Data from Temperature Dependent Activity of F.t. LVS

Today was the final day of my experiment from 4/14/15 where I wanted to see if there will be a difference in LD50s for Francisella tularensis in Blaptica dubia roaches.at four different temperatures. Below are to figures I generated from the results that were observed.

//BEE

Temperature Dependent LD50s of F.t. LVS in B. dubia Roaches

Today I started an experiment to evaluate the potential difference in lethal dose 50% (LD50s) for Francisella tularensis in Blaptica dubia roaches.

I used four different temperature variables: room temp (~20C), 30C, 37C, and 40C. Groups of roaches were acclimated to each temp 7 days prior to initial injections (t=0). They were provided with ample food and water to ensure roaches were healthy and viable before injections.
A 48-hour chocolate agar plate of F.t. LVS struck from -80C freezer stock was used. Due to the time it takes to inject the large amount of roaches needed for this experiment, there was concern regarding the viability of F.t. LVS during the entire experiment. A previous experiment indicated that viable F.t. LVS cells may decrease by one log within 5 hours of being suspended in ddH20, which would skew or LD50 values to be higher with later groups. Therefore, new dilution sets were prepared before each temperature variable for injections in the 5 groups of 10 roaches (as described in the protocol below).

1. Prepare 4 sets of 1:10 dilution tubes with sterile PBS.
2. Suspend on loop of F.t. LVS into PBS and serial dilute 1:10 to the -6 dilution.
3. Inject 20 microL of each dilution (-1 to -5) into a set of 10 roaches.
            a. Wipe roaches with alcohol prior to injections.
            b. Inject with a micro-step repeat pipette, and confirm volume prior to dilutions.
            c. Inject roaches the second to last segment of the abdomen on the dorsal side, under the armor plate.
            d. After injected, store roaches in oversize petri plates--make sure to tape shut to prevent escape.
4. Make titer spot plate using dilutions -1 to -6 on chocolate agar plates, with 10micrL spots; incubate at 37C.

Sample spot-plate, different dilutions were plated in the 4/14 experiment

5. Create a control group with 20micrL injections of PBS into 10 roaches.
            a. Follow same procedure used in step #3.
6. Store roaches at appropriate temperature and check daily for survival. Remove and discard dead roaches.
7. Repeat steps #3-5 for each of the temperature variables.




//BEE

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.

Microscopy Images from Listeria--Hemolymph extraction

Here are the images we obtained from 2/20/15 (Wax worms inoculated with GFP tagged Listeria--Hemolymph extractio). 

Wax worms inoculated with GFP tagged Listeria--Hemolymph extraction

Yesterday (day 1) I helped with a project to view the activity of  Listeria monocytogenes in the hemolymph of Galleria mellonella. The stain of Listeria we are using has a GPF tag. The protocols I used yesterday and today (day2) are described below.

Day 1-Inocculation of worms
1. From agar plate of GFP tagged Listeria, suspend a loopful of bacteria into 500μL of sterile PBS.
2. Dilute the suspesion 1:10 with PBS for two cell suspensions to be used for injections
3. For both the diluted and undiluted solutions, inject a group of 15 wax worms (Galleria mellonella) with 20μL.
     a. Use 70% alcohol to clean worms before injection
     b. Using a microstep-repeat injector with 31G needle and 1mL syringe, inject worms with appropriate suspension in the 2nd to last hind segment.
     c. Incubate worms at 37C groups of 8 per petri plates for 24 hours.

Day 2-Extraction and preparation of hemolymph
1. Prepare the following solutions
     a. 0.05% N-Phenylthiourea in PBS for use as anticoagulant buffer (AC buffer)
     b. 2% Formaldehyde in AC buffer
     c. 0.2% TritonX detergent in PBS
     d. 0.1% DAPI stain in PBS
2. Bleed worms into 1.5mL epp tubes containing10μL AC buffer.
     a. Using forceps to hold worms over tubes, puncture the worm with sterile 16G needle and allow hemolymph to drip into tube (250μLs were collected into each tube, with 3 tubes total)
3. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS and re-pellet.
 4.Discard supernatant and resuspend in 50μL of 2% Formaldehyde.
5. Incubate at room temp for 30 minutes.
6. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS, and re-pellet.
7. Discard supernatant and resuspend in 50μL of 0.2% TritonX detergent
8. Incubate at room temp for 15 minutes.
9. Pellet the cells at 12,000 rcf for 1 minute, wash with PBS and re-pellet.
10.Discard supernatant and resuspend in 50μL of 0.1% DAPI
11.Incubate at room temp for 15 minutes.
12.Pellet the cells at 12,000 rcf for 1 minute, wash in PBS and re-pellet.
13.Discard supernatant and resuspend in 50μL PBS.
14.Pellet again and remove 30μL of supernant.
15. Resuspend the remaining culture and place 10μL onto a slide.
16. Observe under microscope to view Listeria and hemocytes.

 //BEE

Antibiotic testing in Roach after inoculation with F.t. LVS

I have just concluded my recent experiment that is a follow up from 12-12-14. I followed the same protocol, but narrowed the antibiotics to be tested and changed the administration of antibiotics. Instead of feeding the roaches antibiotics, I delivered antibiotics through a systemic injections with a sterile PBS solution.

We found that when streptomycin was delivered directly into the hymolymph, with was effective at rescuing roaches from death by F. tularensis. This gives us evidence that B. dubia roaches have an intact gastrointestinal track that streptomycin cannot cross when given through an oral administration.

Here are the antibiotics that I delivered through needle injections:
Streptomycin (32μg/roach)
Resazurin (11μg/roach)
Doxycycline (32μg/roach)

Azithromycin (100μg/roach)
Ciprofloxacin (1μg/roach)

We saw the following data:
ABX,    Day     0     1      2     3     4     5     6     7
Streptomycin  10    10     9     9     8     8     8     8
Doxycicline    10    10    10   10    9    9      9     9
Ciprofloxacin 10    10     7     7     6     6     6     6
Azithromycin 10    10     9     2     0     0     0     0
Resazurin       10    10     6     2     1     0     0     0
No ABX         10    10    5     2     0     0     0     0
No LVS          10    10   10   10  10   10   10   10

//BEE