Antibiotic Testing with LVS Injected Roaches

Yesterday I began an experiment to study the effects of different antibiotics on the survival of roaches inoculated with a single concentration of Francisella tularensis LVS. I used the following procedure:

LVS dilutions used for injections

1. Streak out F. tularensis LVS on a CHOC II plate, incubate 37C for 48 hours
2. Make cell suspension in PBS (10e+6 cells/ml)
3. Serial dilute 1:10 to -7 
• Create a titter plate using CHOC II, with five 10μL spots for dilutions -4 to -7.
4. Incubate for at least 48 hours at 37C.
5. Inject roaches with single lethal LVS concentration on day 0, 10 roaches per group (one control  group with PBS).
• Use -1 dilution for injections.
• Inject 20μL using small gauge needle with syringe and microstep pipetter.
6. 2 hours post-infection: Feed roaches a 50% sucrose solution with solubilized antibiotics at desired concentration.
• Randomly choose groups of 10 roaches for each antibiotic.
• Place roaches in square containers that allow for air flow.
7. Store roaches at 37C with water crystals and dry dog food.
8. Remove water and food 24 hours before antibiotic feeding.
9. Repeat antibiotic feeding every two days (48 and 96 hours post-infection).



Preparation of abx in sucrose

10. Observe and record survival.

Antibiotics:
Streptomycin (32μg/roach)
Gentamicin (32μg/roach)
Resazurin (11μg/roach)
Vancomycin (15μg/roach)
Ampicillin (100μg/roach)
Chloramphenicol (32μg/roach)
Amikacin (32μg/roach)
Doxycycline (32μg/roach)
Ceftriaxone (8μg/roach)

Feeding of roaches with 50% sucrose solution

Azithromycin (100μg/roach)
Ciprofloxacin (1μg/roach)

Storage of roaches in containers at 37C

 




//BEE