Today I started an assay to observe the growth of Francisella tularensis LVS in hemolymph from Blaptica dubia roaches. I did a previous growth curve in vivo where I inoculated B.dubia roaches with F. tularensis LVS systemically and used gentamicin to determine the relative amounts of intracellular LVS. In the trial run, I want to see if LVS can still grow in the hemolymph in a tube. I planned on using complete hemolymph, cell-free hemolymph, BHI broth, and sheeps blood for different growth conditions but we did not have sheeps blood on hand, and the hemolymph did not pass through the 0.45 micron syringe filter that would have given us the cell-free hemolymph. Therefore, I am only using two growth conditions for this quick assay. I used the following protocol:
1. From a 48-hr chocolate plate, suspend one small loop full of F. tularensis LVS into 1ml sterile PBS.
2. Serial dilute to 1:10 to -6 and plate four 10µl spots per dilution onto CHOC II plates for the titer. Incubate inverted at 37C.
3. For growth conditions, use 500µl of BHI broth and complete hemolymph in sterile 2ml epp tubes. Hemolymph can be extracted using the following method:
- Fill 2ml epp tube with 10µl anticoagulant buffer (0.05% N-Phenylthiourea) by removing
the head of the roach and draining hemolymph into tubes.
- Weigh tubes before and after the addition of hemolymph to calculate exact volume extracted per roach.
- Use sterile scissors for decapitations.
4. At desired time points (5, 24, and 48 hr) post innoculation, serial dilute a small sample (
10µl) 1:10 and plate onto CHOC II using spot method (
plate four 10µl spots per dilution).
5. Count colonies on plates and determine CFU/ml for each timepoint.
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