deletions | additions       diff --git a/section_Methods_subsection_Bacterial_Strains__.tex b/section_Methods_subsection_Bacterial_Strains__.tex  index 8604507..2bcbe22 100644  --- a/section_Methods_subsection_Bacterial_Strains__.tex  +++ b/section_Methods_subsection_Bacterial_Strains__.tex 
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Cockroaches weighing 0.7-1.0 g were transfered to the challenge temperature (usually 37°C) at least seven days prior to challenge in order to allow them to acclimate. We found this acclimation step to be critical--cockroaches that experienced a simultaneous temperature shift and injection trauma tended to have unpredictable deaths in control groups and dose-independent deaths in experimental groups (data not shown). All data reporter here is from temperature-acclimated animals. Bacterial suspensions of 10^6 colony forming units (CFU) were created by suspending 3-4 colonies from a Chocolate II agar plate incubated at 37°C for 48 hours in PBS. These initial suspensions were serial diluted in PBS and aliquots (20 μl) of each dilution were delivered by intrahemoceol injection to the right of the midline at the base of the third tergum (counting from the posterior end) using either a 28-gauge insulin syringe or a sharpened pipette tip (Fig 1B), made by cutting a ~60° bevel into a gel-loading pipette with a razor blade prior to sterilization. We saw no difference in survival of control or \textit{F. tularensis} LVS-challenged animals injected by needle and syringe or sharpened pipette tip (not shown). The latter delivery method is advantageous because is lowers cost and increases safety compared to the use of needle and syringe. In either case, the abdomen was swabbed with 70\% isopropanol prior to injection in order to lessen the risk of external contamination. For each experiment, a control group was injected with PBS to observe effects of trauma alone. Groups of 8 to 10 cockroaches were used for each experiment. Cockroaches were stored at the temperature indicated in Table 1 and observed for survival up to 10 days post-inoculation. Cockroaches were considered dead when they displayed no response to touch. Lethal doses 50\% and corresponding 95\% confidence intervals were estimated by non-linear regression using the drc package in the R programming environment \cite{Ritz}.   \subsection{Antibiotic Administration}  Groups of 10 cockoaches were injected with \textit{F. tularensis} LVS as described, and treated with antibiotics at 2, 48, and 96 hours post infection. Two methods of delivery were used for antibiotic administration; systemic injections and controlled feeding. For systemic injection, 20 μl of each antibiotic suspension was injected into the base of the third terga (abdominal plate) on the ventral side of the body, on the right side halfway between the midline and the spiracle (Fig XA). For the second and third injections, the left side of the same tergum and the right side of the next anterior tergum were used, respectively. For controlled feeding, antibiotics were prepared and diluted to the appropriate concentration in a sterile 50\% sucrose solution. Cockroaches were placed on their back and a 10 μl aliquot of the sucrose solution containing antibiotic was slowly dispensed onto the mouth (Fig XB). 6B).  This resulted in the rapid consumption of the entire dose. Antibiotics were delivered at the following total doses, regardless of route: Streptomycin, 32 μg; Gentamicin, 32 μg; Doxycycline, 32 μg; Ciprofloxacin, 1 μg; Resazurin; 11 μg. \subsection{Enumeration of bacteria in hemolymph}  \textit{B. dubia} roaches were inoculated with 10^6 \textit{F. tularensis} LVS cells as described previously. Roaches were incubated at 37°C with sufficient hydrating foods to prevent dehydration throughout the assay. Hemolymph was extracted from roaches at each designated time point of 6, 12, 24, 50, 72, and 96 hours post initial infection. Two groups were used for each extraction time point. One group of roaches received a 16μg dose of gentamicin 2 hours prior to hemolymph extraction, and a second group received an equal amount of sterile PBS in parallel. The roaches were cleansed with 70\% isopropyl alcohol and decapitated. Hemolymph was immediately drained into a 1.5mL Eppendorf tube containing 10μl chilled PBS with anticoagulant (0.05\% N-Phenylthiourea). Tubes were weighed before and after addition of hemolymph in order to estimate the volume of hemolyph collected from each roach. In order to quantify the number of \textit{F. tularensis} LVS CFU present in the hemolymph, the harvested sample was serial diluted 1:10 in PBS and aliquots of each dilution were plated on Chocolate II agar plates supplemented with ampicillin and trimethoprim. The total number of \textit{F. tularensis} LVS per mL of hemolymph was determined based on the number of CFU observed from PBS-treated cockroaches and the number of intracellular \textit{F. tularensis} LVS per mL of hemolymph was determined based on the number of CFU observed from gentamicin-treated cockroaches.