F.t. LVS survival in 50% sucrose

With concern in my previous experiment regarding the survival of F. tularensis LVS in a 50% sucrose solution that was used for oral administration in B. dubia roaches on 3/10/15, I started an experiment today to assay the survival of LVS in this condition. The following protocol was used to assay survival in the hypertonic solution.

1. Prepare 48hr culture of F.t. LVS on CHOC II plates (37C).
2. Suspend a large loop of colonies in 1ml sterile PBS.
3. Wash cell suspension with centrifuge
4. Resuspend cells with 300µl of PBS
5. Transfer 100µl of cell suspension to 900µl sterile PBS and 900µl sterile 50% sucrose solution in 2ml epp tubes.
•  Use remaining suspension to preform 1:10 serial dilution for titer
6. Incubate tubes at 37C
7. At desired time points, remove 10µl of culture from each condition and serial dilute and plate onto CHOC II.
•  I used 0, 4, 24, 48, and 72 hour timepoints (hours 1 and 6 would have been preferred but my class schedule did not permit.)