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

Launch of my experiment.com campaign

My crowd-funding campaign is up and running! You can view the link here. Make sure to check out the video; don't worry, it's not all about cockroaches.

//BEE

Gentamicin Protection Assay (LVS Growth Curve Inside Tropical Roach)

This week I am using the tropical roach for an in vivo growth curve of Fracisella tularensis LVS. I am using the following protocols and will add updates as the experiment progresses.


Protocol

1. Inject a large number of B. dubia roaches with 10^6 LVS at t=0hr.
• Number of roaches=[(# of time points x 2) x 8] x 1.5
• Extra 50% is for unexpected deaths.
• Store roaches in smaller containers with food and water crystals at 37C.
2. For each time point, randomly select 2 group of 8roaches.
• One group will receive a 16μg dose of gentamycin 2 hours prior to hemolymph extraction.
3. Extract hemolymph from roaches into chilled PBS with anticoagulant (0.05% N-Phenylthiourea) by removing the head of the roach and draining hemolymph into tubes.



Spot plates used for titer of 6hr extraction

.

• Weigh tubes before and after the addition of hemolymph to calculate volume extracted per roach. 
• Use sterile scissors for decapitations.
4. Serial dilute solutions 1:10 using 96-well plate to the -6 dilution.
5. Plate on cysteine heart agar supplemented with isovitalex, ampicillin, and trimethoprim using spot plate method (10micoliter spots) for 0 to -5 dilutions.
6. Repeat at desired time points (6, 12, 24, 48, 72, 96 hours post-infection). 
7. Record CFU on spot plates and determine CFU/ml of hemolymph for LVS in each roach. 

//BEE

Antibiotic Testing with LVS Injected Roaches

Oral administration of antibiotics

Today I started an experiment I tried a few weeks ago (12/4/14). I followed the same protocol and used the same antibiotics at the same concentrations. Roaches will receive antibiotics again after 48 and 96 hours post-infection.

//BEE

McFarland Standard for F.t LVS

Today I used the McFarland standards to find the CFU/ml of Francisella tularensis LVS that corresponds to each number.

I used the 0.5, 1, and 2 standards to create 2mL solutions of LVS that matched the visual optical density. The solutions were then serial diluted 1:10 to -7 and plated on CHOC II plates using the spot plate method (five 10microliter spots for each dilution in a quadrant of the plate).

The plates will be incubated at 37C for 48 hours until CFU can be counted.

//BEE

Antibiotic Testing with LVS Injected Roaches

Yesterday I began an experiment to study the effects of different antibiotics on the survival of roaches inoculated with a single concentration of Francisella tularensis LVS. I used the following procedure:

LVS dilutions used for injections

1. Streak out F. tularensis LVS on a CHOC II plate, incubate 37C for 48 hours
2. Make cell suspension in PBS (10e+6 cells/ml)
3. Serial dilute 1:10 to -7 
• Create a titter plate using CHOC II, with five 10μL spots for dilutions -4 to -7.
4. Incubate for at least 48 hours at 37C.
5. Inject roaches with single lethal LVS concentration on day 0, 10 roaches per group (one control  group with PBS).
• Use -1 dilution for injections.
• Inject 20μL using small gauge needle with syringe and microstep pipetter.
6. 2 hours post-infection: Feed roaches a 50% sucrose solution with solubilized antibiotics at desired concentration.
• Randomly choose groups of 10 roaches for each antibiotic.
• Place roaches in square containers that allow for air flow.
7. Store roaches at 37C with water crystals and dry dog food.
8. Remove water and food 24 hours before antibiotic feeding.
9. Repeat antibiotic feeding every two days (48 and 96 hours post-infection).



Preparation of abx in sucrose

10. Observe and record survival.

Antibiotics:
Streptomycin (32μg/roach)
Gentamicin (32μg/roach)
Resazurin (11μg/roach)
Vancomycin (15μg/roach)
Ampicillin (100μg/roach)
Chloramphenicol (32μg/roach)
Amikacin (32μg/roach)
Doxycycline (32μg/roach)
Ceftriaxone (8μg/roach)

Feeding of roaches with 50% sucrose solution

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

Storage of roaches in containers at 37C

 




//BEE

LD50s of Francisella LVS in Blaptica dubia roaches--Replicate

Storage of roaches in new containers

On wednesday, 11/19/14, I repeated injections of Ft LVS and 4 mutants that were done on 11/14/14. The same protocol was used. Because of the recent concern of roach dehydration affecting survival, the experimental roaches were moved from petri plates to larger contains and were provided water crystals along with dry dog food after 24 hours. Survival count will be recorded in my Onedrive file here.

Water crystals to prevent dehydration

//BEE