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.)

B.dubia challenge with oral innoculation of Lysinibacillus sphaericus

Today I started an assay to see if Lysinibacillus sphaericus and Francisella tularensis LVS are pathogenic towards Blaptica dubia roaches when administered orally. I was supplied with spores stocks of three samples of L. sphaericus at different concentrations (9E+7, 2E+9, and 1E+8) and a plate of F. tularensis LVS on CHOC II agar. I used 600µl of the spore suspensions and spun them into a pellet (10,000rpm for 1 min) and resuspended them to 120µl in a 50% sucrose solution for a 5x concentration. For the F.t LVS, I used a large loop of bacteria and suspended it in 200µl of 50% sucrose solution. The bacterial-sucrose solutions were then fed to groups of 10 roaches similarly to the method used here. The roaches are incubated at 37C with water crystals for hydration. Survival will be checked daily. 

In vitro growth curve of F.t. LVS

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.