50s of Francisella LVS in Blaptica dubia roaches--Replicate

Storage of roaches in new containers

On wednesday, 11/19/14, I repeated injections of Ft LVS and 4 mutants that were done on 11/14/14. The same protocol was used. Because of the recent concern of roach dehydration affecting survival, the experimental roaches were moved from petri plates to larger contains and were provided water crystals along with dry dog food after 24 hours. Survival count will be recorded in my Onedrive file here.

Water crystals to prevent dehydration

//BEE

Dehydration of Roaches affecting Survival

(from 11/14/14 Bd injections of Ft LVS)

Water crystals to prevent dehydration

There was a sudden death in the PBS and non-injected controls after 6 days (12% survival PBS and 50% survival in non-injected). This be attributed to dehydration after a two week incubation at 37C with only dry dog food.

Therefore, I have updated the protocol for roach storage for future experiments. Water crystals will be used as a source of water and larger containers will be used that can hold all 8 roaches for each group and allow for gas exchange (figure shown).

New containers

One concern is the roaches will now be stored in the walk-in 37C incubator used by the department to accommodate for the larger sized containers. This incubator is high-traffic due to teaching labs and the building's exterior door not far down the hallway. There is also a light that is often left on within the incubator. With these precautions in mind, results will be analyzed closely.






//BEE

LVS titer plate on CHOC II agar after 48 hr

(from 11/14/14 Bd injections of Ft LVS)

There were several deaths in Francisella groups. Data will be recorded one my Onedrive file here.
After 24 hours the E.coli spot-titer plates could be read, and all the Francisella plates were read after 48 hours to determine the CFU/dose of each injection group.

E.coli DH5alpha titer plate on LB after 24hr

Roaches contained in petri plates

//BEE

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

From 11/4/14

I have been testing the effects of gentamicin on dubia roaches. Today, I gave each one another dose of the antibiotic solution in the same manner as the original administration. All 10 roaches are still alive.

Observations: Because each roach had an individual piece of dog food in its petri plate, the oral feeding group was less accepting to the sucrose solution compared to the original administration 3 days ago. With enough patience and technique, each roach was given the full 10 microliter dose.

//BEE

Gentamicin dosing of Dubia Roaches

Today I designed an experiment to observe the effects of gentamicin, a non-cell-permeable antibiotic, on the survival of Blaptica dubia roaches. I will be using this antibiotic in the in vivo growth curve of the LVS strain of Francisella to observe intracellular and extracellular growth. This assay is inspired by a similar experiment that uses Drosophila melanogaster flies found here.

This experiment will include two different methods of antibiotic administration--oral feeding and injection. Future experiments will include the administration of different antibiotics using these methods, so this experiment will offer as a preliminary trial for future work.

Procedure:

1. Solutions of gentamycin were made in PBS and a 50% sucrose solution with a concentration of 1mg/mL.
2. A group of 5 roaches (each 0.7-0.9g) were fed 10µl of the gentamicin in 50% sucrose.
3. A group of 5 roaches were injected with 10µl of the gentamicin in PBS on the 2^nd to last segment of the abdomen on the left dorsal side. Site of injection was cleaned with 70% alcohol before. 
4. Each roach was stored in individual small plastic petri plates in the dark 37degC incubator.
5. Roaches will be administered antibiotic with the same dosing method twice a week for two weeks. Survival will be checked daily. 

//BEE