deletions | additions       diff --git a/section_Methods_Bacterial_Strains_and__1.tex b/section_Methods_Bacterial_Strains_and__1.tex  index 7adb04c..dfe5bb5 100644  --- a/section_Methods_Bacterial_Strains_and__1.tex  +++ b/section_Methods_Bacterial_Strains_and__1.tex 
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The wild type Francisella tularensis LVS strain, along with the ΔdsbA and ΔdipA mutants, were provided by Jason Huntley (University of Toledo). The ΔiglC and ΔdeoB mutants were provided by Joseph Horzempa (West Liberty University). Deletion mutants were constructed using the pJH1/pGUTS system. A deletion plasmid was created using the pJH1 backbone and mobilized into F. tularensis LVS via conjugation. Exoconjugates were transformed with pGUTS using electroporation and screened for deletion of target gene. Lastly, pGUTS was cured from the deletion mutant to create the completed deletion mutants.  Bacterial Growth Conditions  Francisella \textit{Francisella tularensis}  LVS deletion mutants were maintained as permanent frozen stocks at -80°C in BHI broth supplemented with 15\% glucose. E.coli DH5α was also maintained as a stock at -80°C in LB broth supplemented with 15\% glucose. Francisella mutants were grown on Chocolate II agar plates (GC base with 1\% isovitalex and 1\% Hemoglobin). For insect infections, Francisella colonies were grown at 37°C for 48 hours. E.coli DH5α was grown in LB broth or agar at 37°C for 20 hours. Cockroach Housing  Blaptica dubia cockroaches were purchased from Backwater Reptiles (www.backwaterreptiles.com) as juveniles. Roaches were stored in vented 32-gallon containers and kept at 30°C in the dark. A Containers were lined with a  two-inch strip of petroleum jellylined the top of each container  to prevent escape. Cardboard was used as substrate for cockroach nesting. Sufficient dry dog food was provided at all times in the containers, and fresh fruits or vegetables including carrots,corn,  oranges, and apples were given periodically as a source of water. Bacterial Infections of Cockroaches  To determine the virulence of each isolate, B. dubia roaches were challenged by intrahemoceol injection. Roaches weighing 0.7-1.0g were acclimated to 37°C at least seven days prior to bacterial infections. Bacterial suspensions of 106 were created from 48 hour colony cultures of each Francisella isolate. The suspensions were serial diluted in PBS immediately prior to injection infection. Injections were performed  on the dorsal side of the second to last abdominal segment using either a sharpened-gel-loading pipet pipette  tip or insulin syringe with a tridac stepper. Roaches were swabbed with alcohol prior to injection to lessen the risk of external contamination. A control group was injected with PBS to observe effects of injection trauma. Ten roaches were used for each experimental group and were housed in plastic containers containing dry dog food and saturated water crystals. Roaches were stored at 37°C and observed for survival for seven days. Roaches were recorded dead when they displayed no response to touch. Antibiotic Administration  Roaches were injected with F. tularensis LVS as described, and treated with antibiotics at 2, 48, and 96 hours post infection at t=0. infection.  The two methods used for antibiotic administration included systemic injections and oral delivery. Antibiotic concentrations were calculated by scaling recommended human dosage to four times the concentration of a roach’s mass (include actual dose concentations?). mass.  Final concentration of antibiotics were prepared in sterile water PBS  or appropriate diluent and a sterile 50\% sucrose solution. The injections of antibiotics were done similarly to bacterial injections, but alternated between left and right-side injections into the last abdominal segment of roaches to prevent repeated trauma. Oral administration was done using a micropipette micropipet  to manually feed roaches a drop precise volume  of the antibiotic sucrose solution to ensure the correct dose was consumed. Hemolymph Extraction  Inject B. dubia roaches as described were inoculated  with 106 CFU of 10^6  F. tularensis LVS cells at  in a PBS solution. Incubate roaches solution as described previously. Roaches were incubated  at 37°C with sufficient water crystals and food hydrating foods  to prevent dehydration. 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 hemplymph hemolymph  to calculate volume of hemolyph collected from each roach. Hemolymph was serial diluted 1:10 in PBS and plated onto BD  Chocolate II agar plates supplemented with Ampicillin ampicillin  and Trimethoprim trimethoprim  using spot plate method (source).  
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