Gentamicin Protection Assay (LVS Growth Curve Inside Tropical Roach)

This week I am using the tropical roach for an in vivo growth curve of Fracisella tularensis LVS. I am using the following protocols and will add updates as the experiment progresses.


Protocol

1. Inject a large number of B. dubia roaches with 10^6 LVS at t=0hr.
• Number of roaches=[(# of time points x 2) x 8] x 1.5
• Extra 50% is for unexpected deaths.
• Store roaches in smaller containers with food and water crystals at 37C.
2. For each time point, randomly select 2 group of 8roaches.
• One group will receive a 16μg dose of gentamycin 2 hours prior to hemolymph extraction.
3. Extract hemolymph from roaches into chilled PBS with anticoagulant (0.05% N-Phenylthiourea) by removing the head of the roach and draining hemolymph into tubes.



Spot plates used for titer of 6hr extraction

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• Weigh tubes before and after the addition of hemolymph to calculate volume extracted per roach. 
• Use sterile scissors for decapitations.
4. Serial dilute solutions 1:10 using 96-well plate to the -6 dilution.
5. Plate on cysteine heart agar supplemented with isovitalex, ampicillin, and trimethoprim using spot plate method (10micoliter spots) for 0 to -5 dilutions.
6. Repeat at desired time points (6, 12, 24, 48, 72, 96 hours post-infection). 
7. Record CFU on spot plates and determine CFU/ml of hemolymph for LVS in each roach. 

//BEE